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A prosleptic syllogism ( / p r ə ˈ s l ɛ p t ɪ k / ; from Greek πρόσληψις proslepsis "taking in addition") is a class of syllogisms that use a prosleptic proposition as one of the premises.
The term originated with Theophrastus . [ 1 ]
Prosleptic syllogisms are classified in three figures, or potential arrangements of the terms according to the figure of the prosleptic proposition used.
Consequently, a third figure prosleptic syllogism would read "A is universally affirmed of everything of which G is universally affirmed; G is universally affirmed of B; therefore, A is universally affirmed of B." ("All G are A; all B are G; therefore, all B are A" or "Statement A is always true of everything for which statement G is always true; statement G is true of all things B; therefore, statement A is true of all things B.")
This linguistics article is a stub . You can help Wikipedia by expanding it . | https://en.wikipedia.org/wiki/Prosleptic_syllogism |
In mathematics , more precisely in algebra , a prosolvable group (less common: prosoluble group ) is a group that is isomorphic to the inverse limit of an inverse system of solvable groups . Equivalently, a group is called prosolvable , if, viewed as a topological group , every open neighborhood of the identity contains a normal subgroup whose corresponding quotient group is a solvable group. | https://en.wikipedia.org/wiki/Prosolvable_group |
Prosoplasia (from Ancient Greek : πρόσω prósō , "forward" + πλάσις plasis , "formation") is the differentiation of cells either to a higher function or to a higher level of organization. [ 1 ]
Assuming an increasing cellular peculiarity from a presupposed stem-cell fate, prosoplasia is therefore a forward differentiation, unlike anaplasia (a backward differentiation).
Examples of prosoplasia include the forward differentiation of cells in the mucosa in Warthin's tumor . [ 2 ]
This oncology article is a stub . You can help Wikipedia by expanding it . | https://en.wikipedia.org/wiki/Prosoplasia |
Prospect theory is a theory of behavioral economics , judgment and decision making that was developed by Daniel Kahneman and Amos Tversky in 1979. [ 1 ] The theory was cited in the decision to award Kahneman the 2002 Nobel Memorial Prize in Economics . [ 2 ]
Based on results from controlled studies , it describes how individuals assess their loss and gain perspectives in an asymmetric manner (see loss aversion ). For example, for some individuals, the pain from losing $1,000 could only be compensated by the pleasure of earning $2,000. Thus, contrary to the expected utility theory (which models the decision that perfectly rational agents would make), prospect theory aims to describe the actual behavior of people.
In the original formulation of the theory, the term prospect referred to the predictable results of a lottery . However, prospect theory can also be applied to the prediction of other forms of behaviors and decisions.
Prospect theory challenges the expected utility theory developed by John von Neumann and Oskar Morgenstern in 1944 and constitutes one of the first economic theories built using experimental methods .
In the draft received by the economist Richard Thaler in 1976, the term "Value Theory" was used instead of Prospect Theory. Later on, Kahneman and Tversky changed the title to Prospect Theory to avoid possible confusions. According to Kahneman, the new title was 'meaningless.' [ 3 ]
Prospect theory stems from loss aversion , where the observation is that agents asymmetrically feel losses greater than that of an equivalent gain. It centralises around the idea that people conclude their utility from gains and losses relative to a certain "neutral" reference point regarding their current individual situation. Thus, rather than making decisions like a rational agent maximizing a fixed expected utility , value decisions are made relative to the current neutral situation, not following any absolute measure of utility. [ 4 ] [ 5 ]
Consider two scenarios;
It is assumed that the agent's individual utility is proportional to the dollar amount (e.g. $1000 would be twice as useful as $500). Prospect theory suggests that;
These two examples are thus in contradiction with the theory of expected utility, which leads only to choices with the maximum utility. Also, the concavity for gains and convexity for losses implies diminishing marginal utility with increasing gains/losses. In other words, someone who has more money has a lower desire for a fixed amount of gain (and lower aversion to a fixed amount of loss) than someone who has less money.
The theory continues with a second concept, based on the observation that people attribute excessive weight to events with low probability and insufficient weight to events with high probability. For example, individuals may unconsciously treat an outcome with a probability of 99% as if its probability were 95%, and an outcome with probability of 1% as if it had a probability of 5%. Under- and over-weighting of probabilities is importantly distinct from under- and over-estimating probabilities, a different type of cognitive bias observed for example in the overconfidence effect .
The theory describes the decision processes in two stages: [ 1 ]
The formula that Kahneman and Tversky assume for the evaluation phase is (in its simplest form) given by:
where V {\displaystyle V} is the overall or expected utility of the outcomes to the individual making the decision, x 1 , x 2 , … , x n {\displaystyle x_{1},x_{2},\ldots ,x_{n}} are the potential outcomes and p 1 , p 2 , … , p n {\displaystyle p_{1},p_{2},\dots ,p_{n}} their respective probabilities and v {\displaystyle v} is a function that assigns a value to an outcome. The value function that passes through the reference point is s-shaped and asymmetrical. Losses hurt more than gains feel good (loss aversion). This differs from expected utility theory , in which a rational agent is indifferent to the reference point. In expected utility theory, the individual does not care how the outcome of losses and gains are framed. The function π {\displaystyle \pi } is a probability weighting function and captures the idea that people tend to overreact to small probability events, but underreact to large probabilities. Let ( x , p ; y , q ) {\displaystyle (x,p;y,q)} denote a prospect with outcome x {\displaystyle x} with probability p {\displaystyle p} and outcome y {\displaystyle y} with probability q {\displaystyle q} and nothing with probability 1 − p − q {\displaystyle 1-p-q} . If ( x , p ; y , q ) {\displaystyle (x,p;y,q)} is a regular prospect (i.e., either p + q < 1 {\displaystyle p+q<1} , or x ≥ 0 ≥ y {\displaystyle x\geq 0\geq y} , or x ≤ 0 ≤ y {\displaystyle x\leq 0\leq y} ), then:
V ( x , p ; y , q ) = π ( p ) ν ( x ) + π ( q ) ν ( y ) {\displaystyle V(x,p;y,q)=\pi (p)\nu (x)+\pi (q)\nu (y)}
However, if p + q = 1 {\displaystyle p+q=1} and either x > y > 0 {\displaystyle x>y>0} or x < y < 0 {\displaystyle x<y<0} , then:
V ( x , p ; y , q ) = ν ( y ) + π ( p ) [ ν ( x ) − ν ( y ) ] {\displaystyle V(x,p;y,q)=\nu (y)+\pi (p)\left[\nu (x)-\nu (y)\right]}
It can be deduced from the first equation that ν ( y ) + ν ( − y ) > ν ( x ) + ν ( − x ) {\displaystyle \nu (y)+\nu (-y)>\nu (x)+\nu (-x)} and ν ( − y ) + ν ( − x ) > ν ( x ) + ν ( − x ) {\displaystyle \nu (-y)+\nu (-x)>\nu (x)+\nu (-x)} . The value function is thus defined on deviations from the reference point, generally concave for gains and commonly convex for losses and steeper for losses than for gains. If ( x , p ) {\displaystyle (x,p)} is equivalent to ( y , p q ) {\displaystyle (y,pq)} then ( x , p r ) {\displaystyle (x,pr)} is not preferred to ( y , p q r ) {\displaystyle (y,pqr)} , but from the first equation it follows that π ( p ) ν ( x ) + π ( p q ) ν ( y ) = π ( p q ) ν ( y ) {\displaystyle \pi (p)\nu (x)+\pi (pq)\nu (y)=\pi (pq)\nu (y)} , which leads to π ( p r ) ν ( x ) ≤ π ( p q r ) ν ( y ) {\displaystyle \pi (pr)\nu (x)\leq \pi (pqr)\nu (y)} , therefore:
π ( p q ) π ( p ) ≤ π ( p q r ) π ( p r ) {\displaystyle {\frac {\pi \left(pq\right)}{\pi \left(p\right)}}\leq {\frac {\pi \left(pqr\right)}{\pi \left(pr\right)}}}
This means that for a fixed ratio of probabilities the decision weights are closer to unity when probabilities are low than when they are high. In prospect theory, π {\displaystyle \pi } is never linear . In the case that x > y > 0 {\displaystyle x>y>0} , p > p ′ {\displaystyle p>p'} and p + q = p ′ + q ′ < 1 , {\displaystyle p+q=p'+q'<1,} prospect ( x , p ′ ; y , q ) {\displaystyle (x,p';y,q)} dominates prospect ( x , p ′ ; y , q ′ ) {\displaystyle (x,p';y,q')} , which means that π ( p ) ν ( x ) + π ( q ) ν ( y ) > π ( p ′ ) ν ( x ) + π ( q ′ ) ν ( y ) {\displaystyle \pi (p)\nu (x)+\pi (q)\nu (y)>\pi (p')\nu (x)+\pi (q')\nu (y)} , therefore:
π ( p ) − π ( p ′ ) π ( q ′ ) − π ( q ) ≤ ν ( y ) ν ( x ) {\displaystyle {\frac {\pi \left(p\right)-\pi (p')}{\pi \left(q'\right)-\pi \left(q\right)}}\leq {\frac {\nu \left(y\right)}{\nu \left(x\right)}}}
As y → x {\displaystyle y\rightarrow x} , π ( p ) − π ( p ′ ) → π ( q ′ ) − π ( q ) {\displaystyle \pi (p)-\pi (p')\rightarrow \pi (q')-\pi (q)} , but since p − p ′ = q ′ − q {\displaystyle p-p'=q'-q} , it would imply that π {\displaystyle \pi } must be linear; however, dominated alternatives are brought to the evaluation phase since they are eliminated in the editing phase. Although direct violations of dominance never happen in prospect theory, it is possible that a prospect A dominates B, B dominates C but C dominates A.
To see how prospect theory can be applied, consider the decision to buy insurance. Assume the probability of the insured risk is 1%, the potential loss is $1,000 and the premium is $15. If we apply prospect theory, we first need to set a reference point. This could be the current wealth or the worst case (losing $1,000). If we set the frame to the current wealth, the decision would be to either
1. Pay $15 for insurance, which yields a prospect-utility of v ( − 15 ) {\displaystyle v(-15)} ,
OR
2. Enter a lottery with possible outcomes of $0 (probability 99%) or −$1,000 (probability 1%), which yields a prospect-utility of π ( 0.01 ) × v ( − 1000 ) + π ( 0.99 ) × v ( 0 ) = π ( 0.01 ) × v ( − 1000 ) {\displaystyle \pi (0.01)\times v(-1000)+\pi (0.99)\times v(0)=\pi (0.01)\times v(-1000)} .
According to prospect theory,
The comparison between π ( 0.01 ) {\displaystyle \pi (0.01)} and v ( − 15 ) / v ( − 1000 ) {\displaystyle v(-15)/v(-1000)} is not immediately evident. However, for typical value and weighting functions, π ( 0.01 ) > v ( − 15 ) / v ( − 1000 ) {\displaystyle \pi (0.01)>v(-15)/v(-1000)} , and hence π ( 0.01 ) × v ( − 1000 ) < v ( − 15 ) {\displaystyle \pi (0.01)\times v(-1000)<v(-15)} . That is, a strong overweighting of small probabilities is likely to undo the effect of the convexity of v {\displaystyle v} in losses, making the insurance attractive.
If we set the frame to -$1,000, we have a choice between v ( 985 ) {\displaystyle v(985)} and π ( 0.99 ) × v ( 1000 ) {\displaystyle \pi (0.99)\times v(1000)} . In this case, the concavity of the value function in gains and the underweighting of high probabilities can also lead to a preference for buying the insurance.
The interplay of overweighting of small probabilities and concavity-convexity of the value function leads to the so-called fourfold pattern of risk attitudes : [ 7 ] risk-averse behavior when gains have moderate probabilities or losses have small probabilities; risk-seeking behavior when losses have moderate probabilities or gains have small probabilities.
Below is an example of the fourfold pattern of risk attitudes. The first item in each quadrant shows an example prospect (e.g. 95% chance to win $10,000 is high probability and a gain). The second item in the quadrant shows the focal emotion that the prospect is likely to evoke. The third item indicates how most people would behave given each of the prospects (either Risk Averse or Risk Seeking). The fourth item states expected attitudes of a potential defendant and plaintiff in discussions of settling a civil suit. [ 8 ]
Probability distortion is that people generally do not look at the value of probability uniformly between 0 and 1. Lower probability is said to be over-weighted (that is, a person is overly concerned with the outcome of the probability) while medium to high probability is under-weighted (that is, a person is not concerned enough with the outcome of the probability). The exact point in which probability goes from over-weighted to under-weighted is arbitrary, but a good point to consider is probability = 0.33. A person values probability = 0.01 much more than the value of probability = 0 (probability = 0.01 is said to be over-weighted). However, a person has about the same value for probability = 0.4 and probability = 0.5. Also, the value of probability = 0.99 is much less than the value of probability = 1, a sure thing (probability = 0.99 is under-weighted). A little more in depth when looking at probability distortion is that π ( p ) + π (1 − p ) < 1 (where π ( p ) is probability in prospect theory). [ 9 ]
Myopic loss aversion (MLA), a concept derived from prospect theory, refers to the natural tendency of humans to focus on short-term losses and gains and to weigh them more heavily than long-term losses and gains. This bias can lead to seemingly poorer decision making, as individuals may focus towards avoiding immediate losses instead of achieving long-term gains.
A prolific study that examined myopic loss aversion was conducted by Gneezy and Potters in 1997.[9] In this study, participants engaged in a straightforward betting game in which they could either place a bet on a coin landing , or they could choose to not bet at all. The participants were provided with a fixed amount of money, and held the task to maximize their earnings over a series of rounds.
The results of the study exhibited that participants were more likely to place a bet when they had just lost money in the previous round, and they were more likely to avoid a bet when they had just won money in the previous round. This behavior is consistent with myopic loss aversion theory, as the participants were placing greater magnitude on their short-term gains and losses instead of their overall earnings over the course of the study.
Additionally, the findings revealed that the participants that were provided with a higher amount of money at the beginning of the study tended to be more risk-averse than those who were given a lower starting amount. This observation supports the concept of diminishing sensitivity to changes in wealth predicted by prospect theory.
Overall, the study by Gneezy and Potters emphasizes the existence of myopic loss aversion, demonstrating how this bias can result in non-optimal decisions. By analyzing how prospect theory and myopic loss aversion influence decision-making, it provides the ability for researchers and policymakers to create interventions that help people make more informed choices and attain their long-term goals.
When referring to investment decisions, myopic loss aversion has the ability to lead to investment decisions that can be of a more conservative approach. For instance, investors potentially overreact to dips in stock prices in their stock portfolio, which causes feelings of fear and anxiety of profit loss. This reaction from investors has the ability to lead in a loss in profit due to selling off their stock. [ 1 ] Studies in behavioral finance analyzed this pattern, observing that there is a tendency to avoid high-reward options in the market, as the risk of short-term loss potentially influences the broker.
Acclaimed behavioral economists Benartzi and Thaler analyzed this concept, calling it the "equity premium puzzle [ 2 ] ." This puzzle refers to the fact that stocks, in terms of historical statistics, exceed profits in comparison to bonds over extended periods of time. More interestingly, they observed that newer investors tend not to emphasize stocks over bonds. This phenomenon has been linked by Benartzi and Thaler to myopic loss aversion due to the lack of emphasis on stocks by young investors, as young investors tended to abandon stocks due to minor dips in the market.
This behavior can lead to a decreases market predictability, as investors act on short-term losses by selling their stocks, there can be a ripple effect that intensifies dips in the economy. As investors that are heavily influenced by the market decline sell their stocks, the now increased amount of shares due to mass sell-offs further lower prices. This hypothetical community of investors react along with falling stock prices, causing them to sell, potentially causing the stock price to lower as a whole. This concept, investor anxiety, can potentially emphasize the want to sell of investments for security reasons, regardless of long-term profit potential. This constant market fluctuation is directly related to market stability.
An example of this effect was seen during economic crises such as the 2008 financial crash, when panic induced sell-offs heavily impacted market stability. The period prior to the Great Recession had a "decade-long expansion in US housing market activity peaked in 2006 [ 4 ] ," which came to a halt in 2007. As the trends prior to 2008 hinted at the fall of mortgage pricing, real-estate investors reacted promptly. The mass sell-offs of mortgaged-backed investments led to a similar instability in other markets, including credit markets, and the stock market.
Some behaviors observed in economics, like the disposition effect or the reversing of risk aversion / risk seeking in case of gains or losses (termed the reflection effect ), can also be explained by referring to the prospect theory.
An important implication of prospect theory is that the way economic agents subjectively frame an outcome or transaction in their mind affects the utility they expect or receive. Narrow framing is a derivative result which has been documented in experimental settings by Tversky and Kahneman, [ 6 ] whereby people evaluate new gambles in isolation, ignoring other relevant risks. This phenomenon can be seen in practice in the reaction of people to stock market fluctuations in comparison with other aspects of their overall wealth; people are more sensitive to spikes in the stock market as opposed to their labor income or the housing market. [ 4 ] It has also been shown that narrow framing causes loss aversion among stock market investors. [ 10 ] And the work of Tversky and Kahneman is largely responsible for the advent of behavioral economics , and is used extensively in mental accounting . [ 11 ]
The digital age has brought the implementation of prospect theory in software. Framing and prospect theory has been applied to a diverse range of situations which appear inconsistent with standard economic rationality: the equity premium puzzle , the excess returns puzzle and long swings/PPP puzzle of exchange rates through the endogenous prospect theory of Imperfect Knowledge Economics, the status quo bias , various gambling and betting puzzles, intertemporal consumption , and the endowment effect . It has also been argued that prospect theory can explain several empirical regularities observed in the context of auctions (such as secret reserve prices) which are difficult to reconcile with standard economic theory. [ 12 ]
Online pay-per bid auction sites are a classic example of decision making under risk. Previous attempts at predicting consumer behavior have shown that utility theory does not sufficiently describe decision making under risk. When prospect theory was added to a previously existing model that was attempting to explain consumer behavior during auctions, out-of-sample predictions were shown to be more accurate than a corresponding expected utility model. Specifically, prospect theory was boiled down to certain elements: preference, loss aversion and probability weighting. These elements were then used to find a backward solution on 537,045 auctions. The greater accuracy may be explained by the new model having the ability to correct for two behavioral irrationalities: The sunk cost fallacy and average auctioneer revenues above current retail price. These findings would also imply that the using prospect theory as a descriptive theory of decision making under risk is also accurate in situations where risk arises through the interactions of different people. [ 13 ]
Given the necessary degree of uncertainty for which prospect theory is applied, it should come as no surprise that it and other psychological models are applied extensively in the context of political decision-making. [ 14 ] Both rational choice and game theoretical models generate significant predictive power in the analysis of politics and international relations (IR). But prospect theory, unlike the alternative models, (1) is "founded on empirical data", (2) allows and accounts for dynamic change, (3) addresses previously-ignored modular elements, (4) emphasizes the situation in the decision-making process, (5) "provides a micro-foundational basis for the explanation of larger phenomena", and (6) stresses the importance of loss in utility and value calculations. [ 15 ] Moreover, again unlike other models, prospect theory "asks different sorts of questions, seeks different evidence, and reaches different conclusions." [ 15 ] However, there exist shortcomings inherent in prospect theory's political application, such as the dilemma regarding an actor's perceived position on the gain-loss domain spectrum, and the discordance between ideological and pragmatic (i.e. 'in the lab' versus 'in the field') assessments of an actor's propensity toward seeking or avoiding risk. [ 16 ]
That said, political scientists have applied prospect theory to a wide range of issues in domestic and comparative politics. For example, they have found that politicians are more likely to phrase a radical economic policy as one ensuring 90% employment rather than 10% unemployment, because framing it as the former puts the citizenry in a "domain of gain," which is thereby conducive to greater populace satisfaction. [ 16 ] On a broader scale: Consider an administration debating the implementation of a controversial reform, and that such a reform yields a small chance for a widespread revolt. "[T]he disutility induced by loss aversion," even with minute probabilities of said insurrection, will dissuade the government from moving forward with the reform. [ 14 ]
Scholars have employed prospect theory to shed light on a number of issue areas in politics. For example, Kurt Weyland finds that political leaders do not always undertake bold and politically risky domestic initiatives when they are at the pinnacle of their power. Instead, such policies often appear to be risky gambits initiated by politically vulnerable regimes. He suggests that in Latin America, politically weakened governments were more likely to implement fundamental and economically painful market-oriented reforms, even though they were more vulnerable to political backlash. [ 17 ] Barbara Vis and Kees van Kersbergen have reached a similar conclusion in their investigation of Italian welfare reforms. [ 18 ]
Maria Fanis uses prospect theory to show how risk acceptance can help domestic groups overcome collective action problems inherent to coalition building. She suggests that collective action is more likely in a perceive domain of loss because individuals become more willing to accept the risk of free riding by others. In Chile, this process led domestic interest groups to form unlikely political coalitions. [ 19 ] Zeynep Somer-Topcu's research suggests that political parties respond more strongly to electoral defeat than to success in the next election cycle. As prospect theory predicts, parties are more likely to shift their policies in response to a vote loss in the previous election cycle compared to a vote gain. [ 20 ] Lawrence Kuznar and James Lutz find that loss frames can increase support of individuals for terrorist groups. [ 21 ]
International relations theorists have applied prospect theory to a wide range of issues in world politics, especially security-related matters. [ 22 ] [ 16 ] For example, in war-time , policy-makers, when in a perceived domain of loss, are more likely to take risks that would otherwise have been avoided, e.g. "gambling on a risky rescue mission", or implementing radical domestic reform to support military efforts. [ 16 ]
Early applications of prospect theory in International Relations emphasized the potential to explain anomalies in foreign policy decision-making that remained difficult to account for on the basis of rational choice theory. They developed detailed qualitative case studies of specific foreign policy decisions to explore the role of framing effects in choice selection. For example, Rose McDermott applied prospect theory to a series of case studies in American foreign policy, including the Suez Crisis in 1956, the U-2 Crisis in 1960, the U.S. decision to admit the Iranian shah to the United States in 1979, and the U.S. decision to carry out a hostage rescue mission in 1980. [ 23 ] Jeffrey Berejikian employed prospect theory to analyze the genesis of the Montreal Protocol , a landmark environmental agreement. [ 24 ]
William Boettcher integrated elements of prospect theory with psychological research on personality dispositions to construct a “Risk Explanation Framework,” which he used to analyze foreign-policy decision making. He then evaluated the framework against six case studies on presidential foreign policy decision-making. [ 25 ]
Applications of prospect theory in the context of insurance seek to explain the consumer choices . Syndor (2010) suggests that the probability weighting aspect of prospect theory aims to explain the behaviour of the consumers who choose a higher premium for a reduced deductible even when the annualised claim rate is very low (approximately 5%). In a study of 50,000 customers, they had four options for the deductibles on their policy; $100, $250, $500, $1000. From this it was found that a $500 deductible resulted in a $715 annual premium and $1000 deductible being $615. The customers that chose the $500 deductible were paying an additional $100 per year even though the chance that a claim will be made is extremely low, and the deductible be paid. Under the expected utility framework, this can only be realised through high levels of risk aversion. Households place a greater weight on the probability that a claim will be made when choosing a policy, thus it is suggested that the reference point of the household significantly influences the decisions when it comes to premiums and deductibles. This is consistent with the theory that people assign excessive weight to scenarios with low probabilities and insufficient weight to events with high probability. [ 12 ] [ 26 ]
The original version of prospect theory gave rise to violations of first-order stochastic dominance . That is, prospect A might be preferred to prospect B even if the probability of receiving a value x or greater is at least as high under prospect B as it is under prospect A for all values of x, and is greater for some value of x. Later theoretical improvements overcame this problem, but at the cost of introducing intransitivity in preferences. A revised version, called cumulative prospect theory overcame this problem by using a probability weighting function derived from rank-dependent expected utility theory. Cumulative prospect theory can also be used for infinitely many or even continuous outcomes (for example, if the outcome can be any real number ). An alternative solution to overcome these problems within the framework of (classical) prospect theory has been suggested as well. [ 27 ]
The reference point in the prospect theory inverse s-shaped graph also could lead to limitations due to it possibly being discontinuous at that point and having a geometric violation. This would lead to limitations in regards to accounting for the zero-outcome effect, the absence of behavioral conditionality in risky decisions as well as limitations in deriving the curve. A transitionary concave-convex universal system was proposed to eliminate this limitation. [ 28 ]
Critics from the field of psychology argued that even if Prospect Theory arose as a descriptive model, it offers no psychological explanations for the processes stated in it. [ 29 ] Furthermore, factors that are equally important to decision making processes have not been included in the model, such as emotion. [ 30 ]
A relatively simple ad hoc decision strategy, the priority heuristic , has been suggested as an alternative model. While it can predict the majority choice in all (one-stage) gambles in Kahneman and Tversky (1979), and predicts the majority choice better than cumulative prospect theory across four different data sets with a total of 260 problems, [ 31 ] this heuristic, however, fails to predict many simple decision situations that are typically not tested in experiments and it also does not explain heterogeneity between subjects. [ 32 ]
An international survey in 53 countries, published in Theory and Decision in 2017, confirmed that prospect theory describes decisions on lotteries well, not only in Western countries, but across many different cultures. [ 33 ] The study also found cultural and economic factors influencing systematically average prospect theory parameters.
A study published in Nature Human Behaviour in 2020 replicated research on prospect theory and concluded that it successfully replicated: "We conclude that the empirical foundations for prospect theory replicate beyond any reasonable thresholds." [ 34 ]
Although Prospect Theory is a largely celebrated idea in behavioural economics it does have limitations. The reference point has been argued to be difficult to precisely determine in any given context. Many external factors can influence what the reference point is and thus makes it difficult to define what a “gain” and a “loss” actually is. Kőszegi and Rabin (2007) present the idea of a personal equilibrium in decision making. This is essentially the premise that expectations and context have a large impact on determining the reference point and therefore the perception of “gains” and “losses”. Considering personal equilibrium and choice with risk creates even more ambiguity about the perception of what the reference point may be. [ 11 ]
Some critics have charged that while prospect theory seeks to predict what people choose, it does not adequately describe the actual process of decision-making. For example, Nathan Berg and Gerd Gigerenzer claim that neither classical economics nor prospect theory provide a convincing explanation of how people actually make decisions. They go so far as to claim that prospect theory is even more demanding of cognitive resources than classical expected utility theory. [ 35 ]
Moreover, scholars have raised doubts about the degree to which framing effects matter. For instance, John List argues that framing effects diminish in complex decision environments. His experimental evidence suggests that as actors gain experience with the consequences of competitive markets, they behave more like rational actors and the impact of prospect theory diminishes. [ 36 ]
Steven Kachelmeier and Mohamed Shehata find little support for prospect theory among experimental subjects in China. They do not, however, make a cultural argument against prospect theory. Rather, they conclude that when payoffs are large relative to net wealth, the effect of prospect theory diminishes. [ 37 ] | https://en.wikipedia.org/wiki/Prospect_theory |
Prostaglandin E is a family of naturally occurring prostaglandins that are used as medications.
Types include:
Both types are on the World Health Organization's List of Essential Medicines . [ 1 ]
Prostaglandin E play an important role in thermoregulation of the human brain. Decreased formation of prostaglandin E through inhibition of cyclooxygenase is the basis for the antipyretic of nonsteroidal anti-inflammatory drugs (NSAIDs).
This biochemistry article is a stub . You can help Wikipedia by expanding it . | https://en.wikipedia.org/wiki/Prostaglandin_E |
The prostaglandin E 2 ( PGE 2 ) receptors are G protein-coupled receptors that bind and are activated by prostaglandin E 2 . They are members of the prostaglandin receptors class of receptors and include the following Protein isoforms :
An antagonist of a prostaglandin E 2 receptor has been shown to serve as an affective contraceptive for female macaques while unaffecting their menstrual cyclicity as well as hormonal patterns. The exact reason behind the reduced amount of successful pregnancies of primates during the study is unclear due a number of possibilities that may affect such result. [ 1 ] Inhibition of the prostaglandin EP 4 receptor has been shown to inhibit tumor growth, angiogenesis , lymphangiogenesis , and metastasis . [ 2 ] [ 3 ]
Prostaglandins are derived from the parent molecule arachidonic acid . The synthesis of prostaglandins can be blocked by anti-inflammatory drugs such as ibuprofen . Nonsteroidal anti-inflammatory drugs (NSAIDs) block the synthesis of cyclooxygenases (COX) which in turn produce prostaglandins. [ 4 ] Prostaglandins (PG) are the result of an enzyme cascade pathway that includes two enzymes cyclooxygenase and PG synthase. Prostaglandin E 2 is produced by PGE synthase via the activation of EP1-4 receptors. Prostaglandin E 2 is associated with the development of vascular diseases that lead to inflammation in the body. In the human body, PGE 2 is involved in the control of the vascular smooth muscle, cell migration, and the division of a cell into two daughter cells. [ 5 ] The process of producing two daughter cells via cell division is called cell proliferation .
This biochemistry article is a stub . You can help Wikipedia by expanding it . | https://en.wikipedia.org/wiki/Prostaglandin_E2_receptor |
Prostaglandin H 2 ( PGH 2 ), or prostaglandin H2 ( PGH2 ), is a type of prostaglandin and a precursor for many other biologically significant molecules. It is synthesized from arachidonic acid in a reaction catalyzed by a cyclooxygenase enzyme. [ 2 ] The conversion from arachidonic acid to prostaglandin H 2 is a two-step process. First, COX-1 catalyzes the addition of two free oxygens to form the 1,2-dioxane bridge and a peroxide functional group to form prostaglandin G 2 (PGG 2 ). [ 3 ] Second, COX-2 reduces the peroxide functional group to a secondary alcohol , forming prostaglandin H 2 . Other peroxidases like hydroquinone have been observed to reduce PGG 2 to PGH 2 . [ 4 ] PGH 2 is unstable at room temperature, with a half life of 90–100 seconds, [ 1 ] so it is often converted into a different prostaglandin.
It is acted upon by:
It rearranges non-enzymatically to:
Functions of prostaglandin H 2 :
Effects of aspirin on prostaglandin H 2 :
This biochemistry article is a stub . You can help Wikipedia by expanding it . | https://en.wikipedia.org/wiki/Prostaglandin_H2 |
In molecular biology , prostanoids are active lipid mediators that regulate inflammatory response . Prostanoids are a subclass of eicosanoids consisting of the prostaglandins (mediators of inflammatory and anaphylactic reactions), the thromboxanes (mediators of vasoconstriction ), and the prostacyclins (active in the resolution phase of inflammation). [ 1 ] Prostanoids are seen to target NSAIDS which allow for therapeutic potential. Prostanoids are present within areas of the body such as the gastrointestinal tract , urinary tract , respiratory and cardiovascular systems , reproductive tract and vascular system . Prostanoids can even be seen with aid to the water and ion transportation within cells.
Prostanoids were discovered through biological research studies conducted in the 1930s. [ 2 ] [ 3 ] The first discovery was seen through semen by a Swedish Physiologist Ulf von Euler , who assumed they originated from the prostate. After intensive study throughout the 1960-1970s Sune K. Bergström and Bengt Ingemar Samuelsson and British biochemist Sir John Robert Vane were able to understand the function and chemical formation of Prostanoids: receiving a Nobel Prize for their analysis of prostanoids.
Cyclooxygenase ( COX ) catalyzes the conversion of the free essential fatty acids to prostanoids by a two-step process.
In the first step, two molecules of O 2 are added as two peroxide linkages and a 5-member carbon ring is forged near the middle of the fatty acid chain. This forms the short-lived, unstable intermediate Prostaglandin G (PGG).
One of the peroxide linkages sheds a single oxygen, forming PGH. (See diagrams and more detail at Cyclooxygenase ). All other prostanoids originate from PGH (as PGH 1 , PGH 2 , or PGH 3 ).
The image at right shows how PGH 2 (derived from Arachidonic acid ) is converted:
Arachidonic acid is made up of a 20-Carbon unnatural poly unsaturated Omega-fatty acid. [ 1 ] Arachidonic acid presents within the phospholipid bi-layer as well as in the plasma membrane of a cell. With Arachidonic acid prostaglandins are formed through synthesis and oxygenation of enzymes. Active lipids in the oxylipin family derive from the synthesis of Cyclooxygenase or Prostaglandins.
The three classes of prostanoids have distinctive rings in the center of the molecule. They differ in their structures and do not share common structure as Thromboxane. The PGH compounds (parents to all the rest) have a 5-carbon ring, bridged by two oxygens (a peroxide .) The derived prostaglandins contain a single, unsaturated 5-carbon ring. In prostacyclins, this ring is conjoined to another oxygen-containing ring. In thromboxanes the ring becomes a 6-member ring with one oxygen.
Production of PGE 2 in bacterial and viral infections appear to be stimulated by certain cytokines, e.g., interleukin-1 . [ 4 ]
This biochemistry article is a stub . You can help Wikipedia by expanding it . | https://en.wikipedia.org/wiki/Prostanoid |
2ZCH , 2ZCK , 2ZCL , 3QUM
354
18048
ENSG00000142515
ENSMUSG00000066513
P07288
P00757
NM_145864
NM_010915
NP_001025218 NP_001025219 NP_001639
NP_035045
Prostate-specific antigen ( PSA ), also known as gamma-seminoprotein or kallikrein-3 ( KLK3 ), P-30 antigen, is a glycoprotein enzyme encoded in humans by the KLK3 gene . PSA is a member of the kallikrein -related peptidase family and is secreted by the epithelial cells of the prostate gland in men and the paraurethral glands in women. [ 5 ]
PSA is produced for the ejaculate , where it liquefies semen in the seminal coagulum and allows sperm to swim freely. [ 6 ] It is also believed to be instrumental in dissolving cervical mucus , allowing the entry of sperm into the uterus . [ 7 ]
PSA is present in small quantities in the serum of men with healthy prostates, but is often elevated in the presence of prostate cancer or other prostate disorders. [ 8 ] PSA is not uniquely an indicator of prostate cancer, but may also detect prostatitis or benign prostatic hyperplasia . [ 9 ]
Clinical practice guidelines for prostate cancer screening vary and are controversial, in part due to uncertainty as to whether the benefits of screening ultimately outweigh the risks of overdiagnosis and overtreatment. [ 10 ] In the United States, the Food and Drug Administration (FDA) has approved the PSA test for annual screening of prostate cancer in men of age 50 and older. [ medical citation needed ] The patient is required to be informed of the risks and benefits of PSA testing prior to performing the test. [ medical citation needed ]
In the United Kingdom, the National Health Service (NHS) as of 2018 [update] does not mandate, nor advise for PSA test, but allows patients to decide based on their doctor's advice. [ 11 ] The NHS does not offer general PSA screening, for similar reasons. [ 12 ]
PSA levels between 4 and 10 ng/mL (nanograms per milliliter) are considered to be suspicious, and consideration should be given to confirming the abnormal PSA with a repeat test. If indicated, prostate biopsy is performed to obtain a tissue sample for histopathological analysis. [ citation needed ]
While PSA testing may help 1 in 1,000 avoid death due to prostate cancer, 4 to 5 in 1,000 would die from prostate cancer after 10 years even with screening. This means that PSA screening may reduce mortality from prostate cancer by up to 25%. Expected harms include anxiety for 100–120 receiving false positives, biopsy pain, and other complications from biopsy for false positive tests. [ medical citation needed ]
Use of PSA screening tests is also controversial due to questionable test accuracy. The screening can present abnormal results even when a man does not have cancer (known as a false-positive result ), or normal results even when a man does have cancer (known as a false-negative result ). [ 13 ] False-positive test results can cause confusion and anxiety in men, and can lead to unnecessary prostate biopsies , a procedure which causes risk of pain, infection, and hemorrhage . False-negative results can give men a false sense of security, though they may actually have cancer. [ medical citation needed ]
Of those found to have prostate cancer, overtreatment is common because most cases of prostate cancer are not expected to cause any symptoms due to low rate of growth of the prostate tumor. Therefore, many will experience the side effects of treatment, such as for every 1000 men screened, 29 will experience erectile dysfunction, 18 will develop urinary incontinence, two will have serious cardiovascular events, one will develop pulmonary embolus or deep venous thrombosis, and one perioperative death. [ failed verification ] Since the expected harms relative to risk of death are perceived by patients as minimal, men found to have prostate cancer usually (up to 90% of cases) elect to receive treatment. [ 14 ] [ 15 ] [ 16 ]
Men with prostate cancer may be characterized as low, intermediate, or high risk for having/developing metastatic disease or dying of prostate cancer. PSA level is one of three variables on which the risk stratification is based; the others are the grade of prostate cancer ( Gleason grading system ) and the stage of cancer based on physical examination and imaging studies. D'Amico criteria for each risk category are: [ 17 ]
Given the relative simplicity of the 1998 D'Amico criteria (above), other predictive models of risk stratification based on mathematical probability constructs exist or have been proposed to allow for better matching of treatment decisions with disease features. [ 18 ] Studies are being conducted into the incorporation of multiparametric MRI imaging results into nomograms that rely on PSA, Gleason grade, and tumor stage. [ 19 ]
PSA levels are monitored periodically (e.g., every 6–36 months) after treatment for prostate cancer – more frequently in patients with high-risk disease, less frequently in patients with lower-risk disease. If surgical therapy (i.e., radical prostatectomy) is successful at removing all prostate tissue (and prostate cancer), PSA becomes undetectable within a few weeks. A subsequent rise in PSA level above 0.2 ng/mL [ 20 ] L [ disputed – discuss ] is generally regarded as evidence of recurrent prostate cancer after a radical prostatectomy; less commonly, it may simply indicate residual benign prostate tissue. [ citation needed ]
Following radiation therapy of any type for prostate cancer, some PSA levels might be detected, even when the treatment ultimately proves to be successful. This makes interpreting the relationship between PSA levels and recurrence/persistence of prostate cancer after radiation therapy more difficult. PSA levels may continue to decrease for several years after radiation therapy. The lowest level is referred to as the PSA nadir. A subsequent increase in PSA levels by 2.0 ng/mL [ disputed – discuss ] above the nadir is the currently accepted definition of prostate cancer recurrence after radiation therapy. [ citation needed ]
Recurrent prostate cancer detected by a rise in PSA levels after curative treatment is referred to as a " biochemical recurrence ". The likelihood of developing recurrent prostate cancer after curative treatment is related to the pre-operative variables described in the preceding section (PSA level and grade/stage of cancer). Low-risk cancers are the least likely to recur, but they are also the least likely to have required treatment in the first place. [ citation needed ]
PSA levels increase in the setting of prostate infection/inflammation (prostatitis), often markedly (> 100).
PSA was first identified by researchers attempting to find a substance in seminal fluid that would aid in the investigation of rape cases. [ 21 ] PSA is used to indicate the presence of semen in forensic serology . [ 22 ] The semen of adult males has PSA levels far in excess of those found in other tissues; therefore, a high level of PSA found in a sample is an indicator that semen may be present. Because PSA is a biomarker that is expressed independently of spermatozoa , it remains useful in identifying semen from vasectomized and azoospermic males. [ 23 ]
PSA can also be found at low levels in other body fluids, such as urine and breast milk, thus setting a high minimum threshold of interpretation to rule out false positive results and conclusively state that semen is present. [ 24 ] While traditional tests such as crossover electrophoresis have a sufficiently low sensitivity to detect only seminal PSA, newer diagnostics tests developed from clinical prostate cancer screening methods have lowered the threshold of detection down to 4 ng/mL. [ 25 ] This level of antigen has been shown to be present in the peripheral blood of males with prostate cancer, and rarely in female urine samples and breast milk. [ 24 ]
PSA is produced in the epithelial cells of the prostate, and can be demonstrated in biopsy samples or other histological specimens using immunohistochemistry . Disruption of this epithelium, for example in inflammation or benign prostatic hyperplasia , may lead to some diffusion of the antigen into the tissue around the epithelium, and is the cause of elevated blood levels of PSA in these conditions. [ 26 ]
More significantly, PSA remains present in prostate cells after they become malignant. Prostate cancer cells generally have variable or weak staining for PSA, due to the disruption of their normal functioning. Thus, individual prostate cancer cells produce less PSA than healthy cells; the raised serum levels in prostate cancer patients is due to the greatly increased number of such cells, not their individual activity. In most cases of prostate cancer, though, the cells remain positive for the antigen, which can then be used to identify metastasis . Since some high-grade prostate cancers may be entirely negative for PSA, however, histological analysis to identify such cases usually uses PSA in combination with other antibodies, such as prostatic acid phosphatase and CD57 . [ 26 ]
The physiological function of KLK3 is the dissolution of the coagulum, the sperm-entrapping gel composed of semenogelins and fibronectin . Its proteolytic action is effective in liquefying the coagulum so that the sperm can be liberated. The activity of PSA is well regulated. In the prostate, it is present as an inactive pro-form, which is activated through the action of KLK2 , another kallikrein-related peptidase. In the prostate, zinc ion concentrations are 10 times higher than in other bodily fluids. Zinc ions have a strong inhibitory effect on the activity of PSA and on that of KLK2, so that PSA is totally inactive. [ 27 ]
Further regulation is achieved through pH variations. Although its activity is increased by higher pH, the inhibitory effect of zinc also increases. The pH of semen is slightly alkaline and the concentrations of zinc are high. On ejaculation, semen is exposed to the acidic pH of the vagina , due to the presence of lactic acid . In fertile couples, the final vaginal pH after coitus approaches the 6-7 levels, which coincides well with reduced zinc inhibition of PSA. At these pH levels, the reduced PSA activity is countered by a decrease in zinc inhibition. Thus, the coagulum is slowly liquefied, releasing the sperm in a well-regulated manner. [ citation needed ]
Prostate-specific antigen (PSA, also known as kallikrein III, seminin, semenogelase, γ-seminoprotein and P-30 antigen) is a 34- kD glycoprotein produced almost exclusively by the prostate gland . It is a serine protease ( EC 3.4.21.77 ) enzyme , the gene of which is located on the 19th chromosome (19q13) in humans. [ 28 ]
The discovery of prostate-specific antigen (PSA) is beset with controversy; as PSA is present in prostatic tissue and semen, it was independently discovered and given different names, thus adding to the controversy. [ 29 ]
Flocks was the first to experiment with antigens in the prostate [ 30 ] and 10 years later Ablin reported the presence of precipitation antigens in the prostate. [ 31 ]
In 1971, Mitsuwo Hara characterized a unique protein in the semen fluid, gamma-seminoprotein. Li and Beling, in 1973, isolated a protein, E1, from human semen in an attempt to find a novel method to achieve fertility control. [ 32 ] [ 33 ]
In 1978, Sensabaugh identified semen-specific protein p30, but proved that it was similar to E1 protein, and that prostate was the source. [ 34 ] In 1979, Wang purified a tissue-specific antigen from the prostate ('prostate antigen'). [ 35 ]
PSA was first measured quantitatively in the blood by Papsidero in 1980, [ 36 ] and Stamey carried out the initial work on the clinical use of PSA as a marker of prostate cancer. [ 29 ]
PSA is normally present in the blood at very low levels. The reference range of less than 4 ng/mL for the first commercial PSA test, the Hybritech Tandem-R PSA test released in February 1986, was based on a study that found 99% of 472 apparently healthy men had a total PSA level below 4 ng/mL. [ 37 ] [ 38 ] [ 39 ] [ 40 ] [ 41 ] [ 42 ] [ 43 ] [ 44 ]
Increased levels of PSA may suggest the presence of prostate cancer. However, prostate cancer can also be present in the complete absence of an elevated PSA level, in which case the test result would be a false negative . [ 45 ]
Obesity has been reported to reduce serum PSA levels. [ 46 ] Delayed early detection may partially explain worse outcomes in obese men with early prostate cancer. [ 47 ] After treatment, higher BMI also correlates to higher risk of recurrence. [ 48 ]
PSA levels can be also increased by prostatitis , irritation, benign prostatic hyperplasia (BPH), and recent ejaculation, [ 49 ] [ 50 ] producing a false positive result. Digital rectal examination (DRE) has been shown in several studies [ 51 ] [ 52 ] [ 53 ] [ 54 ] to produce an increase in PSA. However, the effect is clinically insignificant, since DRE causes the most substantial increases in patients with PSA levels already elevated over 4.0 ng/mL. PSA levels are higher during the summer than during the rest of the year. [ 55 ] [ 56 ]
The "normal" reference ranges for prostate-specific antigen increase with age, as do the usual ranges in cancer (per associated table). [ 57 ] [ 58 ]
Despite earlier findings, [ 59 ] recent research suggests that the rate of increase of PSA (e.g. >0.35 ng/mL/yr, the 'PSA velocity' [ 60 ] ) is not a more specific marker for prostate cancer than the serum level of PSA. [ 61 ]
However, the PSA rate of rise may have value in prostate cancer prognosis. Men with prostate cancer whose PSA level increased by more than 2.0 ng per milliliter during the year before the diagnosis of prostate cancer have a higher risk of death from prostate cancer despite undergoing radical prostatectomy . [ 62 ] PSA velocity (PSAV) was found in a 2008 study to be more useful than the PSA doubling time (PSA DT) to help identify those men with life-threatening disease before start of treatment. [ 63 ]
Men who are known to be at risk for prostate cancer, and who decide to plot their PSA values as a function of time (i.e., years), may choose to use a semi-log plot . An exponential growth in PSA values appears as a straight line [ 64 ] on a semi-log plot, so that a new PSA value significantly above the straight line signals a switch to a new and significantly higher growth rate, [ 64 ] i.e., a higher PSA velocity.
Most PSA in the blood is bound to serum proteins. A small amount is not protein-bound and is called 'free PSA'. In men with prostate cancer, the ratio of free (unbound) PSA to total PSA is decreased. The risk of cancer increases if the free to total ratio is less than 25%. (See graph) The lower the ratio is, the greater the probability of prostate cancer. Measuring the ratio of free to total PSA appears to be particularly promising for eliminating unnecessary biopsies in men with PSA levels between 4 and 10 ng/mL. [ 66 ] However, both total and free PSA increase immediately after ejaculation, returning slowly to baseline levels within 24 hours. [ 49 ]
The PSA test in 1994 failed to differentiate between prostate cancer and benign prostate hyperplasia (BPH) and the commercial assay kits for PSA did not provide correct PSA values. [ 67 ] Thus with the introduction of the ratio of free-to-total PSA, the reliability of the test has improved. Measuring the activity of the enzyme could add to the ratio of free-to-total PSA and further improve the diagnostic value of test. [ 68 ] Proteolytically active PSA has been shown to have an anti-angiogenic effect [ 69 ] and certain inactive subforms may be associated with prostate cancer, as shown by MAb 5D3D11, an antibody able to detect forms abundantly represented in sera from cancer patients. [ 70 ] The presence of inactive proenzyme forms of PSA is another potential indicator of disease. [ 71 ]
PSA exists in serum in the free (unbound) form and in a complex with alpha 1-antichymotrypsin ; research has been conducted to see if measurements of complexed PSA are more specific and sensitive biomarkers for prostate cancer than other approaches. [ 72 ] [ 73 ]
The term prostate-specific antigen is a misnomer : it is an antigen but is not specific to the prostate. Although present in large amounts in prostatic tissue and semen, it has been detected in other body fluids and tissues. [ 24 ]
In women, PSA is found in female ejaculate at concentrations roughly equal to that found in male semen. [ 5 ] Other than semen and female ejaculate, the greatest concentrations of PSA in biological fluids are detected in breast milk and amniotic fluid. Low concentrations of PSA have been identified in the urethral glands, endometrium, normal breast tissue and salivary gland tissue. PSA also is found in the serum of women with breast, lung, or uterine cancer and in some patients with renal cancer. [ 74 ]
Tissue samples can be stained for the presence of PSA in order to determine the origin of malignant cells that have metastasized. [ 75 ]
Prostate-specific antigen has been shown to interact with protein C inhibitor . [ 76 ] [ 77 ] Prostate-specific antigen interacts with and activates the vascular endothelial growth factors VEGF-C and VEGF-D , which are involved in tumor angiogenesis and in the lymphatic metastasis of tumors. [ 78 ] | https://en.wikipedia.org/wiki/Prostate-specific_antigen |
Prosthecate bacteria are a non- phylogenetically related group of Gram-negative bacteria that possess appendages , termed prosthecae . These cellular appendages, also known as stalks , are neither pili nor flagella , as they are extensions of the cellular membrane and contain cytosol . [ 1 ] One notable group of prosthecates is the genus Caulobacter .
Prosthecates are generally chemoorganotrophic aerobes that can grow in nutrient-poor habitats, being able to survive at nutrient levels on the order of parts-per-million for which reason they are often found in aquatic habitats. These bacteria will attach to surfaces with their prosthecae, allowing a greater surface area with which to take up nutrients (and release waste products). [ 1 ] [ 2 ] Some prosthecates will grow in nutrient-poor soils as aerobic heterotrophs .
Oligotrophic
Poindexter, Jeanne S. Dimorphic Prosthecate Bacteria: The Genera Caulobacter , Asticcacaulis , Hyphomicrobium , Pedomicrobium , Hyphomonas and Thiodendron . [1] | https://en.wikipedia.org/wiki/Prosthecate_bacteria |
In medicine , a prosthesis ( pl. : prostheses ; from Ancient Greek : πρόσθεσις , romanized : prósthesis , lit. 'addition, application, attachment'), [ 1 ] or a prosthetic implant , [ 2 ] [ 3 ] is an artificial device that replaces a missing body part, which may be lost through physical trauma , disease, or a condition present at birth ( congenital disorder ). Prostheses may restore the normal functions of the missing body part, [ 4 ] or may perform a cosmetic function.
A person who has undergone an amputation is sometimes referred to as an amputee , however, this term may be offensive. [ 5 ] Rehabilitation for someone with an amputation is primarily coordinated by a physiatrist as part of an inter-disciplinary team consisting of physiatrists, prosthetists, nurses, physical therapists, and occupational therapists. [ 6 ] Prostheses can be created by hand or with computer-aided design (CAD), a software interface that helps creators design and analyze the creation with computer-generated 2-D and 3-D graphics as well as analysis and optimization tools. [ 7 ]
A person's prosthetic device should be designed and assembled to meet their individual appearance and functional needs. Depending on personal circumstances, co-morbidities, budget or health insurance coverage, and access to medical care, decisions may need to balance aesthetics and function. In addition, for some individuals, a myoelectric device, a body-powered device, or an activity-specific device may be appropriate options. The person's future goals and vocational aspirations and potential capabilities may help them choose between one or more devices. [ citation needed ]
Craniofacial prostheses include intra-oral and extra-oral prostheses. Extra-oral prostheses are further divided into hemifacial, auricular (ear), nasal, orbital and ocular . Intra-oral prostheses include dental prostheses , such as dentures , obturators , and dental implants .
Prostheses of the neck include larynx substitutes , trachea and upper esophageal replacements,
Some prostheses of the torso include breast prostheses which may be either single or bilateral, full breast devices or nipple prostheses .
Penile prostheses are used to treat erectile dysfunction , perform phalloplasty procedures in cisgender men, and to build a new penis in female-to-male gender reassignment surgeries .
Limb prostheses include both upper- and lower-extremity prostheses.
Upper-extremity prostheses are used at varying levels of amputation: forequarter, shoulder disarticulation, transhumeral prosthesis, elbow disarticulation, transradial prosthesis, wrist disarticulation, full hand, partial hand, finger, partial finger. A transradial prosthesis is an artificial limb that replaces an arm missing below the elbow.
Upper limb prostheses can be categorized in three main categories: Passive devices, Body Powered devices, and Externally Powered (myoelectric) devices. Passive devices can either be passive hands, mainly used for cosmetic purposes, or passive tools, mainly used for specific activities (e.g. leisure or vocational). An extensive overview and classification of passive devices can be found in a literature review by Maat et.al. [ 8 ] A passive device can be static, meaning the device has no movable parts, or it can be adjustable, meaning its configuration can be adjusted (e.g. adjustable hand opening). Despite the absence of active grasping, passive devices are very useful in bimanual tasks that require fixation or support of an object, or for gesticulation in social interaction. According to scientific data a third of the upper limb amputees worldwide use a passive prosthetic hand. [ 8 ] Body Powered or cable-operated limbs work by attaching a harness and cable around the opposite shoulder of the damaged arm. A recent body-powered approach has explored the utilization of the user's breathing to power and control the prosthetic hand to help eliminate actuation cable and harness. [ 9 ] [ 10 ] [ 11 ] The third category of available prosthetic devices comprises myoelectric arms. This particular class of devices distinguishes itself from the previous ones due to the inclusion of a battery system. This battery serves the dual purpose of providing energy for both actuation and sensing components. While actuation predominantly relies on motor or pneumatic systems, [ 12 ] a variety of solutions have been explored for capturing muscle activity, including techniques such as Electromyography , Sonomyography, Myokinetic, and others. [ 13 ] [ 14 ] [ 15 ] These methods function by detecting the minute electrical currents generated by contracted muscles during upper arm movement, typically employing electrodes or other suitable tools. Subsequently, these acquired signals are converted into gripping patterns or postures that the artificial hand will then execute.
In the prosthetics industry, a trans-radial prosthetic arm is often referred to as a "BE" or below elbow prosthesis.
Lower-extremity prostheses provide replacements at varying levels of amputation. These include hip disarticulation , transfemoral prosthesis, knee disarticulation, transtibial prosthesis, Syme's amputation, foot, partial foot, and toe. The two main subcategories of lower extremity prosthetic devices are trans-tibial (any amputation transecting the tibia bone or a congenital anomaly resulting in a tibial deficiency) and trans-femoral (any amputation transecting the femur bone or a congenital anomaly resulting in a femoral deficiency). [ citation needed ]
A transfemoral prosthesis is an artificial limb that replaces a leg missing above the knee. Transfemoral amputees can have a very difficult time regaining normal movement. In general, a transfemoral amputee must use approximately 80% more energy to walk than a person with two whole legs. [ 16 ] This is due to the complexities in movement associated with the knee. In newer and more improved designs, hydraulics, carbon fiber, mechanical linkages, motors, computer microprocessors, and innovative combinations of these technologies are employed to give more control to the user. In the prosthetics industry, a trans-femoral prosthetic leg is often referred to as an "AK" or above the knee prosthesis.
A transtibial prosthesis is an artificial limb that replaces a leg missing below the knee. A transtibial amputee is usually able to regain normal movement more readily than someone with a transfemoral amputation, due in large part to retaining the knee, which allows for easier movement. Lower extremity prosthetics describe artificially replaced limbs located at the hip level or lower. In the prosthetics industry, a trans-tibial prosthetic leg is often referred to as a "BK" or below the knee prosthesis.
Prostheses are manufactured and fit by clinical prosthetists. Prosthetists are healthcare professionals responsible for making, fitting, and adjusting prostheses and for lower limb prostheses will assess both gait and prosthetic alignment. Once a prosthesis has been fit and adjusted by a prosthetist, a rehabilitation physiotherapist (called physical therapist in America) will help teach a new prosthetic user to walk with a leg prosthesis. To do so, the physical therapist may provide verbal instructions and may also help guide the person using touch or tactile cues. This may be done in a clinic or home. There is some research suggesting that such training in the home may be more successful if the treatment includes the use of a treadmill. [ 17 ] Using a treadmill, along with the physical therapy treatment, helps the person to experience many of the challenges of walking with a prosthesis.
In the United Kingdom, 75% of lower limb amputations are performed due to inadequate circulation (dysvascularity). [ 18 ] This condition is often associated with many other medical conditions ( co-morbidities ) including diabetes and heart disease that may make it a challenge to recover and use a prosthetic limb to regain mobility and independence. [ 18 ] For people who have inadequate circulation and have lost a lower limb, there is insufficient evidence due to a lack of research, to inform them regarding their choice of prosthetic rehabilitation approaches. [ 18 ]
Lower extremity prostheses are often categorized by the level of amputation or after the name of a surgeon: [ 19 ] [ 20 ]
Prosthetic are made lightweight for better convenience for the amputee. Some of these materials include:
Wheeled prostheses have also been used extensively in the rehabilitation of injured domestic animals, including dogs, cats, pigs, rabbits, and turtles. [ 21 ]
Organ prostheses include artificial hearts , and artificial kidneys .
Prosthetics originate from the ancient Near East circa 3000 BCE, with the earliest evidence of prosthetics appearing in ancient Egypt and Iran . The earliest recorded mention of eye prosthetics is from the Egyptian story of the Eye of Horus dated circa 3000 BC, which involves the left eye of Horus being plucked out and then restored by Thoth . Circa 3000-2800 BC, the earliest archaeological evidence of prosthetics is found in ancient Iran, where an eye prosthetic is found buried with a woman in Shahr-i Shōkhta . It was likely made of bitumen paste that was covered with a thin layer of gold. [ 22 ] The Egyptians were also early pioneers of foot prosthetics, as shown by the wooden toe found on a body from the New Kingdom circa 1000 BC. [ 23 ] Another early textual mention is found in South Asia circa 1200 BC, involving the warrior queen Vishpala in the Rigveda . [ 24 ] Roman bronze crowns have also been found, but their use could have been more aesthetic than medical. [ 25 ]
An early mention of a prosthetic comes from the Greek historian Herodotus , who tells the story of Hegesistratus , a Greek diviner who cut off his own foot to escape his Spartan captors and replaced it with a wooden one. [ 26 ]
Pliny the Elder also recorded the tale of a Roman general, Marcus Sergius , whose right hand was cut off while campaigning and had an iron hand made to hold his shield so that he could return to battle. A famous and quite refined [ 28 ] historical prosthetic arm was that of Götz von Berlichingen , made at the beginning of the 16th century. The first confirmed use of a prosthetic device, however, is from 950 to 710 BC. In 2000, research pathologists discovered a mummy from this period buried in the Egyptian necropolis near ancient Thebes that possessed an artificial big toe. This toe, consisting of wood and leather, exhibited evidence of use. When reproduced by bio-mechanical engineers in 2011, researchers discovered that this ancient prosthetic enabled its wearer to walk both barefoot and in Egyptian style sandals. Previously, the earliest discovered prosthetic was an artificial leg from Capua . [ 29 ]
Around the same time, François de la Noue is also reported to have had an iron hand, as is, in the 17th century, René-Robert Cavalier de la Salle . [ 30 ] Henri de Tonti had a prosthetic hook for a hand. During the Middle Ages, prosthetics remained quite basic in form. Debilitated knights would be fitted with prosthetics so they could hold up a shield, grasp a lance or a sword, or stabilize a mounted warrior. [ 31 ] Only the wealthy could afford anything that would assist in daily life. [ 32 ]
One notable prosthesis was that belonging to an Italian man, who scientists estimate replaced his amputated right hand with a knife. [ 33 ] [ 34 ] Scientists investigating the skeleton, which was found in a Longobard cemetery in Povegliano Veronese , estimated that the man had lived sometime between the 6th and 8th centuries AD. [ 35 ] [ 34 ] Materials found near the man's body suggest that the knife prosthesis was attached with a leather strap, which he repeatedly tightened with his teeth. [ 35 ]
During the Renaissance, prosthetics developed with the use of iron, steel, copper, and wood. Functional prosthetics began to make an appearance in the 1500s. [ 36 ]
An Italian surgeon recorded the existence of an amputee who had an arm that allowed him to remove his hat, open his purse, and sign his name. [ 37 ] Improvement in amputation surgery and prosthetic design came at the hands of Ambroise Paré . Among his inventions was an above-knee device that was a kneeling peg leg and foot prosthesis with a fixed position, adjustable harness, and knee lock control. The functionality of his advancements showed how future prosthetics could develop.
Other major improvements before the modern era:
At the end of World War II, the NAS (National Academy of Sciences) began to advocate better research and development of prosthetics. Through government funding, a research and development program was developed within the Army, Navy, Air Force, and the Veterans Administration.
After the Second World War, a team at the University of California, Berkeley including James Foort and C.W. Radcliff helped to develop the quadrilateral socket by developing a jig fitting system for amputations above the knee. Socket technology for lower extremity limbs saw a further revolution during the 1980s when John Sabolich C.P.O., invented the Contoured Adducted Trochanteric-Controlled Alignment Method (CATCAM) socket, later to evolve into the Sabolich Socket. He followed the direction of Ivan Long and Ossur Christensen as they developed alternatives to the quadrilateral socket, which in turn followed the open ended plug socket, created from wood. [ 40 ] The advancement was due to the difference in the socket to patient contact model. Prior to this, sockets were made in the shape of a square shape with no specialized containment for muscular tissue. New designs thus help to lock in the bony anatomy, locking it into place and distributing the weight evenly over the existing limb as well as the musculature of the patient. Ischial containment is well known and used today by many prosthetist to help in patient care. Variations of the ischial containment socket thus exists and each socket is tailored to the specific needs of the patient. Others who contributed to socket development and changes over the years include Tim Staats, Chris Hoyt, and Frank Gottschalk. Gottschalk disputed the efficacy of the CAT-CAM socket- insisting the surgical procedure done by the amputation surgeon was most important to prepare the amputee for good use of a prosthesis of any type socket design. [ 41 ]
The first microprocessor-controlled prosthetic knees became available in the early 1990s. The Intelligent Prosthesis was the first commercially available microprocessor-controlled prosthetic knee. It was released by Chas. A. Blatchford & Sons, Ltd., of Great Britain, in 1993 and made walking with the prosthesis feel and look more natural. [ 42 ] An improved version was released in 1995 by the name Intelligent Prosthesis Plus. Blatchford released another prosthesis, the Adaptive Prosthesis, in 1998. The Adaptive Prosthesis utilized hydraulic controls, pneumatic controls, and a microprocessor to provide the amputee with a gait that was more responsive to changes in walking speed. Cost analysis reveals that a sophisticated above-knee prosthesis will be about $1 million in 45 years, given only annual cost of living adjustments. [ 43 ]
In 2019, a project under AT2030 was launched in which bespoke sockets are made using a thermoplastic, rather than through a plaster cast. This is faster to do and significantly less expensive. The sockets were called Amparo Confidence sockets. [ 44 ] [ 45 ]
In 2005, DARPA started the Revolutionizing Prosthetics program. [ 46 ] [ 47 ] [ 48 ] [ 49 ] [ 50 ] [ 51 ] According to DARPA, the goal of the $100 million program was to "develop an advanced electromechanical prosthetic upper limb with near-natural control that would dramatically enhance independence and quality of life for amputees." [ 52 ] [ 53 ] In 2014, the LUKE Arm developed by Dean Kamen and his team at DEKA Research and Development Corp. became the first prosthetic arm approved by FDA that "translates signals from a person's muscles to perform complex tasks," according to FDA. [ 53 ] [ 54 ] Johns Hopkins University and the U.S. Department of Veteran Affairs also participated in the program. [ 53 ] [ 55 ]
There are many steps in the evolution of prosthetic design trends that are moving forward with time. Many design trends point to lighter, more durable, and flexible materials like carbon fiber, silicone, and advanced polymers. These not only make the prosthetic limb lighter and more durable but also allow it to mimic the look and feel of natural skin, providing users with a more comfortable and natural experience. [ 56 ] This new technology helps prosthetic users blend in with people with normal ligaments to reduce the stigmatism for people who wear prosthetics. Another trend points towards using bionics and myoelectric components in prosthetic design. These limbs utilize sensors to detect electrical signals from the user's residual muscles. The signals are then converted into motions, allowing users to control their prosthetic limbs using their own muscle contractions. This has greatly improved the range and fluidity of movements available to amputees, making tasks like grasping objects or walking naturally much more feasible. [ 56 ] Integration with AI is also on the forefront to the prosthetic design. AI-enabled prosthetic limbs can learn and adapt to the user's habits and preferences over time, ensuring optimal functionality. By analyzing the user's gait, grip, and other movements, these smart limbs can make real-time adjustments, providing smoother and more natural motions. [ 56 ]
A prosthesis is a functional replacement for an amputated or congenitally malformed or missing limb. Prosthetists are responsible for the prescription, design, and management of a prosthetic device.
In most cases, the prosthetist begins by taking a plaster cast of the patient's affected limb. Lightweight, high-strength thermoplastics are custom-formed to this model of the patient. Cutting-edge materials such as carbon fiber, titanium and Kevlar provide strength and durability while making the new prosthesis lighter. More sophisticated prostheses are equipped with advanced electronics, providing additional stability and control. [ 57 ]
Over the years, there have been advancements in artificial limbs. New plastics and other materials, such as carbon fiber , have allowed artificial limbs to be stronger and lighter, limiting the amount of extra energy necessary to operate the limb. This is especially important for trans-femoral amputees. Additional materials have allowed artificial limbs to look much more realistic, which is important to trans-radial and transhumeral amputees because they are more likely to have the artificial limb exposed. [ 58 ]
In addition to new materials, the use of electronics has become very common in artificial limbs. Myoelectric limbs, which control the limbs by converting muscle movements to electrical signals, have become much more common than cable operated limbs. Myoelectric signals are picked up by electrodes, the signal gets integrated and once it exceeds a certain threshold, the prosthetic limb control signal is triggered which is why inherently, all myoelectric controls lag. Conversely, cable control is immediate and physical, and through that offers a certain degree of direct force feedback that myoelectric control does not. Computers are also used extensively in the manufacturing of limbs. Computer Aided Design and Computer Aided Manufacturing are often used to assist in the design and manufacture of artificial limbs. [ 58 ] [ 59 ]
Most modern artificial limbs are attached to the residual limb (stump) of the amputee by belts and cuffs or by suction . The residual limb either directly fits into a socket on the prosthetic, or—more commonly today—a liner is used that then is fixed to the socket either by vacuum (suction sockets) or a pin lock. Liners are soft and by that, they can create a far better suction fit than hard sockets. Silicone liners can be obtained in standard sizes, mostly with a circular (round) cross section, but for any other residual limb shape, custom liners can be made. The socket is custom made to fit the residual limb and to distribute the forces of the artificial limb across the area of the residual limb (rather than just one small spot), which helps reduce wear on the residual limb.
The production of a prosthetic socket begins with capturing the geometry of the residual limb; this process is called shape capture. The goal of this process is to create an accurate representation of the residual limb, which is critical to achieve good socket fit. [ 60 ] The custom socket is created by taking a plaster cast of the residual limb or, more commonly today, of the liner worn over their residual limb, and then making a mold from the plaster cast. The commonly used compound is called Plaster of Paris. [ 61 ] In recent years, various digital shape capture systems have been developed which can be input directly to a computer allowing for a more sophisticated design. In general, the shape capturing process begins with the digital acquisition of three-dimensional (3D) geometric data from the amputee's residual limb. Data are acquired with either a probe, laser scanner, structured light scanner, or a photographic-based 3D scanning system. [ 62 ]
After shape capture, the second phase of the socket production is called rectification, which is the process of modifying the model of the residual limb by adding volume to bony prominence and potential pressure points and remove volume from load bearing area. This can be done manually by adding or removing plaster to the positive model, or virtually by manipulating the computerized model in the software. [ 63 ] Lastly, the fabrication of the prosthetic socket begins once the model has been rectified and finalized. The prosthetists would wrap the positive model with a semi-molten plastic sheet or carbon fiber coated with epoxy resin to construct the prosthetic socket. [ 60 ] For the computerized model, it can be 3D printed using a various of material with different flexibility and mechanical strength. [ 64 ]
Optimal socket fit between the residual limb and socket is critical to the function and usage of the entire prosthesis. If the fit between the residual limb and socket attachment is too loose, this will reduce the area of contact between the residual limb and socket or liner, and increase pockets between residual limb skin and socket or liner. Pressure then is higher, which can be painful. Air pockets can allow sweat to accumulate that can soften the skin. Ultimately, this is a frequent cause for itchy skin rashes. Over time, this can lead to breakdown of the skin. [ 16 ] On the other hand, a very tight fit may excessively increase the interface pressures that may also lead to skin breakdown after prolonged use. [ 65 ]
Artificial limbs are typically manufactured using the following steps: [ 58 ]
Current technology allows body-powered arms to weigh around one-half to one-third of what a myoelectric arm does.
Current body-powered arms contain sockets that are built from hard epoxy or carbon fiber. These sockets or "interfaces" can be made more comfortable by lining them with a softer, compressible foam material that provides padding for the bone prominences. A self-suspending or supra-condylar socket design is useful for those with short to mid-range below elbow absence. Longer limbs may require the use of a locking roll-on type inner liner or more complex harnessing to help augment suspension.
Wrist units are either screw-on connectors featuring the UNF 1/2-20 thread (USA) or quick-release connector, of which there are different models.
Two types of body-powered systems exist, voluntary opening "pull to open" and voluntary closing "pull to close". Virtually all "split hook" prostheses operate with a voluntary opening type system.
More modern "prehensors" called GRIPS utilize voluntary closing systems. The differences are significant. Users of voluntary opening systems rely on elastic bands or springs for gripping force, while users of voluntary closing systems rely on their own body power and energy to create gripping force.
Voluntary closing users can generate prehension forces equivalent to the normal hand, up to or exceeding one hundred pounds. Voluntary closing GRIPS require constant tension to grip, like a human hand, and in that property, they do come closer to matching human hand performance. Voluntary opening split hook users are limited to forces their rubber or springs can generate which usually is below 20 pounds.
An additional difference exists in the biofeedback created that allows the user to "feel" what is being held. Voluntary opening systems once engaged provide the holding force so that they operate like a passive vice at the end of the arm. No gripping feedback is provided once the hook has closed around the object being held. Voluntary closing systems provide directly proportional control and biofeedback so that the user can feel how much force that they are applying.
In 1997, the Colombian Prof. Álvaro Ríos Poveda , a researcher in bionics in Latin America , developed an upper limb and hand prosthesis with sensory feedback . This technology allows amputee patients to handle prosthetic hand systems in a more natural way. [ 66 ]
A recent study showed that by stimulating the median and ulnar nerves, according to the information provided by the artificial sensors from a hand prosthesis, physiologically appropriate (near-natural) sensory information could be provided to an amputee. This feedback enabled the participant to effectively modulate the grasping force of the prosthesis with no visual or auditory feedback. [ 67 ]
In February 2013, researchers from École Polytechnique Fédérale de Lausanne in Switzerland and the Scuola Superiore Sant'Anna in Italy, implanted electrodes into an amputee's arm, which gave the patient sensory feedback and allowed for real time control of the prosthetic. [ 68 ] With wires linked to nerves in his upper arm, the Danish patient was able to handle objects and instantly receive a sense of touch through the special artificial hand that was created by Silvestro Micera and researchers both in Switzerland and Italy. [ 69 ]
In July 2019, this technology was expanded on even further by researchers from the University of Utah , led by Jacob George. The group of researchers implanted electrodes into the patient's arm to map out several sensory precepts. They would then stimulate each electrode to figure out how each sensory precept was triggered, then proceed to map the sensory information onto the prosthetic. This would allow the researchers to get a good approximation of the same kind of information that the patient would receive from their natural hand. Unfortunately, the arm is too expensive for the average user to acquire, however, Jacob mentioned that insurance companies could cover the costs of the prosthetic. [ 70 ]
Terminal devices contain a range of hooks, prehensors, hands or other devices.
Voluntary opening split hook systems are simple, convenient, light, robust, versatile and relatively affordable.
A hook does not match a normal human hand for appearance or overall versatility, but its material tolerances can exceed and surpass the normal human hand for mechanical stress (one can even use a hook to slice open boxes or as a hammer whereas the same is not possible with a normal hand), for thermal stability (one can use a hook to grip items from boiling water, to turn meat on a grill, to hold a match until it has burned down completely) and for chemical hazards (as a metal hook withstands acids or lye, and does not react to solvents like a prosthetic glove or human skin).
Prosthetic hands are available in both voluntary opening and voluntary closing versions and because of their more complex mechanics and cosmetic glove covering require a relatively large activation force, which, depending on the type of harness used, may be uncomfortable. [ 71 ] A recent study by the Delft University of Technology, The Netherlands, showed that the development of mechanical prosthetic hands has been neglected during the past decades. The study showed that the pinch force level of most current mechanical hands is too low for practical use. [ 72 ] The best tested hand was a prosthetic hand developed around 1945. In 2017 however, a research has been started with bionic hands by Laura Hruby of the Medical University of Vienna . [ 73 ] [ 74 ] A few open-hardware 3-D printable bionic hands have also become available. [ 75 ] Some companies are also producing robotic hands with integrated forearm, for fitting unto a patient's upper arm [ 76 ] [ 77 ] and in 2020, at the Italian Institute of Technology (IIT), another robotic hand with integrated forearm (Soft Hand Pro) was developed. [ 78 ]
Hosmer and Otto Bock are major commercial hook providers. Mechanical hands are sold by Hosmer and Otto Bock as well; the Becker Hand is still manufactured by the Becker family. Prosthetic hands may be fitted with standard stock or custom-made cosmetic looking silicone gloves. But regular work gloves may be worn as well. Other terminal devices include the V2P Prehensor, a versatile robust gripper that allows customers to modify aspects of it, Texas Assist Devices (with a whole assortment of tools) and TRS that offers a range of terminal devices for sports. Cable harnesses can be built using aircraft steel cables, ball hinges, and self-lubricating cable sheaths. Some prosthetics have been designed specifically for use in salt water. [ 79 ]
Lower-extremity prosthetics describes artificially replaced limbs located at the hip level or lower. Concerning all ages Ephraim et al. (2003) found a worldwide estimate of all-cause lower-extremity amputations of 2.0–5.9 per 10,000 inhabitants. For birth prevalence rates of congenital limb deficiency they found an estimate between 3.5 and 7.1 cases per 10,000 births. [ 80 ]
The two main subcategories of lower extremity prosthetic devices are trans-tibial (any amputation transecting the tibia bone or a congenital anomaly resulting in a tibial deficiency), and trans-femoral (any amputation transecting the femur bone or a congenital anomaly resulting in a femoral deficiency). In the prosthetic industry, a trans-tibial prosthetic leg is often referred to as a "BK" or below the knee prosthesis while the trans-femoral prosthetic leg is often referred to as an "AK" or above the knee prosthesis.
Other, less prevalent lower extremity cases include the following:
The socket serves as an interface between the residuum and the prosthesis, ideally allowing comfortable weight-bearing, movement control and proprioception . [ 81 ] Socket problems, such as discomfort and skin breakdown, are rated among the most important issues faced by lower-limb amputees. [ 82 ]
This part creates distance and support between the knee-joint and the foot (in case of an upper-leg prosthesis) or between the socket and the foot. The type of connectors that are used between the shank and the knee/foot determines whether the prosthesis is modular or not. Modular means that the angle and the displacement of the foot in respect to the socket can be changed after fitting. In developing countries prosthesis mostly are non-modular, in order to reduce cost. When considering children modularity of angle and height is important because of their average growth of 1.9 cm annually. [ 83 ]
Providing contact to the ground, the foot provides shock absorption and stability during stance. [ 84 ] Additionally it influences gait biomechanics by its shape and stiffness. This is because the trajectory of the center of pressure (COP) and the angle of the ground reaction forces is determined by the shape and stiffness of the foot and needs to match the subject's build in order to produce a normal gait pattern. [ 85 ] Andrysek (2010) found 16 different types of feet, with greatly varying results concerning durability and biomechanics. The main problem found in current feet is durability, endurance ranging from 16 to 32 months [ 86 ] These results are for adults and will probably be worse for children due to higher activity levels and scale effects. Evidence comparing different types of feet and ankle prosthetic devices is not strong enough to determine if one mechanism of ankle/foot is superior to another. [ 87 ] When deciding on a device, the cost of the device, a person's functional need, and the availability of a particular device should be considered. [ 87 ]
In case of a trans-femoral (above knee) amputation, there also is a need for a complex connector providing articulation, allowing flexion during swing-phase but not during stance. As its purpose is to replace the knee, the prosthetic knee joint is the most critical component of the prosthesis for trans-femoral amputees. The function of the good prosthetic knee joint is to mimic the function of the normal knee, such as providing structural support and stability during stance phase but able to flex in a controllable manner during swing phase. Hence it allows users to have a smooth and energy efficient gait and minimize the impact of amputation. [ 88 ] The prosthetic knee is connected to the prosthetic foot by the shank, which is usually made of an aluminum or graphite tube.
One of the most important aspect of a prosthetic knee joint would be its stance-phase control mechanism. The function of stance-phase control is to prevent the leg from buckling when the limb is loaded during weight acceptance. This ensures the stability of the knee in order to support the single limb support task of stance phase and provides a smooth transition to the swing phase. Stance phase control can be achieved in several ways including the mechanical locks, [ 89 ] relative alignment of prosthetic components, [ 90 ] weight activated friction control, [ 90 ] and polycentric mechanisms. [ 91 ]
To mimic the knee's functionality during gait, microprocessor-controlled knee joints have been developed that control the flexion of the knee. Some examples are Otto Bock 's C-leg, introduced in 1997, Ossur 's Rheo Knee, released in 2005, the Power Knee by Ossur, introduced in 2006, the Plié Knee from Freedom Innovations and DAW Industries' Self Learning Knee (SLK). [ 92 ]
The idea was originally developed by Kelly James, a Canadian engineer, at the University of Alberta . [ 93 ]
A microprocessor is used to interpret and analyze signals from knee-angle sensors and moment sensors. The microprocessor receives signals from its sensors to determine the type of motion being employed by the amputee. Most microprocessor controlled knee-joints are powered by a battery housed inside the prosthesis.
The sensory signals computed by the microprocessor are used to control the resistance generated by hydraulic cylinders in the knee-joint. Small valves control the amount of hydraulic fluid that can pass into and out of the cylinder, thus regulating the extension and compression of a piston connected to the upper section of the knee. [ 43 ]
The main advantage of a microprocessor-controlled prosthesis is a closer approximation to an amputee's natural gait. Some allow amputees to walk near walking speed or run. Variations in speed are also possible and are taken into account by sensors and communicated to the microprocessor, which adjusts to these changes accordingly. It also enables the amputees to walk downstairs with a step-over-step approach, rather than the one step at a time approach used with mechanical knees. [ 94 ] There is some research suggesting that people with microprocessor-controlled prostheses report greater satisfaction and improvement in functionality, residual limb health, and safety. [ 95 ] People may be able to perform everyday activities at greater speeds, even while multitasking, and reduce their risk of falls. [ 95 ]
However, some have some significant drawbacks that impair its use. They can be susceptible to water damage and thus great care must be taken to ensure that the prosthesis remains dry. [ 96 ]
A myoelectric prosthesis uses the electrical tension generated every time a muscle contracts, as information. This tension can be captured from voluntarily contracted muscles by electrodes applied on the skin to control the movements of the prosthesis, such as elbow flexion/extension, wrist supination/pronation (rotation) or opening/closing of the fingers. A prosthesis of this type utilizes the residual neuromuscular system of the human body to control the functions of an electric powered prosthetic hand, wrist, elbow or foot. [ 97 ] This is different from an electric switch prosthesis, which requires straps and/or cables actuated by body movements to actuate or operate switches that control the movements of the prosthesis. There is no clear evidence concluding that myoelectric upper extremity prostheses function better than body-powered prostheses. [ 98 ] Advantages to using a myoelectric upper extremity prosthesis include the potential for improvement in cosmetic appeal (this type of prosthesis may have a more natural look), may be better for light everyday activities, and may be beneficial for people experiencing phantom limb pain. [ 98 ] When compared to a body-powered prosthesis, a myoelectric prosthesis may not be as durable, may have a longer training time, may require more adjustments, may need more maintenance, and does not provide feedback to the user. [ 98 ]
Prof. Alvaro Ríos Poveda has been working for several years on a non-invasive and affordable solution to this feedback problem. He considers that: "Prosthetic limbs that can be controlled with thought hold great promise for the amputee, but without sensorial feedback from the signals returning to the brain, it can be difficult to achieve the level of control necessary to perform precise movements. When connecting the sense of touch from a mechanical hand directly to the brain, prosthetics can restore the function of the amputated limb in an almost natural-feeling way." He presented the first Myoelectric prosthetic hand with sensory feedback at the XVIII World Congress on Medical Physics and Biomedical Engineering , 1997, held in Nice, France . [ 99 ] [ 100 ]
The USSR was the first to develop a myoelectric arm in 1958, [ 101 ] while the first myoelectric arm became commercial in 1964 by the Central Prosthetic Research Institute of the USSR , and distributed by the Hangar Limb Factory of the UK . [ 102 ] [ 103 ] The Myoelectric prosthesis are expensive requires regular maintenance, sensitive to sweat and moisture affecting sensor performance.
Robots can be used to generate objective measures of patient's impairment and therapy outcome, assist in diagnosis, customize therapies based on patient's motor abilities, and assure compliance with treatment regimens and maintain patient's records. It is shown in many studies that there is a significant improvement in upper limb motor function after stroke using robotics for upper limb rehabilitation. [ 104 ] In order for a robotic prosthetic limb to work, it must have several components to integrate it into the body's function: Biosensors detect signals from the user's nervous or muscular systems. It then relays this information to a microcontroller located inside the device, and processes feedback from the limb and actuator, e.g., position or force, and sends it to the controller. Examples include surface electrodes that detect electrical activity on the skin, needle electrodes implanted in muscle, or solid-state electrode arrays with nerves growing through them. One type of these biosensors are employed in myoelectric prostheses .
A device known as the controller is connected to the user's nerve and muscular systems and the device itself. It sends intention commands from the user to the actuators of the device and interprets feedback from the mechanical and biosensors to the user. The controller is also responsible for the monitoring and control of the movements of the device.
An actuator mimics the actions of a muscle in producing force and movement. Examples include a motor that aids or replaces original muscle tissue.
Targeted muscle reinnervation (TMR) is a technique in which motor nerves , which previously controlled muscles on an amputated limb, are surgically rerouted such that they reinnervate a small region of a large, intact muscle, such as the pectoralis major . As a result, when a patient thinks about moving the thumb of their missing hand, a small area of muscle on their chest will contract instead. By placing sensors over the reinnervated muscle, these contractions can be made to control the movement of an appropriate part of the robotic prosthesis. [ 105 ] [ 106 ]
A variant of this technique is called targeted sensory reinnervation (TSR). This procedure is similar to TMR, except that sensory nerves are surgically rerouted to skin on the chest, rather than motor nerves rerouted to muscle. Recently, robotic limbs have improved in their ability to take signals from the human brain and translate those signals into motion in the artificial limb. DARPA , the Pentagon's research division, is working to make even more advancements in this area. Their desire is to create an artificial limb that ties directly into the nervous system . [ 107 ]
Advancements in the processors used in myoelectric arms have allowed developers to make gains in fine-tuned control of the prosthetic. The Boston Digital Arm is a recent artificial limb that has taken advantage of these more advanced processors. The arm allows movement in five axes and allows the arm to be programmed for a more customized feel. Recently the I-LIMB Hand , invented in Edinburgh, Scotland, by David Gow has become the first commercially available hand prosthesis with five individually powered digits. The hand also possesses a manually rotatable thumb which is operated passively by the user and allows the hand to grip in precision, power, and key grip modes. [ 108 ]
Another neural prosthetic is Johns Hopkins University Applied Physics Laboratory Proto 1. Besides the Proto 1, the university also finished the Proto 2 in 2010. [ 109 ] Early in 2013, Max Ortiz Catalan and Rickard Brånemark of the Chalmers University of Technology, and Sahlgrenska University Hospital in Sweden, succeeded in making the first robotic arm which is mind-controlled and can be permanently attached to the body (using osseointegration ). [ 110 ] [ 111 ] [ 112 ]
An approach that is very useful is called arm rotation which is common for unilateral amputees which is an amputation that affects only one side of the body; and also essential for bilateral amputees, a person who is missing or has had amputated either both arms or legs, to carry out activities of daily living. This involves inserting a small permanent magnet into the distal end of the residual bone of subjects with upper limb amputations. When a subject rotates the residual arm, the magnet will rotate with the residual bone, causing a change in magnetic field distribution. [ 113 ] EEG (electroencephalogram) signals, detected using small flat metal discs attached to the scalp, essentially decoding human brain activity used for physical movement, is used to control the robotic limbs. This allows the user to control the part directly. [ 114 ]
The research of robotic legs has made some advancement over time, allowing exact movement and control.
Researchers at the Rehabilitation Institute of Chicago announced in September 2013 that they have developed a robotic leg that translates neural impulses from the user's thigh muscles into movement, which is the first prosthetic leg to do so. It is currently in testing. [ 115 ]
Hugh Herr, head of the biomechatronics group at MIT's Media Lab developed a robotic transtibial leg (PowerFoot BiOM). [ 116 ] [ 117 ]
The Icelandic company Össur has also created a robotic transtibial leg with motorized ankle that moves through algorithms and sensors that automatically adjust the angle of the foot during different points in its wearer's stride. Also there are brain-controlled bionic legs that allow an individual to move his limbs with a wireless transmitter. [ 118 ]
The main goal of a robotic prosthesis is to provide active actuation during gait to improve the biomechanics of gait, including, among other things, stability, symmetry, or energy expenditure for amputees. [ 119 ] There are several powered prosthetic legs currently on the market, including fully powered legs, in which actuators directly drive the joints, and semi-active legs, which use small amounts of energy and a small actuator to change the mechanical properties of the leg but do not inject net positive energy into gait. Specific examples include The emPOWER from BionX, the Proprio Foot from Ossur, and the Elan Foot from Endolite. [ 120 ] [ 121 ] [ 122 ] Various research groups have also experimented with robotic legs over the last decade. [ 123 ] Central issues being researched include designing the behavior of the device during stance and swing phases, recognizing the current ambulation task, and various mechanical design problems such as robustness, weight, battery-life/efficiency, and noise-level. However, scientists from Stanford University and Seoul National University has developed artificial nerves system that will help prosthetic limbs feel. [ 124 ] This synthetic nerve system enables prosthetic limbs sense braille , feel the sense of touch and respond to the environment. [ 125 ] [ 126 ]
Prosthetics are being made from recycled plastic bottles and lids around the world. [ 127 ] [ 128 ] [ 129 ] [ 130 ] [ 131 ]
Most prostheses are attached to the exterior of the body in a non-permanent way. The stump and socket method can cause significant pain for the person, which is why direct bone attachment has been explored extensively.
Osseointegration is a method of attaching the artificial limb to the body by a prosthetic implant.
This method is also sometimes referred to as exoprosthesis (attaching an artificial limb to the bone), or endo-exoprosthesis . Endoprosthesis are prosthetic joint implants which remain wholly inside the body such as knee and hip replacement implants.
The method works by inserting a titanium bolt into the bone at the end of the stump. After several months the bone attaches itself to the titanium bolt and an abutment is attached to the titanium bolt. The abutment extends out of the stump and the (removable) artificial limb is then attached to the abutment. Some of the benefits of this method include the following:
The main disadvantage of this method is that amputees with the direct bone attachment cannot have large impacts on the limb, such as those experienced during jogging, because of the potential for the bone to break. [ 16 ]
Cosmetic prosthesis has long been used to disguise injuries and disfigurements. With advances in modern technology, cosmesis , the creation of lifelike limbs made from silicone or PVC , has been made possible. [ 132 ] Such prosthetics, including artificial hands, can now be designed to simulate the appearance of real hands, complete with freckles, veins, hair, fingerprints and even tattoos.
Custom-made cosmeses are generally more expensive (costing thousands of U.S. dollars, depending on the level of detail), while standard cosmeses come premade in a variety of sizes, although they are often not as realistic as their custom-made counterparts. Another option is the custom-made silicone cover, which can be made to match a person's skin tone but not details such as freckles or wrinkles. Cosmeses are attached to the body in any number of ways, using an adhesive, suction, form-fitting, stretchable skin, or a skin sleeve.
Unlike neuromotor prostheses, neurocognitive prostheses would sense or modulate neural function in order to physically reconstitute or augment cognitive processes such as executive function , attention , language, and memory. No neurocognitive prostheses are currently available but the development of implantable neurocognitive brain-computer interfaces has been proposed to help treat conditions such as stroke , traumatic brain injury , cerebral palsy , autism , and Alzheimer's disease . [ 133 ] The recent field of Assistive Technology for Cognition concerns the development of technologies to augment human cognition. Scheduling devices such as Neuropage remind users with memory impairments when to perform certain activities, such as visiting the doctor. Micro-prompting devices such as PEAT, AbleLink and Guide have been used to aid users with memory and executive function problems perform activities of daily living .
In addition to the standard artificial limb for everyday use, many amputees or congenital patients have special limbs and devices to aid in the participation of sports and recreational activities.
Within science fiction, and, more recently, within the scientific community , there has been consideration given to using advanced prostheses to replace healthy body parts with artificial mechanisms and systems to improve function. The morality and desirability of such technologies are being debated by transhumanists , other ethicists, and others in general. [ 134 ] [ 135 ] [ 136 ] [ 137 ] Body parts such as legs, arms, hands, feet, and others can be replaced.
The first experiment with a healthy individual appears to have been that by the British scientist Kevin Warwick . In 2002, an implant was interfaced directly into Warwick's nervous system. The electrode array , which contained around a hundred electrodes , was placed in the median nerve . The signals produced were detailed enough that a robot arm was able to mimic the actions of Warwick's own arm and provide a form of touch feedback again via the implant. [ 138 ]
The DEKA company of Dean Kamen developed the "Luke arm", an advanced nerve-controlled prosthetic . Clinical trials began in 2008, [ 139 ] with FDA approval in 2014 and commercial manufacturing by the Universal Instruments Corporation expected in 2017. The price offered at retail by Mobius Bionics is expected to be around $100,000. [ 140 ]
Further research in April 2019, there have been improvements towards prosthetic function and comfort of 3D-printed personalized wearable systems. Instead of manual integration after printing, integrating electronic sensors at the intersection between a prosthetic and the wearer's tissue can gather information such as pressure across wearer's tissue, that can help improve further iteration of these types of prosthetic. [ 141 ]
In early 2008, Oscar Pistorius , the "Blade Runner" of South Africa, was briefly ruled ineligible to compete in the 2008 Summer Olympics because his transtibial prosthesis limbs were said to give him an unfair advantage over runners who had ankles. One researcher found that his limbs used twenty-five percent less energy than those of a non-disabled runner moving at the same speed. This ruling was overturned on appeal, with the appellate court stating that the overall set of advantages and disadvantages of Pistorius' limbs had not been considered.
Pistorius did not qualify for the South African team for the Olympics, but went on to sweep the 2008 Summer Paralympics , and has been ruled eligible to qualify for any future Olympics. [ citation needed ] He qualified for the 2011 World Championship in South Korea and reached the semi-final where he ended last timewise, he was 14th in the first round, his personal best at 400m would have given him 5th place in the finals. At the 2012 Summer Olympics in London, Pistorius became the first amputee runner to compete at an Olympic Games. [ 142 ] He ran in the 400 metres race semi-finals, [ 143 ] [ 144 ] [ 145 ] and the 4 × 400 metres relay race finals. [ 146 ] He also competed in 5 events in the 2012 Summer Paralympics in London. [ 147 ]
There are multiple factors to consider when designing a transtibial prosthesis. Manufacturers must make choices about their priorities regarding these factors.
Nonetheless, there are certain elements of socket and foot mechanics that are invaluable for the athlete, and these are the focus of today's high-tech prosthetics companies:
The buyer is also concerned with numerous other factors:
A key feature of prosthetics and prosthetic design is the idea of “designing for disabilities.” This might sound like a good idea in which people with disabilities can participate in equitable design but this is unfortunately not true. The idea of designing for disabilities is first problematic because of the underlying meaning of disabilities. It tells amputees that there is a right and wrong way to move and walk and that if amputees are adapted to the surrounding environment by their own means, then that is the wrong way. Along with that underlying meaning of disabilities, many people designing for disabilities are not actually disabled. “Design for disability" from these experiences, takes disability as the object - with the feeling from non-disabled designers that they have properly learned about their job from their own simulation of the experience. The simulation is misleading and does a disservice to disabled people - so the design that flows from this is highly problematic. Engaging in disability design should be… with, ideally, team members who have the relevant disability and are part of communities that matter to the research. [ 148 ] This leads to people, who do not know what the day-to-day personal experiences are, designing materials that do not meet the needs or hinder the needs of people with actual disabilities.
In the USA a typical prosthetic limb costs anywhere between $15,000 and $90,000, depending on the type of limb desired by the patient. With medical insurance, a patient will typically pay 10%–50% of the total cost of a prosthetic limb, while the insurance company will cover the rest of the cost. The percent that the patient pays varies on the type of insurance plan, as well as the limb requested by the patient. [ 149 ] In the United Kingdom, much of Europe, Australia and New Zealand the entire cost of prosthetic limbs is met by state funding or statutory insurance. For example, in Australia prostheses are fully funded by state schemes in the case of amputation due to disease, and by workers compensation or traffic injury insurance in the case of most traumatic amputations. [ 150 ] The National Disability Insurance Scheme , which is being rolled out nationally between 2017 and 2020 also pays for prostheses.
Transradial (below the elbow amputation) and transtibial prostheses (below the knee amputation) typically cost between US $ 6,000 and $8,000, while transfemoral (above the knee amputation) and transhumeral prosthetics (above the elbow amputation) cost approximately twice as much with a range of $10,000 to $15,000 and can sometimes reach costs of $35,000. The cost of an artificial limb often recurs, while a limb typically needs to be replaced every 3–4 years due to wear and tear of everyday use. In addition, if the socket has fit issues, the socket must be replaced within several months from the onset of pain. If height is an issue, components such as pylons can be changed. [ 151 ]
Not only does the patient need to pay for their multiple prosthetic limbs, but they also need to pay for physical and occupational therapy that come along with adapting to living with an artificial limb. Unlike the reoccurring cost of the prosthetic limbs, the patient will typically only pay the $2000 to $5000 for therapy during the first year or two of living as an amputee. Once the patient is strong and comfortable with their new limb, they will not be required to go to therapy anymore. Throughout one's life, it is projected that a typical amputee will go through $1.4 million worth of treatment, including surgeries, prosthetics, as well as therapies. [ 149 ]
Low-cost above-knee prostheses often provide only basic structural support with limited function. This function is often achieved with crude, non-articulating, unstable, or manually locking knee joints. A limited number of organizations, such as the International Committee of the Red Cross (ICRC), create devices for developing countries. Their device which is manufactured by CR Equipments is a single-axis, manually operated locking polymer prosthetic knee joint. [ 152 ]
Table. List of knee joint technologies based on the literature review. [ 86 ]
evidence
A plan for a low-cost artificial leg, designed by Sébastien Dubois, was featured at the 2007 International Design Exhibition and award show in Copenhagen, Denmark, where it won the Index: Award . It would be able to create an energy-return prosthetic leg for US $ 8.00, composed primarily of fiberglass . [ 153 ]
Prior to the 1980s, foot prostheses merely restored basic walking capabilities. These early devices can be characterized by a simple artificial attachment connecting one's residual limb to the ground.
The introduction of the Seattle Foot (Seattle Limb Systems) in 1981 revolutionized the field, bringing the concept of an Energy Storing Prosthetic Foot (ESPF) to the fore. Other companies soon followed suit, and before long, there were multiple models of energy storing prostheses on the market. Each model utilized some variation of a compressible heel. The heel is compressed during initial ground contact, storing energy which is then returned during the latter phase of ground contact to help propel the body forward.
Since then, the foot prosthetics industry has been dominated by steady, small improvements in performance, comfort, and marketability.
With 3D printers , it is possible to manufacture a single product without having to have metal molds , so the costs can be drastically reduced. [ 154 ]
Jaipur foot , an artificial limb from Jaipur , India , costs about US$40.
There is currently an open-design Prosthetics forum known as the " Open Prosthetics Project ". The group employs collaborators and volunteers to advance Prosthetics technology while attempting to lower the costs of these necessary devices. [ 155 ] Open Bionics is a company that is developing open-source robotic prosthetic hands. They utilize 3D printing to manufacture the devices and low-cost 3D scanners to fit them onto the residual limb of a specific patient. Open Bionics' use of 3D printing allows for more personalized designs, such as the "Hero Arm" which incorporates the users favourite colours, textures, and even aesthetics to look like superheroes or characters from Star Wars with the aim of lowering the cost. A review study on a wide range of printed prosthetic hands found that 3D printing technology holds a promise for individualised prosthesis design, is cheaper than commercial prostheses available on the market, and is more expensive than mass production processes such as injection molding. The same study also found that evidence on the functionality, durability and user acceptance of 3D printed hand prostheses is still lacking. [ 156 ]
In the USA an estimate was found of 32,500 children (<21 years) had a major paediatric amputation, with 5,525 new cases each year, of which 3,315 congenital. [ 157 ]
Carr et al. (1998) investigated amputations caused by landmines for Afghanistan, Bosnia and Herzegovina, Cambodia and Mozambique among children (<14 years), showing estimates of respectively 4.7, 0.19, 1.11 and 0.67 per 1000 children. [ 158 ] Mohan (1986) indicated in India a total of 424,000 amputees (23,500 annually), of which 10.3% had an onset of disability below the age of 14, amounting to a total of about 43,700 limb deficient children in India alone. [ 159 ]
Few low-cost solutions have been created specially for children. Examples of low-cost prosthetic devices include:
This hand-held pole with leather support band or platform for the limb is one of the simplest and cheapest solutions found. It serves well as a short-term solution, but is prone to rapid contracture formation if the limb is not stretched daily through a series of range-of motion (RoM) sets. [ 83 ]
This also fairly simple solution comprises a plaster socket with a bamboo or PVC pipe at the bottom, optionally attached to a prosthetic foot. This solution prevents contractures because the knee is moved through its full RoM. The David Werner Collection, an online database for the assistance of disabled village children, displays manuals of production of these solutions. [ 160 ]
This solution is built using a bicycle seat post up side down as foot, generating flexibility and (length) adjustability. It is a very cheap solution, using locally available materials. [ 161 ]
It is an endoskeletal modular lower limb from India, which uses thermoplastic parts. Its main advantages are the small weight and adaptability. [ 83 ]
Monolimbs are non-modular prostheses and thus require more experienced prosthetist for correct fitting, because alignment can barely be changed after production. However, their durability on average is better than low-cost modular solutions. [ 162 ]
A number of theorists have explored the meaning and implications of prosthetic extension of the body. Elizabeth Grosz writes, "Creatures use tools, ornaments, and appliances to augment their bodily capacities. Are their bodies lacking something, which they need to replace with artificial or substitute organs?...Or conversely, should prostheses be understood, in terms of aesthetic reorganization and proliferation, as the consequence of an inventiveness that functions beyond and perhaps in defiance of pragmatic need?" [ 163 ] Elaine Scarry argues that every artifact recreates and extends the body. Chairs supplement the skeleton, tools append the hands, clothing augments the skin. [ 164 ] In Scarry's thinking, "furniture and houses are neither more nor less interior to the human body than the food it absorbs, nor are they fundamentally different from such sophisticated prosthetics as artificial lungs, eyes and kidneys. The consumption of manufactured things turns the body inside out, opening it up to and as the culture of objects." [ 165 ] Mark Wigley , a professor of architecture, continues this line of thinking about how architecture supplements our natural capabilities, and argues that "a blurring of identity is produced by all prostheses." [ 166 ] Some of this work relies on Freud 's earlier characterization of man's relation to objects as one of extension.
Prosthetics play a vital role in how a person perceives themselves and how other people perceive them. The ability to conceal such use enabled participants to ward off social stigmatization that in turn enabled their social integration and the reduction of emotional problems surrounding such disability. [ 167 ] People that lose a limb first have to deal with the emotional result of losing that limb. Regardless of the reasons for amputation, whether due to traumatic causes or as a consequence of illness, emotional shock exists. It may have a smaller or larger amplitude depending on a variety of factors such as patient age, medical culture, medical cause, etc. As a result of amputation, the research participants' reports were loaded with drama. The first emotional response to amputation was one of despair, a severe sense of self-collapse, something almost unbearable. [ 168 ] Emotional factors are just a small part of looking at social implications. Many people who lose a limb may have lots of anxiety surrounding prosthetics and their limbs. After surgery, for an extended period of time, the interviewed patients from the National Library of Medicine noticed the appearance and increase of anxiety. A lot of negative thoughts invaded their minds. Projections about the future were grim, marked by sadness, helplessness, and even despair. Existential uncertainty, lack of control, and further anticipated losses in one's life due to amputation were the primary causes of anxiety and consequently ruminations and insomnia. [ 168 ] From losing a leg and getting a prosthetics there were also many factors that can happen including anger and regret. The amputation of a limb is associated not only with physical loss and change in body image but also with an abrupt severing in one's sense of continuity. For participants with amputation as a result of physical trauma the event is often experienced as a transgression and can lead to frustration and anger. [ 168 ]
There are also many ethical concerns about how the prosthetics are made and produced. A wide range of ethical issues arise in connection with experiments and clinical usage of sensory prostheses: animal experimentation; informed consent, for instance, in patients with a locked-in syndrome that may be alleviated with a sensory prosthesis; unrealistic expectations of research subjects testing new devices. [ 169 ] How prosthetics come to be and testing of the usability of the device is a major concern in the medical world. Although many positives come when a new prosthetic design is announced, how the device got to where it is leads to some questioning the ethics of prosthetics.
There are also many debates among the prosthetic community about whether they should wear prosthetics at all. This is sparked by whether prosthetics help in day-to-day living or make it harder. Many people have adapted to their loss of limb making it work for them and do not need a prosthesis in their life. Not all amputees will wear a prosthesis. In a 2011 national survey of Australian amputees, Limbs 4 Life found that 7 percent of amputees do not wear a prosthesis, and in another Australian hospital study, this number was closer to 20 percent. [ 170 ] Many people report being uncomfortable in prostheses and not wanting to wear them, even reporting that wearing a prosthetic is more cumbersome than not having one at all. These debates are natural among the prosthetic community and help us shed light on the issues that they are facing. | https://en.wikipedia.org/wiki/Prosthesis |
The Protein Common Interface Database ( ProtCID ) is a database of similar protein-protein interfaces in crystal structures of homologous proteins . [ 1 ] [ 5 ]
Its main goal is to identify and cluster homodimeric and heterodimeric interfaces observed in multiple crystal forms of homologous proteins. Such interfaces, especially of non-identical proteins or protein complexes, have been associated with biologically relevant interactions. [ 6 ]
A common interface in ProtCID indicates chain-chain or domain-domain interactions that occur in different crystal forms. All protein sequences of known structure in the Protein Data Bank (PDB) [ 7 ] are assigned a ” Pfam chain architecture”, which denotes the ordered Pfam [ 8 ] assignments for that sequence, e.g. (Pkinase) or (Cyclin_N)_(Cyclin_C). Homodimeric interfaces in all crystals that contain particular domain or chain architectures are compared, regardless of whether there are other protein types in the crystals. All interfaces between two different Pfam domains or Pfam architectures in all PDB entries that contain them are also compared (e.g., (Pkinase) and (Cyclin_N)_(Cyclin_C) ). For both homodimers and heterodimers, the interfaces are clustered into common interfaces based on a similarity score.
ProtCID reports the number of crystal forms that contain a common interface, the number of PDB entries, the number of PDB and PISA [ 9 ] biological assembly annotations that contain the same interface, the average surface area, and the minimum sequence identity of proteins that contain the interface. ProtCID provides an independent check on publicly available annotations of biological interactions for PDB entries.
ProtCID also contains interface clusters between protein domains and peptides, nucleic acids, and ligands. | https://en.wikipedia.org/wiki/ProtCID |
Angiogenesis is the process of forming new blood vessels from existing blood vessels, formed in vasculogenesis . It is a highly complex process involving extensive interplay between cells, soluble factors, and the extracellular matrix (ECM). Angiogenesis is critical during normal physiological development, but it also occurs in adults during inflammation , wound healing, ischemia , and in pathological conditions such as rheumatoid arthritis , hemangioma , and tumor growth. [ 1 ] [ 2 ] Proteolysis has been indicated as one of the first and most sustained activities involved in the formation of new blood vessels. Numerous proteases including matrix metalloproteinases (MMPs), a disintegrin and metalloproteinase domain ( ADAM ), a disintegrin and metalloproteinase domain with throbospondin motifs ( ADAMTS ), and cysteine and serine proteases are involved in angiogenesis. This article focuses on the important and diverse roles that these proteases play in the regulation of angiogenesis.
Matrix metalloproteinases (MMPs) are a large multigene family of zinc-dependent endopeptidases . The collective MMP family is capable of degrading all known ECM macromolecules. MMP activity is regulated at the level of transcription, post-translationally by proteolytic cleavage, and by endogenous inhibitors known as tissue inhibitors of metalloproteinases (TIMPs). The role of matrix metalloproteinases and TIMPs in several pathological conditions including angiogenesis, tumor growth, and metastasis has been investigated and very well described.
Matrix metalloproteinases contain five conserved domains /sequence motifs:
There is also a subfamily of the matrix metalloproteinases, the membrane-type MMPs (MT-MMPs) which contain an additional transmembrane domain and a short cytoplasmic domain. After activation of MMPs by removal of the propeptide domain, their activity is regulated by TIMPs. TIMPs specifically and reversibly inhibit the activity of MMPs. So far there have been identified four members of the family, TIMP1–4. All TIMPs contain twelve conserved cystein residues, which form six disulfide bonds. The C-terminal domains of TIMPs are highly variable and confer their specificity towards preferred MMP targets. [ 3 ] [ 4 ]
ADAMs comprise a family of integral membrane as well as secreted glycoproteins which are related to snake venom metalloproteinases and MMPs. Like MMPs, ADAMs are composed of multiple conserved domains. They contain propeptide, metalloproteinase, disintegrin-like, cystein-rich, and epidermal growth factor like domains, although variations in domain composition have been observed in non-animal organisms. [ 5 ] Membrane anchored ADAMs contain a transmembrane and cytoplasmic domain. The domains contained within the ADAMs family have been characterized, uncovering their functional and structural roles. [ 6 ] ADAMs contain a consensus sequence which has three histidine residues that bind to the catalytically essential zinc ion. The propeptide is removed through cleavage by a furin type protease yielding the active enzyme. The propeptide of most MMPs is cleavable by proteases such as trypsin , plasmin , chymotrypsin and other MMPs. [ 7 ] ADAMs participate in a wide variety of cell surface remodeling processes, including ectodomain shedding, regulation of growth factor availability and mediating cell-matrix interactions. ADAM17 and ADAM15 have recently been identified in endothelial cells (EC). [ 8 ]
ADAMTS are a subfamily of ADAM related metalloproteinases that contain at least one thrombospondin type I sequence repeat motif (TSR). They are secreted proteins; and the TSR facilitates their localization to the ECM placing it in close proximity to their substrates. Functionally, ADAMTS can be divided into three groups: procollagen aminopeptidase, aggrecanase, and ADAMTS13 which cleaves von Willebrand factor . Unlike with MMPs, TIMPs are more selective in their ability to inhibit ADAMs and ADAMTSs. TIMP3 is able to inhibit ADAM17 and 12 as well as ADAMTS4 and 5 . ADAM8 and ADAM9 are not susceptible to inhibition by TIMPs.
Many additional classes of enzymes have been identified that facilitate angiogenesis. They include serine, aspartic, and cysteine-type proteases. A highly characterized example of the serine protease family is the plasminogen activator - plasmin system, which has been shown to be involved in vascular remodelling . Tissue plasminogen activator (tPA), and urokinase plasminogen activator (urokinase, uPA) are serine proteases which cleave and activate plasminogen. The activated form of plasminogen , plasmin, is a wide-ranging protease capable of acting on various ECM components including fibrin , collagens , laminin , fibronectin , and proteoglycans . [ 9 ] Additionally, plasmin also is able to activate various other MMPs.
In humans, the group of cathepsin cysteine proteases or cysteine cathepsins comprises 11 family members, cathepsins B , C , F , H , L1 , L2 , K , O , S , W , and X/Z . [ 10 ] Cysteine cathepsins are synthesized as inactive zymogens and activated by proteolytic removal of their propeptide. These enzymes are primarily localized in lysosomes and function in terminal protein degradation and processing. Cathepsins also can be secreted by cells, associate with the cell surface, and degrade the ECM. A study of all 11 members of the cathepsin family highlights their importance in tumorigenesis and tumor associated angiogenesis. [ 11 ] Examination of cathepsin activity by using chemical probes and in vivo imaging techniques demonstrated an increase in cathepsin activity in the angiogenic blood vessels and invasive fronts of carcinoma in the RIP-Tag2 transgenic mouse model of pancreatic islet tumor genesis.
Aminopeptidases function as exopeptidases which remove amino acids from the amino-terminus of proteins. Aminopeptidase N (CD13/APN) is highly expressed on the endothelium of growing vessels. [ 12 ] Inhibitors of CD13/APN dramatically impair tumor growth.
It has become clear in the past years that ectodomain shedding is an initial step for the activation of specific receptors such as Notch , ErbB-4 and the angiopoietin receptor Tie-1. Notch-1 signaling is essential for endothelial differentiation, and tumor angiogenesis, while the angiopoietin receptor Tie-1 facilitates embryonic blood vessel formation. [ 13 ] [ 14 ] Upon binding of their ligands, Notch-1 and Tie-1 undergo proteolytic cleavage of the ectodomains by ADAM17 and ADAM10 . This cleavage frees the cytoplasmic fragment for cellular signaling. In the case of Notch-1, it transfers to the nucleus.
Many cytokines and growth factors are synthesized as membrane-bound proforms which undergo proteolytic shedding for activation. The ephrins EPH receptor A2 and A3 are shed by ADAM10, creating cleaved soluble Eph receptors , which inhibit tumor angiogenesis in mice. [ 15 ] Additional examples are the proteolytic shedding of soluble E-selectin , [ 16 ] shedding of urokinase receptor (uPAR) by MMP-12 creating soluble uPAR which has chemotactic properties for leukocytes and progenitor cells, and the shedding of interleukin-6 receptors by ADAM10 and ADAM17 which facilitates interleukin-6 signaling in endothelial cells. [ 17 ] Semaphorin 4D is cleaved from its membrane-bound form by MT1-MMP (MMP-14) in tumor cells; it then interacts with plexin B1 on endothelial cells, promoting pro-angiogenic chemotaxis. [ 18 ] Shedding of a membrane-anchored cytokine or growth factor by ADAM proteinases may be relevant for various signal transduction events. Alternatively, shedding may be required for the ligand to diffuse to distant receptors. Shedding may be required for the down regulation of signals by removing signaling ligands, or cleavage and release of receptors. Release of the receptor may also generate soluble receptors which act as decoys by sequestering ligands. These findings indicate that ectodomain shedding is a ubiquitous process facilitating a wide variety of cellular events involved in angiogenesis. Because potent biological modifiers are generated, it is likely controlled by highly regulated mechanism. Along with ADAMs and MT-MMPs, membrane-bound serine proteases also may play a role in ectodomain shedding.
The formation of capillaries from pre-existing blood vessels requires the remodeling of both the peicapillary membrane of the parent venule , as well as the local and distal ECM. At the onset of angiogenesis endothelial cells (EC) must remodel three different barriers in order to migrate and invade the target tissue. First is the basement membrane between the endothelium and vascular smooth muscle cells or pericytes , followed by the fibrin gel formed from fibrinogen that is leaked from the vasculature, and finally the extracellular matrix in the target tissue. The vascular basement membrane is composed of type IV collagen , type XV collagen , type XVIII collagen , laminins , entactin , heparan sulfate proteoglycans, perlecan , and osteonectin . All of these components of the basement membrane are substrates for MMP-2 , 3 , 7 , and 9 , among others. Inhibitors of MMP activity have spotlighted the importance of these proteins in controlling angiogenesis. Recently, it has been discovered that small interfering RNA (siRNA) mediated target RNA degradation of urokinase receptor and MMP-9 inhibits the formation of capillary like structures in both in vitro and in vivo models of angiogenesis. [ 19 ] After working their way through the basement membrane, EC must invade through a dense fibrin gel which is polymerized from fibrinogen derived from the vascular bed. [ 20 ] Plasmin, an effective fibrinolysin produced by tPA or uPA , was thought to be essential in this process, but plasminogen deficient mice do not display major defects of neovascularization in fibrin rich tissues. [ 21 ] These findings highlight the diverse amount of proteolytic enzymes ECs use to remodel the ECM. For example, MMP-3, 7, 8 , 12 and 13 can cleave fibrinogen. [ 22 ]
MMP activity is one of the earliest and most sustained processes that take place during angiogenesis. By studying the transition from an avascular to a vascular tumor Fang et al. were able to identify the key role of MMP-2 in angiogenesis. MMP-2 expression and activity was increased in angiogenic tumors as compared with avascular tumors, and the addition of antisense oligonucleotides targeting MMP-2 inhibits the initiation of angiogenesis maintaining the avascular phenotype. This data along with other reports suggest that MMP activity is necessary to initiate the earliest stages of angiogenesis and tumor development. The creation of MMP deficient mice has provided important insight into the role of MMPs in the regulation of angiogenesis. For example, MMP-2 knockout mice develop normally but display significant inhibition of corneal angiogenesis. [ 23 ]
Numerous proteolytic fragments or domains of ECM proteins have been reported to exert positive or negative activity on angiogenesis. Native proteins which contain such domains with regulatory activity are normally inactive, most likely because they are cryptic segments hidden in the native protein structure. Angiostatin is a 38 kDa plasminogen fragment with angiogenesis inhibitor activity. Angiostatin fragments contain kringle domains which exert their inhibitory activity at several different levels; they inhibit endothelial cell migration and proliferation , increase apoptosis , and modulate the activity of focal adhesion kinase (FAK). Endostatin is a 20 kDa fragment of collagen XVIII. The major role of endostatin is in its ability to potently inhibit endothelial cell migration and induce apoptosis. [ 24 ] These effects are mediated by interacting and interfering with various angiogenic related proteins such as integrins and serine/threonine-specific protein kinases . Numerous studies have demonstrated that tropoelastin , the soluble precursor of elastin , or proteolytic elastin fragments have diverse biological properties. Nackman et al. demonstrated that elastase generated elastin fragments mediate several characteristic features of aneurysmal disease which correlated to angiogenesis. Osteonectin is a metal binding glycoprotein produced by many cell types including ECs. Lastly, endorepellin is a recently described inhibitor of angiogenesis derived from the carboxy terminus of perlecan. [ 25 ] Nanomolar concentrations of endorepellin inhibits EC migration and angiogenesis in different in vitro and in vivo models by blocking EC adhesion to various substrate such as fibronectin and type I collagen .
Endogenous inhibitors or activators generated by proteolytic degradation of larger proteins mostly from the ECM have proven to contribute to the regulation of tumor growth and angiogenesis. This article mentions only a small fraction of the known proteolytic fragments which alter EC behavior and function during angiogenesis. This abundance has garnered increased attention because of their potential for anti-angiogenic and anti-cancer therapies.
Proteases not only modulate cell-matrix interactions but also can control the onset and progression of angiogenesis by activating angiogenic growth factors and cytokines. Hepatocyte growth factor (HGF), an angiogenesis promoting growth factor, is activated by HGF activation factor , a serine protease related to plasminogen. [ 26 ] Several growth factors such as basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) are trapped in the ECM by various proteoglycans. The proteolytic degradation of these proteoglycans liberates the growth factors allowing them to reach their receptors and influence cellular behavior. Growth factors that indirectly affect angiogenesis are also targets of proteolytic activation. For example, plasminogen activators drive the activation of latent transforming growth factor beta (TGF-β) from bone ECM and thus modulate angiogenesis in bone. [ 27 ]
Proteases not only have the ability change the availability of growth factors, but can also modify their properties. This ability was shown for VEGF 165 that is cleaved by MMP-3 or MMP-9 to a smaller molecule with properties similar to VEGF 121 . [ 28 ] These two isoforms of VEGF have very different properties. VEGF 165 induces a regular vessel pattern during tumor neovascularization. VEGF 121 and the truncated VEGF 165 , in contrast, cause irregular patterns of neovascularization, most likely due to their inability to bind heparan sulfates, wherefore they do not provide any spatial information that is buried in the ECM. Another important factor in angiogenesis, stromal cell-derived factor-1 (SDF-1), is also modified by the aminodipeptidase dipeptidyl peptidase-4 (DPP4). Cleavage of SDF-1 reduces it heparan sulfate affinity and interactions with its receptor CXCR4 are reduced. [ 29 ] The ADAM family of proteases is receiving increased attention for their ability to alter the balance between pro-and anti-angiogenic factors. ADAM17 is able to release active tumor necrosis factor-alpha (TNFα) and heparin-binding EGF-like growth factor (HB-EGF) from their membrane bound precursors which can indirectly affect angiogenesis. [ 30 ]
Proteases not only facilitate angiogenesis, but they also have the ability to put the brakes on the process. One example of this is the processing of angiogenesis inhibitors by MMPs. As previously described, MMPs have been shown to cleave plasminogen and collagen XVIII into the endogenous angiogenesis inhibitors angiostatin and endostatin. MMP-2 itself possesses anti-angiogenic properties that are independent of its catalytic domain. Interactions between integrin α v β 3 and MMP-2 on the EC cell surface may be necessary for MMP-2 activity during angiogenesis. The hemopexin like domain in the carboxy terminus of MMP-2 is able to block this interaction of active MMP-2 and integrin α v β 3 on the EC surface which lead to inhibition of MMP-2 activity. [ 31 ]
During angiogenesis ADAM15 is preferentially expressed on EC. ADAM15 is able to interact with integrins α v β 3 and α 5 β 1 through its disintegrin domain via an RGD ( arginine - glycine - aspartic acid ) motif. Most disintegrins contain this conserved RGD motif, but ADAM15 is the only member of the ADAM family to contain this motif. A recombinant disintegrin domain of human ADAM15 inhibits a variety of EC functions in vitro including proliferation, adhesion, migration, and capillary formation. [ 32 ] Overexpression of ADAM15 disintegrin domain resulted in inhibition of angiogenesis, tumor growth, and metastasis. On the other hand, it has not been shown whether full length ADAM15 plays an inhibitory role in vivo . ADAMTS1 and ADAMTS8 inhibit angiogenesis in vitro in two functional angiogenesis assays. Both enzymes inhibit bFGF induced vascularization in the corneal pocket assay and inhibit VEGF induced angiogenesis in the chorioallantoic membrane assay. [ 33 ] All together, these data indicate that proteases can function as both positive and negative regulators of angiogenesis.
Angiogenesis requires the migration and invasive growth of cells. This is facilitated by a balanced interplay between detachment and formation of cell adhesions which enable the cell to crawl forward through the ECM. [ 34 ] The cell uses limited proteolytic activity at sites of individual focal adhesions via the formation of multiprotein complexes. Multiprotein complexes are localized in lipid rafts on the cell surface, where membrane bound proteases are often incorporated. For example, leukocytes complex urokinase (uPA), urokinase receptor (uPAR), and integrins which participate in cell adhesion and invasion. [ 35 ] In these complexes, uPAR acts as an organizing center forming noncovalent complexes with integrins, LRP -like proteins, and urokinase. Similar complexes also are found on ECs.
The proteolytic activities that take place during angiogenesis require precise spatial and temporal regulation. If not for this control excessive proteolysis could lead to damage of the tissue and the loss of anchorage points for migrating cells. This is illustrated by mice which are deficient for plasminogen activator inhibitor-1 (PAI-1). [ 36 ] [ 37 ] PAI-1 inhibits plasminogen activators and thus plasmin activation; therefore it could be assumed that PAI-1 deficiency would increase angiogenesis and tumor growth. Unexpectedly, when PAI-1 deficient mice were challenged with cancer cells on a collagenous matrix, angiogenesis and vascular stabilization was inhibited, hampering tumor growth. This finding was credited to the protective properties PAI-1 imparts against excessive degradation of the surrounding ECM by plasmin. Without this protection the footholds used by endothelial cells to migrate and form capillary structures are destroyed. Uncontrolled proteolysis also is attributed to the disruption of vascular development and premature deaths in murine embryos deficient of the inhibitor reversion-inducing-cysteine-rich protein with kazal motifs (RECK). This is most likely due to uncontrolled MMP activity, because a partial rescue was obtained by simultaneously knocking out RECK and MMP-2. [ 38 ]
Leukocytes and endothelial progenitor cells (EPCs) contribute to the initiation and guidance of new blood vessels. [ 39 ] Monocytes produce a variety of pro-angiogenic factors. There is also a population of CD34 positive cells that can express endothelial associated proteins, such as VE-cadherin and kinase insert domain receptor (KDR, VEGF receptor 2) which aid in influencing the progression of angiogenesis. [ 40 ] The absence or dysfunction of these cells is implicated in impaired vascularization in cardiac and diabetes patients. [ 41 ] MMP-9 plays a key role in mobilizing EPCs from the bone marrow. Heissig et al. have proposed a mechanism for how MMP-9 facilitates the availability of EPCs for angiogenesis. First, circulating VEGF induces MMP-9 expression in the bone marrow, MMP-9 then is able to cleave and release c-kit ligand. Activated c-kit is then able to recruit hematopoietic , endothelial and mast cell progenitor cells, these cells are then accumulated in the angiogenic area and produce large amounts of VEGF tipping the scales in favor of angiogenesis. [ 42 ]
MMP-9 is not the only protease shown to be involved in EPC enhanced angiogenesis. Cathepsin L1 is active at neutral pH by associating with a p41 splice variant of the MHC class II-associated invariant chain which is strongly expressed in EPCs. [ 43 ] This ability to stay active at neutral pH may facilitate EPC invasion, remodeling of matrix collagens and gelatin, and neovascularization. Knock out of cathepsin L1 in mice exhibited impaired blood flow restoration in ischemic limbs, indicating impaired neovascularization. Neovascularization also is impaired in mice treated with bone marrow derived cells deficient of cathepsin L1 as compared with wild type cells. The target by which cathepsin L1 stimulates angiogenesis is not yet identified.
It has been well established that smooth muscle-like pericytes play an important role in stabilizing newly formed blood vessels. Pericytes present in the stroma of tumors of breast cancer patients express MMP-9. [ 44 ] Animal models deficient of MMP-9 display disturbed recruitment of pericytes. [ 45 ] The inability to recruit pericytes severely affects the stability of vessels and the degree of vascularization of neuroblastomas . Aminopeptidase A also may be involved in pericyte recruitment due to its increased expression by activated pericytes in various pathological conditions associated with angiogenesis. [ 46 ] The mechanism by which this protease facilitates vessel maturation has not yet been determined. Angiogenesis requires a fine balance between proteolytic activity and proteinase inhibition. Pericytes secrete TIMP-3 which inhibits MT1-MMP dependent MMP-2 activation on endothelial cell, thus facilitating stabilization of newly formed microvessels. Co-cultures consisting of pericytes and endothelial cells induce the expression of TIMP-3 by pericytes, while endothelial cells produce TIMP-2. [ 47 ] Together, these inhibitors stabilize the vasculature by inhibiting a variety of MMPs, ADAMs, and VEGF receptor 2.
Immature vessels remain dependent on continuous exposure the angiogenic growth factors without pericyte coverage. [ 48 ] As the reservoir of growth factors is removed the endothelial cells do not survive, and undergo caspases induced apoptosis, while other proteases participate in the degradation and removal of the remaining cell debris.
Proteases play numerous roles in angiogenesis, both in development and especially in pathological conditions. Because they are important regulators of tissue degradation and cell migration, it is expected that their inhibition would be beneficial for inhibiting tumor growth and vascularization. Promising results have been observed in animal studies, but clinical trials have failed to demonstrate similar results and are often accompanied by unacceptable side effects. [ 49 ] This has influenced continued research which has identified new families of proteases, such as ADAM, ADAMTS, and MT-MMPs. Perhaps more significantly, a new paradigm has emerged for proteases being essential for modulating growth factors and cytokines, generating biologically active fragments from the matrix, facilitating recruitment of bone marrow derived cells, and stabilization of mature blood vessels. Better understanding of the various activities of proteases and their inhibitors will aid in more tailor made treatments for numerous disorders. | https://en.wikipedia.org/wiki/Proteases_in_angiogenesis |
Protechnik was a front company established on 24 June 1987 by the South African Defence Force to perform quality assurance testing of chemical protective materials and equipment within a covert operation known as Project Coast . [ 1 ]
Founded by Dr. Jan Lourens, a bio-engineering consultant at Roodeplaat Research Laboratories , as Systems Research and Development (SRD), with funding provided by the Medical arm of the SADF. Protechnik's shareholders would include Medchem Consolidated Investments (MCI), WPW Investments (a firm belonging to Wouter Basson ) the Luxembourg-based Charburn Enterprises. According to testimony provided in the Wouter Basson trial, Protechnik was involved in reverse engineering Chemical Agent Monitors . [ 2 ]
The company is now a subsidiary of Armscor . | https://en.wikipedia.org/wiki/Protechnik |
Protected Streaming [ 1 ] is a DRM technology by Adobe . The aim of the technology is to protect digital content (video or audio) from unauthorized use.
Protected Streaming consists of many different techniques; basically there are two main components: encryption and SWF verification.
This technique is used by the Hulu desktop player and the RTÉ Player . Fifa.com also uses this technique to serve the videos on the official site. Some videos on YouTube also use RTMPE, including those uploaded there by BBC Worldwide.
Streamed content is encrypted by the Flash Media Server "on the fly", so that the source file itself does not need to be encrypted (a significant difference from Microsoft 's DRM). For transmission ("streaming"), a special protocol is required, either RTMPE or RTMPS . [ citation needed ]
RTMPS uses SSL -encryption. In contrast, RTMPE is designed to be simpler than RTMPS, by removing the need to acquire a SSL Certificate. RTMPE makes use of well-known industry standard cryptographic primitives, consisting of Diffie–Hellman key exchange and HMACSHA256, generating a pair of RC4 keys, one of which is then used to encrypt the media data sent by the server (the audio or video stream), while the other key is used to encrypt any data sent to the server. RTMPE caused less CPU -load than RTMPS on the Flash Media Server. [ citation needed ]
Adobe fixed the security issue in January 2009, but did not fix the security holes in the design of the RTMPE algorithm itself. [ 2 ] Analysis of the algorithm shows that it relies on security through obscurity . For example, this renders RTMPE vulnerable to Man in the Middle attacks . [ citation needed ]
Tools which have a copy of the well-known constants extracted from the Adobe Flash Player are able to capture RTMPE streams, a form of the trusted client problem. Adobe issued DMCA takedowns on RTMPE recording tools, including rtmpdump , to try to limit their distribution. In the case of rtmpdump , however, this led to a Streisand effect . [ 3 ]
The Adobe Flash Player uses a well-known constant, appended to information derived from the SWF file (a hash of the file and its size), as input to HMACSHA256. The HMACSHA256 key is the last 32 bytes of the server's first handshake packet. The Flash Media Server uses this to limit access to those clients which have access to the SWF file (or have been given a copy of the hash and the size of the SWF file).
All officially allowed clients (which are in fact *.swf files) need to be placed on the Flash Media Server streaming the file. Any other client requesting a connection will receive a "connection reject".
The combination of both techniques is intended to ensure streams cannot be sniffed and stored into a local file, as SWF verification is intended to prevent third party clients from accessing the content. However, it does not achieve this goal. Third party clients are free to write the decrypted content to a local file simply by knowing the hash of the SWF file and its size. In practice, therefore, Adobe's own implementation of the Macromedia Flash Player is the only client which does not allow saving to a local file.
The only possible way to restrict connections to a Flash Media Server is to use a list of known hosts, to avoid the whole player (the Flash client) being placed on an unauthorised website. Even this has no real benefit for mass-distributed files, as any one of the known hosts could take a copy of the data and re-distribute it at will. Thus "known host" security is only useful when the known hosts can be trusted not to re-distribute the data. | https://en.wikipedia.org/wiki/Protected_Streaming |
The Chemical Facility Anti-Terrorism Standards Program Authorization and Accountability Act of 2014 ( H.R. 4007 ) is a bill that proposed making permanent the United States Department of Homeland Security ’s (DHS’s) authority to regulate security at certain chemical facilities in the United States. [ 1 ] Under the Chemical Facility Anti-Terrorism Standards (CFATS) program, DHS collects and reviews information from chemical facilities in the United States to determine which facilities present security risks and then requires them to write and enact security plans. [ 1 ]
The bill was introduced into the United States House of Representatives during the 113th United States Congress , and passed on July 8, 2014. An amended version of the bill passed the Senate on December 10 and was sent back to the House, which voted to pass on December 11. The bill was signed into law on December 18, 2014 as the Protecting and Securing Chemicals Facilities from Terrorist Attacks Act of 2014 by President Barack Obama . [ 2 ]
The Chemical Facility Anti-Terrorism Standards ( CFATS ), also known as 6 CFR, Part 27, are a set of US government security regulations for high-risk chemical facilities such as chemical plants , electrical generating facilities, refineries, and universities . [ 3 ] The US Department of Homeland Security promulgated the Final Rule on April 9, 2007. [ 4 ] The regulations came into effect on June 8, 2007, apart from material covered in Appendix A, which took effect upon its publication in the Federal Register on November 20, 2007. [ 5 ]
The response from the US chemical community to the initial legislation was rather critical, [ 6 ] but the revisions introduced in November appear to have addressed many of the concerns of both industry and academia. [ 7 ] For example, certain routine chemicals of low toxicity, such as acetone or urea , have been removed from the list, since record-keeping for such common compounds was considered an excessive burden.
This summary is based largely on the summary provided by the Congressional Research Service , a public domain source. [ 8 ]
The Chemical Facility Anti-Terrorism Standards Program Authorization and Accountability Act of 2014 would reestablish the Chemical Facility Anti-Terrorism Standards (CFATS) Program, under which the United States Secretary of Homeland Security (DHS) is required to: (1) establish risk-based performance standards designed to protect covered chemical facilities from acts of terrorism ; (2) require such facilities to submit security vulnerability assessments and develop and implement site security plans; (3) review and approve or disapprove each such assessment and plan; (4) arrange for the audit and inspection of covered chemical facilities to determine compliance with this Act; and (5) notify, and issue an order to comply to, the owner or operator of a facility not in compliance. [ 8 ]
The bill would authorize the Secretary to: (1) issue an order assessing a civil penalty or to cease operations if an owner or operator fails to comply; and (2) approve an alternative security program established by a private sector entity or a federal, state, or local authority that meets the requirements of this Act. [ 8 ]
The bill would authorize a covered chemical facility, in order to satisfy the requirements of a risk-based performance standard that addresses personnel surety by identifying individuals with terrorist ties, to utilize any federal screening program that periodically vets individuals against the Terrorist Screening Database . [ 8 ]
The bill would require the Secretary to: (1) consult with the heads of other federal agencies, states and political subdivisions, and relevant business associations to identify all chemical facilities of interest; and (2) develop a risk assessment approach and corresponding tiering methodology that incorporates all relevant elements of risk, including threat, vulnerability, and consequence. Defines "covered chemical facility" to mean a chemical facility that the Secretary designates as a facility of interest and determines presents a high level of security risk, with specified exceptions. [ 8 ]
The bill would require: (1) information developed pursuant to this Act to be protected from public disclosure, but permits the sharing of such information with state and local government officials possessing the necessary security clearances; and (2) information submitted to or obtained by the Secretary under this Act to be treated as classified material. [ 8 ]
The bill would set forth civil penalties for violations of orders issued under this Act. [ 8 ]
The bill would terminate this Act two years after its enactment. [ 8 ]
This summary is based largely on the summary provided by the Congressional Budget Office , as ordered reported by the House Committee on Homeland Security on April 30, 2014. This is a public domain source. [ 1 ]
H.R. 4007 would make permanent the United States Department of Homeland Security ’s (DHS’s) authority to regulate security at certain chemical facilities in the United States. Under the Chemical Facility Anti-Terrorism Standards (CFATS) program, DHS collects and reviews information from chemical facilities in the United States to determine which facilities present security risks. Facilities determined to present a high level of security risk are then required to develop a Site Security Plan (SSP). DHS in turn conducts inspections to validate the adequacy of a facility’s SSP and their compliance with it. The program is set to end on October 4, 2014. [ 1 ]
The Chemical Facility Anti-Terrorism Standards Program Authorization and Accountability Act of 2014 was introduced into the United States House of Representatives on February 6, 2014 by Rep. Patrick Meehan (R, PA-7) . [ 9 ] It was referred to the United States House Committee on Homeland Security , the United States House Committee on Energy and Commerce , the United States House Homeland Security Subcommittee on Cybersecurity, Infrastructure Protection, and Security Technologies , and the United States House Energy Subcommittee on Environment and Economy . [ 9 ] The DHS National Protection and Programs Directorate 's Office of Infrastructure Protection Assistant Secretary Caitlin Durkovich testified in favor of the bill before the United States House Homeland Security Subcommittee on Cybersecurity, Infrastructure Protection, and Security Technologies . [ 10 ] On June 23, 2014, it was reported (amended) alongside House Report 113-491 part 1 . On July 8, 2014, the House voted in a voice vote to pass the bill. [ 9 ]
Rep. Meehan, who introduced the bill, argued that "the fertilizer plant explosion in West, Texas less than a year ago tragically demonstrated the potential for catastrophe at one of our country's thousands of chemical facilities. The Chemical Facility Anti-Terrorism Standards program is an important part of ensuring these facilities take reasonable precautions to protect against terrorist attack." [ 11 ]
The Department of Homeland Security (DHS) supported the bill, arguing that "CFATS is making the Nation more secure by reducing the risks associated with our Nation’s chemical infrastructure." [ 10 ] The DHS also argued that the reauthorization was necessary because "uncertainty about the future of CFATS also has provided an incentive for potentially regulated facilities storing large quantities of dangerous chemicals to ignore their obligations under CFATS in hopes that the program will be allowed to sunset." [ 10 ]
The Fertilizer Institute supported the bill, arguing that it would allow the DHS to "effectively establish programs and make necessary changes to existing ones without worrying about whether or not the resources to administer them will be available in the future. The legislation would allow industry to be able to plan for investments with the certainty of knowing the program will be in place." [ 12 ]
The National Association of Chemical Distributors supported the bill, arguing that it was important to reauthorize the program for two years to avoid situations like the one resulting from the United States federal government shutdown of 2013 , when the law lapsed. [ 13 ]
This article incorporates public domain material from websites or documents of the United States government . | https://en.wikipedia.org/wiki/Protecting_and_Securing_Chemicals_Facilities_from_Terrorist_Attacks_Act_of_2014 |
A protecting group or protective group is introduced into a molecule by chemical modification of a functional group to obtain chemoselectivity in a subsequent chemical reaction. It plays an important role in multistep organic synthesis . [ 1 ]
In many preparations of delicate organic compounds , specific parts of the molecules cannot survive the required reagents or chemical environments. These parts (functional groups) must be protected . For example, lithium aluminium hydride is a highly reactive reagent that usefully reduces esters to alcohols . It always reacts with carbonyl groups, and cannot be discouraged by any means. When an ester must be reduced in the presence of a carbonyl, hydride attack on the carbonyl must be prevented. One way to do so converts the carbonyl into an acetal , which does not react with hydrides. The acetal is then called a protecting group for the carbonyl. After the hydride step is complete, aqueous acid removes the acetal, restoring the carbonyl. This step is called deprotection .
Protecting groups are more common in small-scale laboratory work and initial development than in industrial production because they add additional steps and material costs. However, compounds with repetitive functional groups – generally, biomolecules like peptides , oligosaccharides or nucleotides – may require protecting groups to order their assembly. Also, cheap chiral protecting groups may often shorten an enantioselective synthesis (e.g. shikimic acid for oseltamivir ).
As a rule, the introduction of a protecting group is straightforward. The difficulties honestly lie in their stability and in selective removal. Apparent problems in synthesis strategies with protecting groups are rarely documented in the academic literature. [ 2 ]
Orthogonal protection is a strategy allowing the specific deprotection of one protective group in a multiply-protected structure. For example, the amino acid tyrosine could be protected as a benzyl ester on the carboxyl group, a fluorenylmethylenoxy carbamate on the amine group, and a tert -butyl ether on the phenol group. The benzyl ester can be removed by hydrogenolysis, the fluorenylmethylenoxy group (Fmoc) by bases (such as piperidine), and the phenolic tert -butyl ether cleaved with acids (e.g. with trifluoroacetic acid).
A common example for this application, the Fmoc peptide synthesis, in which peptides are grown in solution and on solid phase, is very important. [ 3 ] The protecting groups in solid-phase synthesis regarding the reaction conditions such as reaction time, temperature and reagents can be standardized so that they are carried out by a machine, while yields of well over 99% can be achieved. Otherwise, the separation of the resulting mixture of reaction products is virtually impossible (see also § Industrial applications ). [ 4 ]
A further important example of orthogonal protecting groups occurs in carbohydrate chemistry. As carbohydrates or hydroxyl groups exhibit very similar reactivities, a transformation that protects or deprotects a single hydroxy group must be possible for a successful synthesis.
Many reaction conditions have been established that will cleave protecting groups. One can roughly distinguish between the following environments: [ 5 ]
Various groups are cleaved in acid or base conditions, but the others are more unusual.
Fluoride ions form very strong bonds to silicon ; thus silicon protecting groups are almost invariably removed by fluoride ions. Each type of counterion, i.e. cleavage reagent, can also selectively cleave different silicon protecting groups depending on steric hindrance . The advantage of fluoride-labile protecting groups is that no other protecting group is attacked by the cleavage conditions.
Lipases and other enzymes cleave ethers at biological pH (5-9) and temperatures (30–40 °C). Because enzymes have very high substrate specificity, the method is quite rare, but extremely attractive.
Catalytic hydrogenation removes a wide variety of benzyl groups : ethers, esters, urethanes, carbonates, etc.
Only a few protecting groups can be detached oxidatively: the methoxybenzyl ethers, which oxidize to a quinomethide . They can be removed with ceric ammonium nitrate (CAN) or dichlorodicyanobenzoquinone (DDQ).
Allyl compounds will isomerize to a vinyl group in the presence of noble metals . The residual enol ether (from a protected alcohol) or enamine (resp. amine) hydrolyzes in light acid.
Photolabile protecting groups bear a chromophore , which is activated through radiation with an appropriate wavelength and so can be removed. [ 6 ] For examples the o -nitrobenzylgroup ought be listed here.
The rare double-layer protecting group is a protected protecting group, which exemplify high stability.
The classical protecting groups for alcohols are esters , deprotected by nucleophiles ; triorganosilyl ethers , deprotected by acids and fluoride ions; and (hemi)acetals , deprotected by weak acids. In rarer cases, a carbon ether might be used.
The most important esters with common protecting-group use are the acetate , benzoate , and pivalate esters , for these exhibit differential removal. Sterically hindered esters are less susceptible to nucleophilic attack:
Triorganosilyl sources have quite variable prices, and the most economical is chlorotrimethylsilane (TMS-Cl), a Direct Process byproduct. The trimethylsilyl ethers are also extremely sensitive to acid hydrolysis (for example silica gel suffices as a proton donator) and are consequently rarely used nowadays as protecting groups.
Aliphatic methyl ethers cleave with difficulty and only under drastic conditions, so that these are in general only used with quinonic phenols. However, hemiacetals and acetals are much easier to cleave.
Esters:
Silyl ethers:
Benzyl ethers:
Acetals:
Other ethers:
The 1,2‑diols ( glycols ) present for protecting-group chemistry a special class of alcohols. One can exploit the adjacency of two hydroxy groups, e.g. in sugars , in that one protects both hydroxy groups codependently as an acetal . Common in this situation are the benzylidene , isopropylidene and cyclohexylidene or cyclopentylidene acetals.
An exceptional case appears with the benzylideneprotecting group,which also admits reductive cleavage. This proceeds either through catalytic hydrogenation or with the hydride donor diisobutyl aluminum hydride (DIBAL). The cleavage with DIBAL deprotects one alcohol group, for the benzyl moiety stays as a benzyl ether on the second, sterically hindered hydroxy group. [ 45 ] [ 46 ]
Amines have a special importance in peptide synthesis , but are a quite potent nucleophile and also relatively strong bases . These characteristics imply that new protecting groups for amines are always under development. [ 47 ]
Amine groups are primarily protected through acylation , typically as a carbamate . When a carbamate deprotects, it evolves carbon dioxide . The commonest-used carbamates are the tert -butoxycarbonyl, benzoxycarbonyl, fluorenylmethylenoxycarbonyl, and allyloxycarbonyl compounds.
Other, more exotic amine protectors are the phthalimides , which admit reductive cleavage, [ 48 ] and the trifluoroacetamides, which hydrolyze easily in base. Indoles , pyrroles und imidazoles — verily any aza-heterocycle — admit protection as N ‑sulfonylamides,which are far too stable with aliphatic amines. [ 49 ] N ‑benzylated amines can be removed through catalytic hydrogenation or Birch reduction, but have a decided drawback relative to the carbamates or amides: they retain a basic nitrogen.
Carbamates:
Other amides:
Benzylamines:
The most common protecting groups for carbonyls are acetals and typically cyclic acetals with diols. The runners-up used are also cyclic acetals with 1,2‑hydroxythiols or dithioglycols – the so-called O , S – or S , S -acetals.
Overall, trans-acetalation plays a lesser role in forming protective acetals; they are formed as a rule from glycols through dehydration. Normally a simple glycol like ethylene glycol or 1,3-propadiol is used for acetalation.Modern variants also use glycols, but with the hydroxyl hydrogens replaced with a trimethylsilyl group. [ 60 ] [ 61 ]
Acetals can be removed in acidic aqueous conditions. For those ends, the mineral acids are appropriate acids. Acetone is a common cosolvent, used to promote dissolution. For a non-acidic cleavage technique, a palladium(II) chloride acetonitrile complex in acetone [ 62 ] or iron(III) chloride on silica gel can be performed with workup in chloroform. [ 63 ]
Cyclic acetals are very much more stable against acid hydrolysis than acyclic acetals. Consequently acyclic acetals are used practically only when a very mild cleavage is required or when two different protected carbonyl groups must be differentiated in their liberation. [ 64 ]
Besides the O , O -acetals, the S , O - and S , S -acetals also have an application, albeit scant, as carbonyl protecting groups too. Thiols , which one begins with to form these acetals, have a very unpleasant stench and are poisonous, which severely limit their applications. Thioacetals and the mixed S , O -acetals are, unlike the pure O , O -acetals, very much stabler against acid hydrolysis. This enables the selective cleavage of the latter in the presence of sulfur -protected carbonyl groups.
The formation of S , S -acetals normally follows analogously to the O , O -acetals with acid catalysis from a dithiol and the carbonyl compound. Because of the greater stability of thioacetals, the equilibrium lies on the side of the acetal. In contradistinction to the O , O ‑acetal case, it is not needed to remove water from the reaction mixture in order to shift the equilibrium. [ 65 ]
S , O -Acetals are hydrolyzed a factor of 10,000 times faster than the corresponding S , S -acetals. Their formation follows analogously from the thioalcohol. Also their cleavage proceeds under similar conditions and predominantly through mercury(II) compounds in wet acetonitrile. [ 66 ]
For aldehydes, a temporary protection of the carbonyl group the presence of ketones as hemiaminal ions is shown below. Here it is applied, that aldehydes are very much more activated carbonyls than ketones and that many addition reactions are reversible. [ 67 ] [ 68 ]
The most important protecting groups for carboxylic acids are the esters of various alcohols. Occasionally, esters are protected as ortho-esters or oxazolines . [ 69 ]
Many groups can suffice for the alcoholic component, and the specific cleaving conditions are contrariwise generally quite similar: each ester can be hydrolyzed in a basic water-alcohol solution. Instead, most ester protecting groups vary in how mildly they can be formed from the original acid.
Alkenes rarely need protection or are protected. They are as a rule only involved in undesired side reactions with electrophilic attack, isomerization or catalytic hydration. For alkenes two protecting groups are basically known:
For alkynes there are in any case two types of protecting groups. For terminal alkynes it is sometimes important to mask the acidic hydrogen atom. This normally proceeds from deprotonation (via a strong base like methylmagnesium bromide or butyllithium in tetrahydrofuran/ dimethylsulfoxide ) and subsequently reaction with chlorotrimethylsilane to a terminally TMS-protected alkyne. [ 95 ] Cleavage follows hydrolytically – with potassium carbonate in methanol – or with fluoride ions like for example with tetrabutylammonium fluoride . [ 96 ]
In order to protect the triple bond itself, sometimes a transition metal-alkyne complex with dicobalt octacarbonyl is used. The release of the cobalt then follows from oxidation. [ 97 ] [ 98 ] [ 99 ] [ 100 ] [ 101 ]
The use of protective groups is pervasive but not without criticism. [ 103 ] In practical terms their use adds two steps (protection-deprotection sequence) to a synthesis, either or both of which can dramatically lower chemical yield . Crucially, added complexity impedes the use of synthetic total synthesis in drug discovery . In contrast biomimetic synthesis does not employ protective groups. As an alternative, Baran presented a novel protective-group free synthesis of the compound hapalindole U. The previously published synthesis [ 104 ] [ 105 ] [ 106 ] according to Baran, contained 20 steps with multiple protective group manipulations (two confirmed):
Although the use of protecting groups is not preferred in industrial syntheses, they are still used in industrial contexts, e.g. sucralose (sweetener) or the Roche synthesis of oseltamivir (Tamiflu, an antiviral drug)
An important example of industrial applications of protecting group theory is the synthesis of ascorbic acid (Vitamin C) à la Reichstein .
In order to prevent oxidation of the secondary alcohols with potassium permanganate , they are protected via acetalation with acetone and then deprotected after the oxidation of the primary alcohols to carboxylic acids. [ 107 ]
A very spectacular example application of protecting groups from natural product synthesis is the 1994 total synthesis of palytoxin acid by Yoshito Kishi 's research group. [ 108 ] Here 42 functional groups (39 hydroxyls, one diol, an amine group, and a carboxylic acid) required protection. These proceeded through 8 different protecting groups (a methyl ester, five acetals, 20 TBDMS esters, nine p ‑methoxybenzyl ethers, four benzoates, a methyl hemiacetal, an acetone acetal and an SEM ester). [ 109 ]
The introduction or modification of a protecting group occasionally influences the reactivity of the whole molecule. For example, diagrammed below is an excerpt of the synthesis of an analogue of Mitomycin C by Danishefsky . [ 110 ]
The exchange of a protecting group from a methyl ether to a MOM-ether inhibits here the opening of an epoxide to an aldehyde .
Protecting group chemistry finds itself an important application in the automated synthesis of peptides and nucleosides. The technique was introduced in the field of peptide synthesis by Robert Bruce Merrifield in 1977. [ 111 ] For peptide synthesis via automated machine, the orthogonality of the Fmoc group (basic cleavage), the tert ‑butyl group (acidic cleavage) and diverse protecting groups for functional groups on the amino acid side-chains are used. [ 112 ] Up to four different protecting groups per nucleobase are used for the automated synthesis of DNA and RNA sequences in the oligonucleotide synthesis . The procedure begins actually with redox chemistry at the protected phosphorus atom. A tricoordinate phosphorus, used on account of the high reactivity, is tagged with a cyanoethyl protecting group on a free oxygen. After the coupling step follows an oxidation to phosphate, whereby the protecting group stays attached. Free OH-groups, which did not react in the coupling step, are acetylated in an intermediate step. These additionally-introduced protecting groups then inhibit, that these OH-groups might couple in the next cycle. [ 113 ] | https://en.wikipedia.org/wiki/Protecting_group |
A protective colloid is a lyophilic colloid that when present in small quantities keeps lyophobic colloids from precipitating under the coagulating action of electrolytes .
When a small amount of hydrophilic colloid is added to hydrophobic colloids it may coagulate the latter. This is due to neutralisation of the charge on the hydrophobic colloidal particles. However, the addition of large amount of hydrophilic colloid increases the stability of the hydrophobic colloidal system. This is due to adsorption .
When lyophilic sols are added to lyophobic sols, depending on their sizes, either lyophobic sol is adsorbed in the surface of lyophilic sol or lyophilic sol is adsorbed on the surface of lyophobic sol. The layer of the protective colloid prevents direct collision between the hydrophobic colloidal particles and thus prevents coagulation. [ 1 ]
Lyophilic sols like starch and gelatin act as protective colloids. [ 2 ]
For a comparative study Zsigmondy introduced a scale of protective
action for different protective colloids in terms of gold number . The gold number is the weight in milligrams of a protective colloid which checks the coagulation of 10ml of a given gold sol on adding 1 ml of 10% sodium chloride.
Thus smaller the gold number, greater is the protective action. [ 3 ] Gold numbers of some materials
Gelatin 0.005-0.01
Albumin 0.1
Acacia 0.1-0.2
Sodium oleate 1-5
Tragacanth 2 [4] | https://en.wikipedia.org/wiki/Protective_colloid |
A protective distribution system ( PDS ), also called protected distribution system , is a US government term for wireline or fiber-optic telecommunication system that includes terminals and adequate acoustical , electrical , electromagnetic , and physical safeguards to permit its use for the unencrypted transmission of classified information . At one time these systems were called " approved circuits ".
A complete protected distribution system includes the subscriber and terminal equipment and the interconnecting lines.
The purpose of a PDS is to deter, detect and/or make difficult physical access to the communication lines carrying national security information.
A specification called the National Security Telecommunications and Information Systems Security Instruction (NSTISSI) 7003 was issued in December 1996 by the Committee on National Security Systems . [ 1 ] Approval authority, standards, and guidance for the design, installation, and maintenance for PDS are provided by NSTISSI 7003 to U.S. government departments and agencies and their contractors and vendors. This instruction describes the requirements for all PDS installations within the U.S. and for low and medium threat locations outside the U.S.
PDS is commonly used to protect SIPRNet and JWICS networks.
The document superseded one numbered NASCI 4009 on Protected Distribution Systems, dated December 30, 1981, and part of a document called NACSEM 5203, that covered guidelines for facility design, using the designations "red" and "black". [ 1 ]
There are two types of PDS: hardened distribution systems and simple distribution systems.
Hardened distribution PDSs provide significant physical protection and can be implemented in three forms: hardened carrier PDSs, alarmed carrier PDSs and continuously viewed carrier PDSs.
In a hardened carrier PDS, the data cables are installed in a carrier constructed of electrical metallic tubing ( EMT ), ferrous conduit or pipe, or rigid sheet steel ducting. All of the connections in a Hardened Carrier System are permanently sealed completely around all surfaces with welds, epoxy or other such sealants. If the hardened carrier is buried under ground, to secure cables running between buildings for example, the carrier containing the cables is encased in concrete.
With a hardened carrier system , detection is accomplished via human inspections that are required to be performed periodically. Therefore, hardened carriers are installed below ceilings or above flooring so they can be visually inspected to ensure that no intrusions have occurred. These periodic visual inspections (PVIs) occur at a frequency dependent upon the level of threat to the environment, the security classification of the data, and the access control to the area.
As an alternative to conducting human visual inspections, an alarmed carrier PDS may be constructed to automate the inspection process through electronic monitoring with an alarm system. In an Alarmed Carrier PDS, the carrier system is “alarmed” with specialized optical fibers deployed within the conduit for the purpose of sensing acoustic vibrations that usually occur when an intrusion is being attempted on the conduit in order to gain access to the cables.
Alarmed carrier PDS offers several advantages over hardened carrier PDS:
Legacy alarmed carrier systems monitor the carrier containing the cables being protected. More advanced systems monitor the fibers within, or intrinsic to, the cables being protected to turn those cables into sensors, which detect intrusion attempts.
Depending on the government organization, utilizing an alarmed carrier PDS in conjunction with interlocking armored cable may, in some cases, allow for the elimination of the carrier systems altogether. In these instances, the cables being protected can be installed in existing conveyance (wire basket, ladder rack) or suspended cabling (on D-rings, J-Hooks, etc.).
A Continuously Viewed Carrier PDS is one that is under continuous observation, 24 hours per day (including when operational). Such circuits may be grouped together, but should be separated from all non-continuously viewed circuits ensuring an open field of view. Standing orders should include the requirement to investigate any attempt to disturb the PDS. Appropriate security personnel should investigate the area of attempted penetration within 15 minutes of discovery. This type of hardened carrier is not used for Top Secret or special category information for non-U.S. UAA. [ clarification needed ] UAA is an Uncontrolled Access Area (UAA). Like definitions include Controlled Access Area (CAA) and Restricted Access Area (RAA). A Secure Room (SR) offers the highest degree of protection.
Therefore, from the least protected (least secure) to the most protected is as follows:
UAA
RAA
CAA
SR
Simple distribution PDSs are afforded a reduced level of physical security protection as compared to a hardened distribution PDS. They use a simple carrier system and the following means are acceptable under NSTISSI 7003: | https://en.wikipedia.org/wiki/Protective_distribution_system |
Within the field of molecular biology , a protein-fragment complementation assay , or PCA, is a method for the identification and quantification of protein–protein interactions . In the PCA, the proteins of interest ("bait" and "prey") are each covalently linked to fragments of a third protein (e.g. DHFR, which acts as a "reporter"). Interaction between the bait and the prey proteins brings the fragments of the reporter protein in close proximity to allow them to form a functional reporter protein whose activity can be measured. This principle can be applied to many different reporter proteins and is also the basis for the yeast two-hybrid system , an archetypical PCA assay.
Any protein that can be split into two parts and reconstituted non-covalently to form a functional protein may be used in a PCA. The two fragments however have low affinity for each other and must be brought together by other interacting proteins fused to them (often called "bait" and "prey" since the bait protein can be used to identify a prey protein, see figure ). The protein that produces a detectable readout is called "reporter". Usually enzymes which confer resistance to nutrient deprivation or antibiotics, such as dihydrofolate reductase or beta-lactamase respectively, or proteins that give colorimetric or fluorescent signals are used as reporters. When fluorescent proteins are reconstituted the PCA is called Bimolecular fluorescence complementation assay . The following proteins have been used in split protein PCAs:
The methods mentioned above have been applied to whole genomes , e.g. yeast [ 3 ] or syphilis bacteria. [ 20 ] | https://en.wikipedia.org/wiki/Protein-fragment_complementation_assay |
The Protein Data Bank ( PDB ) [ 1 ] is a database for the three-dimensional structural data of large biological molecules such as proteins and nucleic acids , which is overseen by the Worldwide Protein Data Bank (wwPDB). This structural data is obtained and deposited by biologists and biochemists worldwide through the use of experimental methodologies such as X-ray crystallography , NMR spectroscopy , and, increasingly, cryo-electron microscopy . All submitted data are reviewed by expert biocurators and, once approved, are made freely available on the Internet under the CC0 Public Domain Dedication. [ 2 ] Global access to the data is provided by the websites of the wwPDB member organizations (PDBe, [ 3 ] PDBj, [ 4 ] RCSB PDB, [ 5 ] and BMRB [ 6 ] ).
The PDB is a key in areas of structural biology , such as structural genomics . Most major scientific journals and some funding agencies now require scientists to submit their structure data to the PDB. Many other databases use protein structures deposited in the PDB. For example, SCOP and CATH classify protein structures, while PDBsum provides a graphic overview of PDB entries using information from other sources, such as Gene Ontology . [ 7 ] [ 8 ]
Two forces converged to initiate the PDB: a small but growing collection of sets of protein structure data determined by X-ray diffraction; and the newly available (1968) molecular graphics display, the Brookhaven RAster Display (BRAD), to visualize these protein structures in 3-D. In 1969, with the sponsorship of Walter Hamilton at the Brookhaven National Laboratory , Edgar Meyer ( Texas A&M University ) began to write software to store atomic coordinate files in a common format to make them available for geometric and graphical evaluation. By 1971, one of Meyer's programs, SEARCH, enabled researchers to remotely access information from the database to study protein structures offline. [ 9 ] SEARCH was instrumental in enabling networking, thus marking the functional beginning of the PDB.
The Protein Data Bank was announced in October 1971 in Nature New Biology [ 10 ] as a joint venture between Cambridge Crystallographic Data Centre , UK and Brookhaven National Laboratory, US.
Upon Hamilton's death in 1973, Tom Koetzle took over direction of the PDB for the subsequent 20 years. In January 1994, Joel Sussman of Israel's Weizmann Institute of Science was appointed head of the PDB. In October 1998, [ 11 ] the PDB was transferred to the Research Collaboratory for Structural Bioinformatics (RCSB); [ 12 ] the transfer was completed in June 1999. The new director was Helen M. Berman of Rutgers University (one of the managing institutions of the RCSB, the other being the San Diego Supercomputer Center at UC San Diego ). [ 13 ] In 2003, with the formation of the wwPDB, the PDB became an international organization. The founding members are PDBe (Europe), [ 3 ] RCSB (US), and PDBj (Japan). [ 4 ] The BMRB [ 6 ] joined in 2006. Each of the four members of wwPDB can act as deposition, data processing and distribution centers for PDB data. The data processing refers to the fact that wwPDB staff review and annotate each submitted entry. [ 14 ] The data are then automatically checked for plausibility (the source code [ 15 ] for this validation software has been made available to the public at no charge).
The PDB database is updated weekly ( UTC +0 Wednesday), along with its holdings list. [ 16 ] As of 10 January 2023 [update] , the PDB comprised:
Most structures are determined by X-ray diffraction, but about 7% of structures are determined by protein NMR . When using X-ray diffraction, approximations of the coordinates of the atoms of the protein are obtained, whereas using NMR, the distance between pairs of atoms of the protein is estimated. The final conformation of the protein is obtained from NMR by solving a distance geometry problem. After 2013, a growing number of proteins are determined by cryo-electron microscopy .
For PDB structures determined by X-ray diffraction that have a structure factor file, their electron density map may be viewed. The data of such structures may be viewed on the three PDB websites.
Historically, the number of structures in the PDB has grown at an approximately exponential rate, with 100 registered structures in 1982, 1,000 structures in 1993, 10,000 in 1999, 100,000 in 2014, and 200,000 in January 2023. [ 18 ] [ 19 ]
The file format initially used by the PDB was called the PDB file format. The original format was restricted by the width of computer punch cards to 80 characters per line. Around 1996, the "macromolecular Crystallographic Information file" format, mmCIF, which is an extension of the CIF format was phased in. mmCIF became the standard format for the PDB archive in 2014. [ 20 ] In 2019, the wwPDB announced that depositions for crystallographic methods would only be accepted in mmCIF format. [ 21 ]
An XML version of PDB, called PDBML, was described in 2005. [ 22 ] The structure files can be downloaded in any of these three formats, though an increasing number of structures do not fit the legacy PDB format. Individual files are easily downloaded into graphics packages from Internet URLs :
The " 4hhb " is the PDB identifier. Each structure published in PDB receives a four-character alphanumeric identifier, its PDB ID. (This is not a unique identifier for biomolecules, because several structures for the same molecule—in different environments or conformations—may be contained in PDB with different PDB IDs.)
The structure files may be viewed using one of several free and open source computer programs , including Jmol , Pymol , VMD , Molstar and Rasmol . Other non-free, shareware programs include ICM-Browser, [ 23 ] MDL Chime , UCSF Chimera , Swiss-PDB Viewer, [ 24 ] StarBiochem [ 25 ] (a Java-based interactive molecular viewer with integrated search of protein databank), Sirius , and VisProt3DS [ 26 ] (a tool for Protein Visualization in 3D stereoscopic view in anaglyph and other modes), and Discovery Studio . The RCSB PDB website contains an extensive list of both free and commercial molecule visualization programs and web browser plugins. | https://en.wikipedia.org/wiki/Protein_Data_Bank |
The Protein Data Bank (PDB) file format is a textual file format describing the three-dimensional structures of molecules held in the Protein Data Bank , now succeeded by the mmCIF format. The PDB format accordingly provides for description and annotation of protein and nucleic acid structures including atomic coordinates, secondary structure assignments, as well as atomic connectivity. In addition experimental metadata are stored. The PDB format is the legacy file format for the Protein Data Bank which has kept data on biological macromolecules in the newer PDBx/mmCIF file format since 2014. [ 1 ]
The PDB file format was invented in 1972 [ 2 ] [ 3 ] as a human-readable file that would allow researchers to exchange the atomic coordinates in a given protein through a database system. Its fixed-column width format is limited to 80 or 140 [ 4 ] columns, which was based on the width of the computer punch cards that were previously used to exchange the coordinates. [ 5 ] Through the years the file format has undergone many changes and revisions. The final update to the PDB file format was in November 2012 with version 3.30. [ 6 ]
A typical PDB file describing a protein consists of hundreds to thousands of lines like the following (taken from a file describing the structure of a synthetic collagen-like peptide ): | https://en.wikipedia.org/wiki/Protein_Data_Bank_(file_format) |
Protein L was first isolated from the surface of bacterial species Peptostreptococcus magnus and was found to bind immunoglobulins through L chain interaction, from which the name was suggested. [ 2 ] It consists of 719 amino acid residues. [ 3 ] The molecular weight of protein L purified from the cell walls of Peptostreptoccus magnus was first estimated as 95kD by SDS-PAGE in the presence of reducing agent 2-mercaptoethanol, while the molecular weight was determined to 76kD by gel chromatography in the presence of 6 M guanidine HCl. Protein L does not contain any interchain disulfide loops, nor does it consist of disulfide-linked subunits. It is an acidic molecule with a pI of 4.0. [ 4 ] Unlike protein A and protein G , which bind to the Fc region of immunoglobulins ( antibodies ), protein L binds antibodies through light chain interactions. Since no part of the heavy chain is involved in the binding interaction, Protein L binds a wider range of antibody classes than protein A or G. Protein L binds to representatives of all antibody classes, including IgG , IgM , IgA , IgE and IgD . Single chain variable fragments ( scFv ) and Fab fragments also bind to protein L.
Despite this wide binding range, protein L is not a universal antibody-binding protein . Protein L binding is restricted to those antibodies that contain kappa light chains. In humans and mice, most antibody molecules contain kappa (κ) light chains and the remainder have lambda (λ) light chains. Protein L is only effective in binding certain subtypes of kappa light chains. For example, it binds human VκI, VκIII and VκIV subtypes but does not bind the VκII subtype. Binding of mouse immunoglobulins is restricted to those having VκI light chains. [ 5 ]
Given these specific requirements for effective binding, the main application for immobilized protein L is purification of monoclonal antibodies from ascites or cell culture supernatant that are known to have the kappa light chain . Protein L is extremely useful for purification of VLκ-containing monoclonal antibodies from culture supernatant because it does not bind bovine immunoglobulins, which are often present in the media as a serum supplement. Also, protein L does not interfere with the antigen-binding site of the antibody, making it useful for immunoprecipitation assays, even using IgM.
The gene for protein L contains five components: a signal sequence of 18 amino acids; a NH2-terminal region ("A") of 79 residues; five homologous "B" repeats of 72-76 amino acids each; a COOH terminus region of two additional "C" repeats (52 amino acids each); a hydrophilic, proline-rich putative cell wall-spanning region ("W") after the C repeats; a hydrophobic membrane anchor ("M"). The B repeats (36kD) were found to be responsible for the interaction with Ig light chains. [2]
In addition to protein L, other immunoglobulin-binding bacterial proteins such as protein A , protein G and protein A/G are all commonly used to purify, immobilize or detect immunoglobulins. Each of these immunoglobulin-binding proteins has a different antibody binding profile in terms of the portion of the antibody that is recognized and the species and type of antibodies it will bind. | https://en.wikipedia.org/wiki/Protein_L |
Protein Local Optimization Program ( PLOP ) is computer software , [ 3 ] a molecular dynamics simulation package written in the programming language Fortran . It was developed originally by Matthew P. Jacobson and Richard A. Friesner of the Friesner lab at Columbia University , and then moved to the Jacobson lab at University of California, San Francisco (UCSF), and Schrödinger , LLC.
This computational chemistry -related article is a stub . You can help Wikipedia by expanding it . | https://en.wikipedia.org/wiki/Protein_Local_Optimization_Program |
The Protein Society is an international, not-for-profit , scholarly society with the mission to provide forums for the advancement of research into protein structure , function, design and applications.
The Protein Society was founded in 1986, with the leadership of Ralph Bradshaw, Finn Wold, David Eisenberg , Ken Walsh, Hans Neurath , and other protein researchers from diverse fields. [ 1 ]
Ralph Bradshaw was the society's first president, [ 2 ] followed by David Eisenberg , Finn Wold, Mark Hermodson, Joseph Villafranca, Brian Matthews , Robert Sauer , Christopher Dobson , Wiliam DeGrado , C. Robert Matthews , Jeffery Kelly , Arthur Palmer , Daniel Raleigh, Lynne Regan , James U. Bowie , Carol Post , Charles L. Brooks III , Amy E. Keating , Chuck R. Sanders , Elizabeth Meiering
In 1987, the Society began publishing the trans-disciplinary academic journal Protein Science , [ 2 ] with Hans Neurath serving as editor-in-chief. The journal covers research on the structure, function, and biochemical significance of proteins, their role in molecular and cell biology, genetics, and evolution, and their regulation and mechanisms of action. As of 2024, the editor-in-chief is John Kuriyan . [ 3 ]
The society organizes an annual symposium which hosts hundreds of participants from all over the world, features research presentations by leaders from the diverse fields involved in protein science, a graduate student poster competition, networking opportunities , free undergraduate registration, educational workshops, and the annual presentation of the Protein Society's Awards.
The Protein Society presents eight awards each year: | https://en.wikipedia.org/wiki/Protein_Society |
The Protein Structure Initiative (PSI) was a USA based project that aimed at accelerating discovery in structural genomics and contribute to understanding biological function. [ 1 ] Funded by the U.S. National Institute of General Medical Sciences (NIGMS) between 2000 and 2015, its aim was to reduce the cost and time required to determine three-dimensional protein structures and to develop techniques for solving challenging problems in structural biology, including membrane proteins. Over a dozen research centers have been supported by the PSI for work in building and maintaining high-throughput structural genomics pipelines, developing computational protein structure prediction methods, organizing and disseminating information generated by the PSI, and applying high-throughput structure determination to study a broad range of important biological and biomedical problems.
The project has been organized into three separate phases. The first phase of the Protein Structure Initiative (PSI-1) spanned from 2000 to 2005, and was dedicated to demonstrating the feasibility of high-throughput structure determination, solving unique protein structures, and preparing for a subsequent production phase. [ 2 ] The second phase, PSI-2, focused on implementing the high-throughput structure determination methods developed in PSI-1, as well as homology modeling and addressing bottlenecks like modeling membrane proteins . [ 3 ] The third phase, PSI:Biology, began in 2010 and consisted of networks of investigators applying high-throughput structure determination to study a broad range of biological and biomedical problems. [ 4 ] PSI program ended on 7/1/2015, [ 5 ] even that some of the PSI centers continue structure determination supported by other funding mechanisms.
The first phase of the Protein Structure Initiative (PSI-1) lasted from June 2000 until September 2005, and had a budget of $270 million funded primarily by NIGMS with support from the National Institute of Allergy and Infectious Diseases . [ 2 ] PSI-1 saw the establishment of nine pilot centers focusing on structural genomics studies of a range of organisms, including Arabidopsis thaliana , Caenorhabditis elegans and Mycobacterium tuberculosis . [ 2 ] During this five-year period over 1,100 protein structures were determined, over 700 of which were classified as "unique" due to their < 30% sequence similarity with other known protein structures. [ 2 ]
The primary goal of PSI-1, to develop methods to streamline the structure determination process, resulted in an array of technical advances. Several methods developed during PSI-1 enhanced expression of recombinant proteins in systems like Escherichia coli , Pichia pastoris and insect cell lines. New streamlined approaches to cell cloning , expression and protein purification were also introduced, in which robotics and software platforms were integrated into the protein production pipeline to minimize required manpower, increase speed, and lower costs. [ 6 ]
The second phase of the Protein Structure Initiative (PSI-2) lasted from July 2005 to June 2010. Its goal was to use methods introduced in PSI-1 to determine a large number of proteins and continue development in streamlining the structural genomics pipeline. PSI-2 had a five-year budget of $325 million provided by NIGMS with support from the National Center for Research Resources . By the end of this phase, the Protein Structure Initiative had solved over 4,800 protein structures; over 4,100 of these were unique. [ 7 ]
The number of sponsored research centers grew to 14 during PSI-2. Four centers were selected as Large Scale centers, with a mandate to place 15% effort on targets nominated by the broader research community, 15% on targets of biomedical relevance, and 70% on broad structural coverage; these centers were the Joint Center for Structural Genomics (JCSG), the Midwest Center for Structural Genomics (MCSG), the Northeast Structural Genomics Consortium (NESG), and the New York SGX Research Center for Structural Genomics (NYSGXRC). The new centers participating in PSI-2 included four specialized centers: Accelerated Technologies Center for Gene to 3D Structure (ATCG3D), the Center for Eukaryotic Structural Genomics (CESG), the Center for High-Throughput Structural Biology (CHTSB), a branch of the Structural Genomics of Pathogenic Protozoa Consortium taking that institution's place), the Center for Structures of Membrane Proteins (CSMP), and the New York Consortium on Membrane Protein Structure (NYCOMPS). Two homology modeling centers, the Joint Center for Molecular Modeling (JCMM) and New Methods for High-Resolution Comparative Modeling (NMHRCM) were also added, as well as two resource centers, the PSI Materials Repository (PSI-MR) and the PSI Structural Biology Knowledgebase (SBKB). [ 8 ] The TB Structural Genomics Consortium was removed from the roster of supported research centers in the transition from PSI-1 to PSI-2. [ 2 ]
Originally launched in February 2008, the SBKB is a free resource that provides information on protein sequence and keyword searching, as well as modules describing target selection, experimental protocols, structure models, functional annotation, metrics on overall progress, and updates on structure determination technology. Like the PDB , it is directed by Dr. Helen M. Berman and hosted at Rutgers University .
The PSI Materials Repository, established in 2006 at the Harvard Institute of Proteomics, stores and ships PSI-generated plasmid clones . [ 9 ] Clones are sequence-verified, annotated and stored in the DNASU Plasmid Repository , [ 10 ] currently located at the Biodesign Institute at Arizona State University. As of September 2011, there are over 50,000 PSI-generated plasmid clones and empty vectors available for request through DNASU in addition to over 147,000 clones generated from non-PSI sources. Plasmids are distributed to researchers worldwide. Now called the PSI:Biology Materials Repository, this resource has a five-year budget of $5.4 million and is under the direction of Dr. Joshua LaBaer, [ 11 ] who moved to Arizona State University in the middle of 2009, taking the PSI:Biology-MR with him.
The third phase of the PSI was called PSI:Biology and was intended to reflect the emphasis on the biological relevance of the work. [ 4 ] During this phase, highly organized networks of investigators were applying the new paradigm of high-throughput structure determination, which was successfully developed during the earlier phases of the PSI, to study a broad range of important biological and biomedical problems. The network included centers for high-throughput structure determination, centers for membrane protein structure determination, consortia for high-throughput-enabled structural biology partnerships, the SBKB and the PSI-MR. In September 2013 NIH announced that PSI would not be renewed after its third phase would end in 2015.
As of January 2006, about two thirds of worldwide structural genomics (SG) output was made by PSI centers. [ 12 ] Of these PSI contributions over 20% represented new Pfam families, compared to the non-SG average of 5%. [ 12 ] Pfam families represent structurally distinct groups of proteins as predicted from sequenced genomes. Not targeting homologs of known structure was accomplished by using sequence comparison tools like BLAST and PSI-BLAST . [ 12 ] Like the difference in novelty as determined by discovery of new Pfam families, the PSI also discovered more SCOP folds and superfamilies than non-SG efforts. In 2006, 16% of structures solved by the PSI represented new SCOP folds and superfamilies, while the non-SG average was 4%. [ 12 ] Solving such novel structures reflects increased coverage of protein fold space, one of the PSI's main goals. [ 1 ] Determining the structure a novel protein allows homology modeling to more accurately predict the fold of other proteins in the same structural family.
While most of the structures solved by the four large-scale PSI centers lack functional annotation, many of the remaining PSI centers determine structures for proteins with known biological function. The TB Structural Genomics Consortium, for example, focused exclusively on functionally characterized proteins. During its term in PSI-1, it deposited structures for over 70 unique proteins from Mycobacterium tuberculosis , which represented more than 35% of total unique M. tuberculosis structures solved through 2007. [ 13 ] In following with its biomedical theme to increase coverage of phosphotomes, the NYSGXRC has determined structures for about 10% of all human phosphatases . [ 14 ]
The PSI consortia have provided the overwhelming majority of targets for the Critical Assessment of Techniques for Protein Structure Prediction (CASP), a community-wide, biannual experiment to determine the state and progress of protein structure prediction . [ 15 ] [ 16 ] [ 17 ]
A major goal during the PSI:Biology phase is to utilize the high-throughput methods developed during the initiative's first decade to generate protein structures for functional studies, broadening the PSI's biomedical impact. It is also expected to advance knowledge and understanding of membrane proteins. [ citation needed ]
The PSI has received notable criticism from the structural biology community. Among these charges is that the main product of the PSI – PDB files of proteins' atomic coordinates as determined by X-ray crystallography or NMR spectroscopy – are not useful enough to biologists to justify the project's $764 million cost. [ 18 ] [ 19 ] Critics note that money currently spent on the PSI could have otherwise funded what they consider worthier causes:
The $60 million a year in public money that is being spent – I would say, wasted – on the PSI is enough to fund approximately 100–200 individual investigator-initiated research grants. These hypothesis-driven proposals are the lifeblood of the scientific enterprise, and as I have discussed recently in other columns, they are being sucked dry by, among other things, an increasing trend to fund large initiatives at their expense. That $60 million a year would raise the payline at a typical NIH institute by about 6 percentile points, enough to make a huge difference to peer review and to the continuance of a lot of important science. [ 19 ]
A short response to this was published: [ 20 ]
In conclusion, it should be kept in mind that scientific research, and the cutting- edge technologies that both drive and are driven by it, are constantly and rapidly evolving. Some of Petsko’s criticisms are constructive, and should be noted by policy-makers. But one should not throw the baby out with the bathwater, rather tune the scope and objectives of the PSI to the needs of the life-science community as a whole, much in the spirit of SPINE, the SGC and other European structural genomics/ proteomics projects. [ 21 ] If such a constructive approach is adopted, we feel confident that the structural data provided by the PSI and its cousins will serve as no less valuable a resource than genome sequences.
In October 2008 the NIGMS hosted a meeting concerning the future of structural genomics efforts and invited speakers from the PSI Advisory Committee, members of the NIGMS Advisory Council, and interested scientists who had no previous involvement with the PSI. Representatives of other genomics, proteomics, and structural genomics initiatives, as well as scientists from academia, government, and industry were also included. Based on this meeting and the subsequent recommendations from the PSI Advisory Committee, [ 22 ] [ 23 ] a concept-clearance document was released in January 2009 describing what a third phase of the PSI might entail. Most notable was a large emphasis on partnerships and collaborations to ensure that the majority of PSI research is focused on proteins of interest to the broader research community as well as efforts to make PSI products more accessible to the research community. [ 24 ]
Grant applications for PSI:Biology were submitted by October 29, 2009. See Phase 3 section above. | https://en.wikipedia.org/wiki/Protein_Structure_Initiative |
Adsorption (not to be mistaken for absorption ) is the accumulation and adhesion of molecules, atoms, ions, or larger particles to a surface, but without surface penetration occurring. The adsorption of larger biomolecules such as proteins is of high physiological relevance , and as such they adsorb with different mechanisms than their molecular or atomic analogs. Some of the major driving forces behind protein adsorption include: surface energy, intermolecular forces, hydrophobicity, and ionic or electrostatic interaction. By knowing how these factors affect protein adsorption, they can then be manipulated by machining, alloying, and other engineering techniques to select for the most optimal performance in biomedical or physiological applications.
Many medical devices and products come into contact with the internal surfaces of the body, such as surgical tools and implants. When a non-native material enters the body, the first step of the immune response takes place and host extracellular matrix and plasma proteins aggregate to the material in attempts to contain, neutralize, or wall-off the injurious agent. [ 1 ] These proteins can facilitate the attachment of various cell types such as osteoblasts and fibroblasts that can encourage tissue repair. [ 2 ] Taking this a step further, implantable devices can be coated with a bioactive material to encourage adsorption of specific proteins, fibrous capsule formation, and wound healing. This would reduce the risk of implant rejection and accelerate recovery by selecting for the necessary proteins and cells necessary for endothelialization. After the formation of the endothelium , the body will no longer be exposed to the foreign material, and will stop the immune response.
Proteins such as collagen or fibrin often serve as scaffolds for cell adhesion and cell growth. This is an integral part to the structural integrity of cell sheets and their differentiation into more complex tissue and organ structures. The adhesion properties of proteins to non-biological surfaces greatly influences whether or not cells can indirectly attach to them via scaffolds. An implant like a hip-stem replacement necessitates integration with the host tissues, and protein adsorption facilitates this integration.
Surgical tools can be designed to be sterilized more easily so that proteins do not remain adsorbed to a surface, risking cross-contamination. Some diseases such as Creutzfeldt–Jakob disease and kuru (both related to mad cow disease ) are caused by the transmission of prions , which are errant or improperly folded forms of a normally native protein. Surgical tools contaminated with prions require a special method of sterilization to completely eradicate all trace elements of the misfolded protein, as they are resistant to many of the normally used cleansing methods.
However, in some cases, protein adsorption to biomaterials can be an extremely unfavorable event. The adhesion of clotting factors may induce thrombosis , which may lead to stroke or other blockages. [ 3 ] Some devices are intended to interact with the internal body environment such as sensors or drug-delivery vehicles, and protein adsorption would hinder their effectiveness.
Proteins are biomolecules that are composed of amino acid subunits. Each amino acid has a side chain that gains or loses charge depending on the pH of the surrounding environment, as well as its own individual polar/nonpolar qualities. [ 4 ]
Charged regions can greatly contribute to how that protein interacts with other molecules and surfaces, as well as its own tertiary structure (protein folding). As a result of their hydrophilicity, charged amino acids tend to be located on the outside of proteins, where they are able to interact with surfaces. [ 5 ] It is the unique combination of amino acids that gives a protein its properties. In terms of surface chemistry , protein adsorption is a critical phenomenon that describes the aggregation of these molecules on the exterior of a material. The tendency for proteins to remain attached to a surface depends largely on the material properties such as surface energy, texture, and relative charge distribution. Larger proteins are more likely to adsorb and remain attached to a surface due to the higher number of contact sites between amino acids and the surface (Figure 1).
The fundamental idea behind spontaneous protein adsorption is that adsorption occurs when more energy is released than gained according to Gibbs law of free energy.
This is seen in the equation:
where:
In order for the protein adsorption to occur spontaneously, ∆ ads G must be a negative number.
Proteins and other molecules are constantly in competition with one another over binding sites on a surface. The Vroman Effect , developed by Leo Vroman, postulates that small and abundant molecules will be the first to coat a surface. However, over time, molecules with higher affinity for that particular surface will replace them. This is often seen in materials that contact the blood where fibrinogen will bind to the surface first and over time will be replaced by kininogen . [ 6 ]
In order for proteins to adsorb, they must first come into contact with the surface through one or more of these major transport mechanisms: diffusion , thermal convection , bulk flow , or a combination thereof. When considering the transport of proteins, it is clear how concentration gradients, temperature, protein size and flow velocity will influence the arrival of proteins to a solid surface. Under conditions of low flow and minimal temperature gradients, the adsorption rate can be modeled after the diffusion rate equation. [ 5 ]
where:
A higher bulk concentration and/or higher diffusion coefficient (inversely proportional to molecular size) results in a larger number of molecules arriving at the surface. The consequential protein surface interactions result in high local concentrations of adsorbed protein, reaching concentrations of up to 1000 times higher than in the bulk solution. [ 5 ] However, the body is much more complex, containing flow and convective diffusion, and these must be considered in the rate of protein adsorption.
and
where:
This equation [ 5 ] is especially applicable to analyzing protein adsorption to biomedical devices in arteries, e.g. stents .
The four fundamental classes of forces and interaction in protein adsorption are: 1) ionic or electrostatic interaction, 2) hydrogen bonding , 3) hydrophobic interaction (largely entropically driven), and 4) interactions of charge-transfer or particle electron donor/acceptor type. [ 7 ]
The charge of proteins is determined by the pKa of its amino acid side chains, and the terminal amino acid and carboxylic acid. Proteins with isoelectric point (pI) above physiological conditions have a positive charge and proteins with pI below physiological conditions have a negative charge. The net charge of the protein, determined by the sum charge of its constituents, results in electrophoretic migration in a physiologic electric field. These effects are short-range because of the high di-electric constant of water, however, once the protein is close to a charged surface, electrostatic coupling becomes the dominant force. [ 8 ]
Water has as much propensity to form hydrogen bonds as any group in a polypeptide . During a folding and association process, peptide and amino acid groups exchange hydrogen bonds with water. Thus, hydrogen bonding does not have a strong stabilizing effect on protein adsorption in an aqueous medium. [ 9 ]
Hydrophobic interactions are essentially entropic interactions basically due to order/disorder phenomena in an aqueous medium. The free energy associated with minimizing interfacial areas is responsible for minimizing the surface area of water droplets and air bubbles in water. This same principle is the reason that hydrophobic amino acid side chains are oriented away from water, minimizing their interaction with water. The hydrophilic groups on the outside of the molecule result in protein water solubility. Characterizing this phenomenon can be done by treating these hydrophobic relationships with interfacial free energy concepts. Accordingly, one can think of the driving force of these interactions as the minimization of total interfacial free energy, i.e. minimization of surface area. [ 10 ]
Charge-transfer interactions are also important in protein stabilization and surface interaction. In general donor-acceptor processes, one can think of excess electron density being present which can be donated to an electrophilic species. In aqueous media, these solute interactions are primarily due to pi orbital electron effects. [ 11 ]
Temperature has an effect on both, the equilibrium state and kinetics of protein adsorption. The amount of protein adsorbed at high temperature is usually higher than that at room temperature. Temperature variation causes conformational changes in protein influencing adsorption. These conformational rearrangements in proteins results in an entropy gain which acts as a major driving force for protein adsorption. The temperature effect on protein adsorption can be seen in food manufacturing processes, especially liquid foods such as, milk which causes severe fouling on the wall surfaces of equipment where thermal treatment is carried out. [ 12 ] [ 13 ]
Ionic strength determines the Debye length that correlates with the damping distance of the electric potential of a fixed charge in an electrolyte. So, higher the ionic strength the shorter are electrostatic interactions between charged entities. As a result, the adsorption of charged proteins to oppositely charged substrates is hindered whereas the adsorption to like charged substrates is enhanced, thereby influencing adsorption kinetics. Also, high ionic strength increases the tendency of proteins to aggregate. [ 12 ]
When a surface is exposed to a multi-protein solution, adsorption of certain protein molecules are favored over the others. Protein molecules approaching the surface compete for binding sites. In multi-protein system attraction between molecules can occur, whereas in single-protein solutions intermolecular repulsive interactions dominate. In addition, there is a time-dependent protein spreading, where protein molecules initially make contact with minimal binding sites on the surface. With the increase in protein's residence time on the surface, the protein may unfold for interaction with additional binding sites. This results in a time-dependent increase in the contact points between protein and surface. This further makes desorption less likely. [ 5 ]
This technique measures a concentration change of proteins in bulk solution before and after adsorption , Δc p . Any protein concentration change is attributed to the adsorbed layer, Γ p .
Γ p = Δc p V/A tot
where:
This method also requires a high surface area material such as particulate and beaded adsorbents. [ 14 ]
Ellipsometry has been used widely for measuring protein adsorption kinetics as well as the structure of the adsorbed protein layer. It is an optical technique that measures the change of the polarization of light after reflection from a surface. This technique requires planar, reflecting surfaces, preferably quartz, silicon or silica, and a strong change in refractive index upon protein adsorption. [ 12 ]
Atomic-force microscopy (AFM) is a powerful microscopy technique used for studying samples at a nanoscale and is often used to image protein distribution on a surface. It consists of a cantilever with a tip to scan over the surface. It is a valuable tool for measuring protein-protein and protein-surface interaction. However, the limiting factor of many AFM studies is that imaging is often performed after drying the surface which might affect protein folding and the structure of the protein layer. Moreover, the cantilever tip can dislodge a protein or corrugate the protein layer. [ 12 ] [ 15 ]
Surface plasmon resonance (SPR) has been widely used for measuring protein adsorption with high sensitivity. This technique is based on the excitation of surface plasmons, longitudinal electromagnetic waves originated at the interface between metals and dielectrics. The deposition on the conducting surface of molecules and thin layers within 200 nm modifies the dielectric properties of the system and thus the SPR response, signaling the presence of molecules on a metal surface. [ 16 ]
Quartz crystal microbalance (QCM) is an acoustic sensor built around a disk shaped quartz crystal. It makes use of the converse piezoelectric effect. QCM, and extended versions such as QCM-D , has been widely used for protein adsorption studies, especially, real time monitoring of label-free protein adsorption. In addition to the adsorption studies, QCM-D also provides information regarding elastic moduli, viscosity , and conformational changes [ 17 ]
Optical waveguide lightmode spectroscopy (OWLS) is a device that relies on a thin-film optical waveguide, enclosing a discrete number of guided electromagnetic waves. Guidance is achieved by means of a grating coupler. It is based on the measurements of effective refractive index of a thin-film layer above the waveguide. This technique works only on highly transparent surfaces. [ 17 ]
Other methods widely used for measuring the amount of protein adsorbed on surfaces include radio-labelling, Lowry assay , scanning angle reflectometry, total internal reflection fluorescence , bicinchoninic acid assay etc.
Metallic bonding refers to the specific bonding between positive metal ions and surrounding valence electron clouds. [ 18 ] This intermolecular force is relatively strong, and gives rise to the repeated crystalline orientation of atoms, also referred to as its lattice system . There are several types of common lattice formations, and each has its own unique packing density and atomic closeness. The negatively charged electron clouds of the metal ions will sterically hinder the adhesion of negatively charged protein regions due to charge repulsion , thus limiting the available binding sites of a protein to a metal surface.
The lattice formation can lead to connection with exposed potential metal-ion-dependent adhesion sites (MIDAS) which are binding sites for collagen and other proteins. [ 19 ] The surface of the metal has different properties than the bulk since the normal crystalline repeating subunits is terminated at the surface. This leaves the surface atoms without a neighboring atom on one side, which inherently alters the electron distribution. This phenomenon also explains why the surface atoms have a higher energy than the bulk, often simply referred to as surface energy . This state of higher energy is unfavorable, and the surface atoms will try to reduce it by binding to available reactive molecules. [ 20 ]
This is often accomplished by protein adsorption, where the surface atoms are reduced to a more advantageous energy state.
The internal environment of the body is often modeled to be an aqueous environment at 37 °C at pH 7.3 with plenty of dissolved oxygen, electrolytes, proteins, and cells. [ 5 ] When exposed to oxygen for an extended period of time, many metals may become oxidized and increase their surface oxidation state by losing electrons. [ 21 ] This new cationic state leaves the surface with a net positive charge, and a higher affinity for negatively charged protein side groups. Within the vast diversity of metals and metal alloys, many are susceptible to corrosion when implanted in the body. Elements that are more electronegative are corroded faster when exposed to an electrolyte-rich aqueous environment such as the human body. [ 22 ] Both oxidation and corrosion will lower the free energy, thus affecting protein adsorption as seen in Eq. 1. [ 23 ]
Surface roughness and texture has an undeniable influence on protein adsorption on all materials, but with the ubiquity of metal machining processes, it is useful to address how these impact protein behavior. The initial adsorption is important, as well as maintained adhesion and integrity. Research has shown that surface roughness can encourage the adhesion of scaffold proteins and osteoblasts, and results in an increase in surface mineralization. [ 24 ] Surfaces with more topographical features and roughness will have more exposed surface area for proteins to interact with. [ 5 ] In terms of biomedical engineering applications, micromachining techniques are often used to increase protein adhesion to implants in the hopes of shortening recovery time. The technique of laserpatterning introduces grooves and surface roughness that will influence adhesion, migration and alignment. Grit-blasting, a method analogous to sand blasting, and chemical etching have proven to be successful surface roughening techniques that promote the long-term stability of titanium implants. [ 25 ] The increase in stability is a direct result of the observed increase in extracellular matrix and collagen attachment, which results in increased osteoblast attachment and mineralization when compared to non-roughened surfaces. [ 26 ] Adsorption is not always desirable, however. Machinery can be negatively affected by adsorption, particularly with Protein adsorption in the food industry .
Source: [ 27 ]
Polymers are of great importance when considering protein adsorption in the biomedical arena. Polymers are composed of one or more types of "mers" bound together repeatedly, typically by directional covalent bonds. As the chain grows by the addition of mers, the chemical and physical properties of the material are dictated by the molecular structure of the monomer. By carefully selecting the type or types of mers in a polymer and its manufacturing process, the chemical and physical properties of a polymer can be highly tailored to adsorb specific proteins and cells for a particular application.
Protein adsorption often results in significant conformational changes, which refers to changes in the secondary , tertiary , and quartary structures of proteins. In addition to adsorption rates and amounts, orientation and conformation are of critical importance. These conformational changes can affect protein interaction with ligands , substrates , and antigens which are dependent on the orientation of the binding site of interest. These conformational changes, as a result of protein adsorption, can also denature the protein and change its native properties.
Tissue engineering is a relatively new field that utilizes a scaffolding as a platform upon which the desired cells proliferate. It is not clear what defines an ideal scaffold for a specific tissue type. The considerations are complex and protein adsorption only adds to the complexity. Although architecture, structural mechanics, and surface properties play a key role, understanding degradation and rate of protein adsorption are also key. In addition to the essentials of mechanics and geometry, a suitable scaffold construct will possess surface properties that are optimized for the attachment and migration of the cell types of particular interest.
Generally, it has been found that scaffolds that closely resemble the natural environments of the tissue being engineered are the most successful. As a result, much research has gone into investigating natural polymers that can be tailored, through processing methodology, toward specific design criteria. Chitosan is currently one of the most widely used polymers as it is very similar to naturally occurring glycosaminoglycan (GAGs) and it is degradable by human enzymes . [ 28 ]
Chitosan is a linear polysaccharide containing linked chitin-derived residues and is widely studied as a biomaterial due to its high compatibility with numerous proteins in the body. Chitosan is cationic and thus electrostatically reacts with numerous proteoglycans , anionic GAGs, and other molecules possessing a negative charge. Since many cytokines and growth factors are linked to GAG, scaffolds with the chitosan-GAG complexes are able to retain these proteins secreted by the adhered cells. Another quality of chitosan that gives it good biomaterial potential is its high charge density in solutions. This allows chitosan to form ionic complexes with many water-soluble anionic polymers, expanding the range of proteins that are able to bind to it and thus expanding its possible uses. [ 29 ]
Due to their amphiphilic chemistry proteins are surface active and adsorb at fluid interfaces. In multiphase systems like emulsions or foams proteins adsorb at the oil-water or air-water interface, respectively, and reduce the interface tension , thereby increasing their stability. [ 36 ] Proteins are pH-dependent polyampholytes that undergo structural rearrangements upon adsorption, wherefore their adsorption depends on the protein thermodynamic stability, [ 37 ] the pH and ionic strength of the aqueous phase, [ 38 ] and the polarity of the respective phases. [ 39 ] Protein adsorption at fluid interfaces plays a critical role in the production and stability of many food emulsions and foams like mayonnaise , whipped cream , or meringue [ 40 ] and in physiological fluids like tear film , lipid droplets , or pulmonary surfactant . [ 41 ] Certain enzymes like lipases involved in fat digestion act by adsorbing at the oil-water interface of ingested fat. [ 42 ] Some animals exploit the foaming of secreted proteins, such as the sea snail Janthina janthina for passive flotation or certain species of spittlebug ( Cercopidae ) nymphs for protection from predators, moisture loss, and UV-radiation . [ 43 ]
Protein adsorption on solid surfaces plays an important role in many applications, including medical and dental practice (e.g., implants and catheters), biomedical research (e.g., drug delivery and release), and devices for diagnostics and drug discovery (e.g., assays, microarrays, and lab-on-a-chip ). Therefore, the accurate prediction of protein adsorption is critical in the progression of these sectors. Unfortunately, due to the inherent complexity of both the adsorption process and the protein molecular surface in general, protein adsorption prediction has continued to frustrate researchers. However, the Protein Adsorption Predictor is an application which aims to forecast protein concentration on surfaces using semi-empirical relationships. [ 44 ]
In an effort to aid in the prediction of protein adsorption, researchers have created the Biomolecular Adsorption Database (BAD). BAD is a freely available online database with experimental protein adsorption data collected from the literature. The database can be used for the selection of materials for microfluidic device fabrication and for the selection of optimum operating conditions of lab-on-a-chip devices. The first version was published in 2009. [ 45 ]
In 2024, an upgraded version, BAD2.0, was made publically available here . This database comprises 865 protein adsorption records (flat solid surfaces) from peer-reviewed literature. [ 46 ] BAD 2.0 comprises only data that quantitatively report, completely, the descriptors of all three classes of descriptors relevant to protein adsorption: (i) the adsorbed protein, preferably identifiable in the Protein Data Bank (PDB); to allow further derivation of protein descriptors; (ii) the flat surface (type, water contact angle or surface tension); and (iii) fluid descriptors, i.e., protein concentration in solution, pH, ionic strength, and temperature. BAD2.0 also comprises auxiliary data, i.e., method of measurement, and the DOI of the relevant references for easy retrieval. | https://en.wikipedia.org/wiki/Protein_adsorption |
Protein adsorption refers to the adhesion of proteins to solid surfaces. This phenomenon is an important issue in the food processing industry , particularly in milk processing and wine and beer making . Excessive adsorption, or protein fouling, can lead to health and sanitation issues, as the adsorbed protein is very difficult to clean and can harbor bacteria, as is the case in biofilms . Product quality can be adversely affected if the adsorbed material interferes with processing steps, like pasteurization . However, in some cases protein adsorption is used to improve food quality, as is the case in fining of wines.
Protein adsorption and protein fouling can cause major problems in the food industry (particularly the dairy industry ) when proteins from food adsorb to processing surfaces, such as stainless steel or plastic (e.g. polypropylene ). Protein fouling is the gathering of protein aggregates on a surface. This is most common in heating processes that create a temperature gradient between the equipment and the bulk substance being heated. [ 1 ] In protein-fouled heating equipment, adsorbed proteins can create an insulating layer between the heater and the bulk material, reducing heating efficiency. This leads to inefficient sterilization and pasteurization. Also, proteins stuck to the heater may cause a burned taste or color in the bulk material. [ 1 ] Additionally, in processes that employ filtration, protein aggregates that gather on the surface of the filter can block the flow of the bulk material and greatly reduce filter efficiency. [ 2 ]
Beerstone is a buildup that forms when oxalate, proteins, and calcium or magnesium salts from the grains and water in the beer brewing process precipitate and form scale on kegs, barrels and tap lines. The minerals adsorb to the surface of the container first, driven by charge attractions. Proteins are often coordinated to these minerals in the solution and can bind with them to the surface. In other cases proteins also adsorb to the minerals on the surface, making deposits difficult to remove, [ 3 ] as well as providing a surface that can easily harbor microorganisms. If built-up beer stone inside tap lines flakes off, it can negatively affect the quality of the finished product by making beer hazy and contributing "off" flavors. It is also harmful from a nutritional standpoint: oxalates can decrease absorption of calcium in the body, in addition to increasing risk of kidney stone formation. [ 4 ]
Grape and wine proteins tend to aggregate and form hazes and sediment in finished wines, especially white wines. [ 5 ] Haze-causing proteins can persist in wine due to low settling velocities or charge repulsion on individual particles. Fining agents, such as bentonite clays, are used to clarify wine by removing these proteins. Also, proteinaceous agents such as albumin, casein, or gelatin are used in wine clarification to remove tannins or other phenols. [ 6 ]
A biofilm is a community of microorganisms adsorbed to a surface. Microorganisms in biofilms are enclosed in a polymeric matrix consisting of exopolysaccharides, extracellular DNA and proteins. Seconds after a surface (usually metal) is placed in a solution, inorganic and organic molecules adsorb onto the surface. These molecules are attracted mainly by Coulombic forces (see above section), and can adhere very strongly to the surface. This first layer is called the conditioning layer, and is necessary for the microorganisms to bind to the surface. These microorganisms then attach reversibly by Van der Waals forces , followed by irreversible adhesion through self-produced attachment structures such as pili or flagella. [ 7 ] Biofilms form on solid substrates such as stainless steel. A biofilm's enclosing polymeric matrix offers protection to its microbes, increasing their resistance to detergents and cleaning agents. Biofilms on food processing surfaces can be a biological hazard to food safety. Increased chemical resistance in biofilms can lead to a persistent contamination condition. [ 8 ]
Thermal treatment of milk by indirect heating (e.g. pasteurization) to reduce microbial load and increase shelf life is generally performed by a plate heat exchanger . Heat exchanger surfaces can become fouled by adsorbed milk protein deposits. Fouling is initiated by formation of a protein monolayer at room temperature, followed by heat induced aggregation and deposition of whey protein and calcium phosphate deposits. [ 9 ] Adsorbed proteins decrease efficiency of heat transfer and potentially affect product quality by preventing adequate heating of milk.
The common trend in all examples of protein adsorption in the food industry is that of adsorption to minerals adsorbed to the surface first. This phenomenon has been studied but it is not well understood. Spectroscopy of proteins adsorbed onto clay-like minerals show variations in the C=O and N-H bond stretches, meaning that these bonds are involved in the protein binding. [ 10 ]
In some cases proteins are attracted to surfaces by an excessive surface charge . When a surface in a fluid has a net charge, ions in the fluid will adsorb to the surface. Proteins also have charged surfaces due to charge amino acid residues on the surface of the protein. The surface and the protein are then attracted by Coulombic forces. [ 11 ]
The attraction a protein feels from a charged surface ( ϕ {\displaystyle \phi } ) depends exponentially on the surface's charge, as described by the following formula: [ 12 ]
Where
A protein's surface's potential is given by the number of charged amino acids it has and its isoelectric point , pI.
Protein adsorption can also occur as a direct result of heating a mixture. Protein adsorption in milk processing is often used as a model for this type of adsorption in other situations. Milk is composed mainly of water, with less than 20% of suspended solids or dissolved proteins. Proteins make up only 3.6% of milk in total, and only 26% of the components that are not water. [ 13 ] These proteins are all responsible for fouling that occurs during pasteurization .
As milk is heated during pasteurization many of the proteins in the milk are denatured. Pasteurization temperatures can reach 161 °F (71.7 °C). This temperature is high enough to denature the proteins below, lowering the nutritional value of the milk and causing fouling. Milk is heated to these high temperatures for a short time (15–20 seconds) to reduce the amount of denaturization . However fouling from denatured proteins is still a significant problem.
Denaturation exposes hydrophobic amino acid residues in the protein, which had been previously protected by the protein. The exposed hydrophobic amino acids decrease the entropy of the water surrounding them, making it favorable for surface adsorption. Some of the β-lactoglobulin (β-lg) will adsorb directly onto the surface of a heat exchanger or container. Other denatured β-lg molecules adsorb to casein micelles , which are also present in the milk. As more and more β-lg proteins bind to the casein micelle it forms an aggregate, which will then diffuse to the heat exchanger and/or surface of the container.
While the aggregates can explain much of the protein fouling found in milk processing, this does not account for it all. A third type of fouling has been discovered that is explained by the chemical interactions of the denatured β-lg proteins. [ 15 ]
β-lg contains 5 cysteine residues, four of which are covalently bonded to each other, forming an S-S bond. When β-lg is denatured, the fifth cysteine residue is exposed to the water. This residue then bonds to other β-lg proteins, including those already adsorbed to the surface. This produces a strong interaction between the denatured proteins and the surface of the container.
Isotherms are used to quantify the amount of adsorbed protein on a surface at a constant temperature, depending on the concentration of protein above the surface. Researchers have used a Langmuir-type isotherm model to describe experimental values for protein adsorption. [ 14 ]
In this equation
This equation has been applied to a laboratory setting of protein adsorption at temperatures higher than 50 °C from a model solution of protein and water. It is especially useful for modeling protein fouling in milk processing.
Adsorbed proteins are among the most difficult food soils to remove from food contact surfaces. In particular, heat-denatured proteins (such as those found in dairy industry applications) adhere tightly to surfaces and require strong alkaline cleaners for removal. [ 16 ] It is important that cleaning methods are capable of removing both visible and non-visible protein soils. Nutrients for bacterial growth must be removed as well as biofilms that may have built up on the food contact surface. Proteins are water-insoluble, slightly soluble in acidic solutions and soluble in alkaline solutions, which limits the type of cleaner that can be used to remove protein from the surface. [ 16 ] Generally speaking, highly alkaline cleaners with peptizing and wetting agents are most effective in protein removal on food contact surfaces. [ 17 ] Cleaning temperature is also a concern for effective protein removal. As temperature increases, the activity of the cleaning compound increases, making soil removal easier. However, at higher temperatures (> 55 °C) proteins denature and cleaning efficacy is reduced. [ 16 ]
Alkaline cleaners are classified as compounds with pH 7-14. Proteins are most effectively removed from surfaces by cleaners with a pH of 11 or higher. [ 16 ] An example of a strong alkaline cleaning agent is sodium hydroxide , also called caustic soda. Although sodium hydroxide (NaOH) can cause corrosion on food contact surfaces such as stainless steel, it is the preferred cleaning agent for protein removal due to its efficacy in dissolving proteins and dispersing/emulsifying food soils. Silicates are often added to these cleaners to reduce corrosion on metal surfaces. The mechanism of alkaline cleaning action in proteins follows a three-step process: [ 18 ]
Hypochlorite is often added to alkaline cleaners to peptize proteins. Chlorinated cleansers work by oxidizing sulfide crosslinks in proteins. [ 16 ] Cleaning speed and efficiency is improved due to increased diffusion of the cleaner into the soil matrix, now composed of smaller, more soluble proteins.
Enzyme-based cleaners are especially useful for biofilm removal. Bacteria are somewhat difficult to remove with traditional alkaline or acid cleaners. [ 19 ] Enzyme cleaners are more effective on biofilms since they work as proteases by breaking down proteins at bacterial attachment sites. They work at maximum efficiency at high pH and at temperatures below 60 °C. [ 16 ] Enzyme cleaners are an increasingly attractive alternative to traditional chemical cleaners because of biodegradability and other environmental factors, such as reduced wastewater generation and energy savings from using cold water. [ 20 ] However, they are typically more expensive than alkaline or acid cleaners. | https://en.wikipedia.org/wiki/Protein_adsorption_in_the_food_industry |
Protein Arginine Phosphatase (PAPs) , also known as Phosphoarginine Phosphatase, is an enzyme that catalyzes the dephosphorylation of phosphoarginine residues in proteins. [ 1 ] Protein phosphatases (PPs) are "obligatory heteromers [ 2 ] " made up of two maximum catalytic subunits attached to a non-catalytic subunit. Arginine modification is a post-translational protein modification in gram-positive bacteria . McsB and YwIE were recently identified as phosphorylating enzymes in Bacillus Subtilis (B.Subtilis). [ 3 ] YwIE was thought to be a protein-tyrosine-phosphatase, and McsB a tyrosine-kinase, [ 4 ] however in 2012 Elsholz et al. [ 3 ] showed that McsB is a protein-arginine-kinase (PAK) and YwlE is a phosphatase-arginine-phosphatase (PAP).
Many proteins rely on protein phosphatase activity for regulating their stability, localization, and interaction with other proteins. [ 3 ] Arginine modification is a post-translational protein modification in gram-positive bacteria, and protein arginine phosphorylation regulates transcription factors, in addition to tagging rogue proteins for degradation in gram-positive bacteria. [ 5 ] Like phosphorylation, dephosphorylation is a reversible post-translational event. It is reversible through the action of kinases (enzymes that adds a phosphate group to a protein via phosphorylation), and this antagonist activity of phosphorylation and dephosphorylation of proteins controls all aspect of prokaryotic and eukaryotic life. [ 5 ] In general, protein phosphatases play a crucial role in cell signaling regulation in both eukaryotes and prokaryotes . They act by removing a phosphate group from proteins, and their activity counteracts that of protein kinases. [ 6 ]
YwIE is a member of the low-molecular-weight protein tyrosine phosphatase (LMW-PTP). [ 7 ] It is the only active PAP present in B.subtilis, and PAPs exhibits almost no activity against Protein Serine, Protein Tyrosine , and Protein Threonine peptides. [ 3 ] Also, YwIE has been shown to play a role in B.Subtilis's resistance to stress. Elsholz et al. [ 3 ] (2012), reported in their paper that protein arginine phosphorylation likely plays a critical physiological and regulatory role in bacteria. They showed that protein arginine phosphorylation is involved in the regulation of homeostasis, biofilm formation, motility, competence, stress, and stringent responses by regulating gene expression and protein activity in Bacillus Subtilis. [ 3 ] Their results suggested that the combined action of protein arginine phosphatase and kinase allows for rapid and reversible regulation of protein activity. Also, that protein-arginine-phosphatases reverse the effect of protein arginine kinases (PAKs) in living organisms. [ 3 ] In B.Subtilis, YwIE, a PAP, counteracts the action of McsB, a protein arginine kinase (PAK). McsB phosphorylates arginine residues in the winged helix-turn-helix domain of CtsR4, preventing it from binding to DNA, allowing for the expression of the repressed gene. However, YwIE is capable of restoring the DNA-binding ability of the CtsR repressor, a stress response & heat shock regulator in B.Subtilis, by reversing the McsB-mediated phosphorylation4. It accomplishes this by dephosphorylating the CtsR Protein. Additionally, McsB and YwIE are capable of differentiating between phosphoarginine and other amino acid residues [ 5 ] [ 8 ]
As of 2020, YwIE is the only known active PAP in B.Subtilis , although Fuhrmann et al. (2013). [ 9 ] identified a YwIE homolog in Drosophila, but its role in the species is still unknown. In contrast, Suzuki et al. (2013) identified the presence of McsB in over 150 bacteria species [ 5 ]
The specific molecular mechanism of action of the ywIE protein is currently unknown. [ 1 ] However, YwIE is believed to dephosphorylate phosphoarginine residues using a concerted, 2-step process via SN2 reactions . [ 1 ] Step 1 involves a nucleophilic attack of Cys7 on the phosphorus atom of the phosphoric group. [ 1 ] Then a thiophosphate intermediate is formed. In the second step, a phosphorylation-enzyme intermediate is hydrolyzed following the deprotonation of a water molecule by Asp118. [ 1 ] Fuhrmann et al. [ 1 ] (2016) believe that Asp118 likely promotes the reaction through the stabilization of the positive charge of the amino group via electrostatic interaction.
Sample general dephosphorylation reaction equation: H 2 O + C 6 H 15 N 4 O 5 P ⟶ C 6 H 14 N 4 O 2 + PO 4 3 − {\displaystyle {\ce {H2O + C6H15N4O5P-> C6H14N4O2 + PO4^3-}}}
In 2005, Suskiewicz et al. [ 1 ] classified the enzyme YwIE as a tyrosine phosphatase. And Kirstein et al. [ 4 ] (2005) found that McsB is a tyrosine kinase that needs McsA to become activated. They also found that the interaction of McsA and McsB with CtsR results in the formation of a 3-protein complex that stops the binding of CtsR to its target DNA and leads to subsequent phosphorylation of McsB, McsA, and CtsR.
In their study, Fuhrmann et al. [ 10 ] (2009), performed a biochemical and structural analysis of the bacterial transcriptional regulators CtsR/McsB stress response. They sort to clarify and outline the exact function of CtsR and McsB in bacterial stress response. So, they screened proteins from various gram-negative bacteria for recombinant production and succeeded in reconstituting the Bacillus stearothermophilus CtsR/McsB system in vitro. Subsequently, they identified McsB as a protein kinase that targets arginine.
Elsholz et al. [ 3 ] (2012), showed that McsB and YwlE are a protein arginine kinase and phosphatase, rather than a tyrosine kinase and phosphatase because they observed only an McsB/YwlE-dependent detection of protein arginine phosphorylation or dephosphorylation in vivo. Specifically, they suggested that YwIE acts as a PAP in vivo.
McsB and YwlE were thought to be tyrosine kinases and phosphatases. [ 4 ] However, in 2012, Elsholz et al. [ 3 ] detected 121 arginine phosphorylation sites in 87 proteins in living Bacillus Subtilis (B.subtillis), a gram-positive bacterium present in soil and human gastrointestinal tract. Their observations led them to believe that protein arginine phosphorylation exists in vivo as a posttranslational modification in bacteria. The arginine-phosphorylated proteins they detected were distributed among "distinct physiological classes of proteins" such as regulators, metabolic enzymes, stress, and ribosomal proteins. This result suggested that YwlE acts as a protein arginine phosphatase that explicitly dephosphorylates arginine residues both in vitro and in vivo [ 3 ]
Secondly, Elsholz et al. [ 3 ] (2012) were only able to detect protein arginine phosphorylation in a YwIE mutant gene and not the wild-type strain. But protein phosphorylates on either serine, threonine, or tyrosine were detected in both wild-type and a YwIE mutant strain in equal amounts. Therefore, they thought that YwIE might solely act as a protein arginine phosphatase. That is, the detection of protein arginine phosphorylation depended on the presence of YwIE. They confirmed this hypothesis after failing to detect protein arginine phosphorylation after (1) analyzing a mutant extract treated in vitro with purified YwIE protein before conducting mass spectroscopy analysis; and (2) overexpressing the YwIE in trans in a YwIE mutant in-vivo . The close interaction of the arginine phosphorylated proteins with YwIE suggested that the stability of the modifications was indeed influenced by the YwIE protein. | https://en.wikipedia.org/wiki/Protein_arginine_phosphatase |
Protein biosynthesis , or protein synthesis , is a core biological process, occurring inside cells , balancing the loss of cellular proteins (via degradation or export ) through the production of new proteins. Proteins perform a number of critical functions as enzymes , structural proteins or hormones . Protein synthesis is a very similar process for both prokaryotes and eukaryotes but there are some distinct differences. [ 1 ]
Protein synthesis can be divided broadly into two phases: transcription and translation . During transcription, a section of DNA encoding a protein, known as a gene , is converted into a molecule called messenger RNA (mRNA). This conversion is carried out by enzymes, known as RNA polymerases , in the nucleus of the cell . [ 2 ] In eukaryotes, this mRNA is initially produced in a premature form ( pre-mRNA ) which undergoes post-transcriptional modifications to produce mature mRNA . The mature mRNA is exported from the cell nucleus via nuclear pores to the cytoplasm of the cell for translation to occur. During translation, the mRNA is read by ribosomes which use the nucleotide sequence of the mRNA to determine the sequence of amino acids . The ribosomes catalyze the formation of covalent peptide bonds between the encoded amino acids to form a polypeptide chain . [ citation needed ]
Following translation the polypeptide chain must fold to form a functional protein; for example, to function as an enzyme the polypeptide chain must fold correctly to produce a functional active site . To adopt a functional three-dimensional shape, the polypeptide chain must first form a series of smaller underlying structures called secondary structures . The polypeptide chain in these secondary structures then folds to produce the overall 3D tertiary structure . Once correctly folded, the protein can undergo further maturation through different post-translational modifications , which can alter the protein's ability to function, its location within the cell (e.g. cytoplasm or nucleus) and its ability to interact with other proteins . [ 3 ]
Protein biosynthesis has a key role in disease as changes and errors in this process, through underlying DNA mutations or protein misfolding , are often the underlying causes of a disease. DNA mutations change the subsequent mRNA sequence, which then alters the mRNA encoded amino acid sequence. Mutations can cause the polypeptide chain to be shorter by generating a stop sequence which causes early termination of translation. Alternatively, a mutation in the mRNA sequence changes the specific amino acid encoded at that position in the polypeptide chain. This amino acid change can impact the protein's ability to function or to fold correctly. [ 4 ] Misfolded proteins have a tendency to form dense protein clumps , which are often implicated in diseases, particularly neurological disorders including Alzheimer's and Parkinson's disease . [ 5 ]
Transcription occurs in the nucleus using DNA as a template to produce mRNA . In eukaryotes , this mRNA molecule is known as pre-mRNA as it undergoes post-transcriptional modifications in the nucleus to produce a mature mRNA molecule. However, in prokaryotes post-transcriptional modifications are not required so the mature mRNA molecule is immediately produced by transcription. [ 1 ]
Initially, an enzyme known as a helicase acts on the molecule of DNA. DNA has an antiparallel , double helix structure composed of two, complementary polynucleotide strands, held together by hydrogen bonds between the base pairs. The helicase disrupts the hydrogen bonds causing a region of DNA – corresponding to a gene – to unwind, separating the two DNA strands and exposing a series of bases. Despite DNA being a double-stranded molecule, only one of the strands acts as a template for pre-mRNA synthesis; this strand is known as the template strand. The other DNA strand (which is complementary to the template strand) is known as the coding strand. [ 6 ]
Both DNA and RNA have intrinsic directionality , meaning there are two distinct ends of the molecule. This property of directionality is due to the asymmetrical underlying nucleotide subunits, with a phosphate group on one side of the pentose sugar and a base on the other. The five carbons in the pentose sugar are numbered from 1' (where ' means prime) to 5'. Therefore, the phosphodiester bonds connecting the nucleotides are formed by joining the hydroxyl group on the 3' carbon of one nucleotide to the phosphate group on the 5' carbon of another nucleotide. Hence, the coding strand of DNA runs in a 5' to 3' direction and the complementary, template DNA strand runs in the opposite direction from 3' to 5'. [ 1 ]
The enzyme RNA polymerase binds to the exposed template strand and reads from the gene in the 3' to 5' direction. Simultaneously, the RNA polymerase synthesizes a single strand of pre-mRNA in the 5'-to-3' direction by catalysing the formation of phosphodiester bonds between activated nucleotides (free in the nucleus) that are capable of complementary base pairing with the template strand. Behind the moving RNA polymerase the two strands of DNA rejoin, so only 12 base pairs of DNA are exposed at one time. [ 6 ] RNA polymerase builds the pre-mRNA molecule at a rate of 20 nucleotides per second enabling the production of thousands of pre-mRNA molecules from the same gene in an hour. Despite the fast rate of synthesis, the RNA polymerase enzyme contains its own proofreading mechanism. The proofreading mechanisms allows the RNA polymerase to remove incorrect nucleotides (which are not complementary to the template strand of DNA) from the growing pre-mRNA molecule through an excision reaction. [ 1 ] When RNA polymerases reaches a specific DNA sequence which terminates transcription, RNA polymerase detaches and pre-mRNA synthesis is complete. [ 6 ]
The pre-mRNA molecule synthesized is complementary to the template DNA strand and shares the same nucleotide sequence as the coding DNA strand. However, there is one crucial difference in the nucleotide composition of DNA and mRNA molecules. DNA is composed of the bases: guanine , cytosine , adenine and thymine (G, C, A and T). RNA is also composed of four bases: guanine, cytosine, adenine and uracil . In RNA molecules, the DNA base thymine is replaced by uracil which is able to base pair with adenine. Therefore, in the pre-mRNA molecule, all complementary bases which would be thymine in the coding DNA strand are replaced by uracil. [ 7 ]
Once transcription is complete, the pre-mRNA molecule undergoes post-transcriptional modifications to produce a mature mRNA molecule. [ citation needed ]
There are 3 key steps within post-transcriptional modifications: [ citation needed ]
The 5' cap is added to the 5' end of the pre-mRNA molecule and is composed of a guanine nucleotide modified through methylation . The purpose of the 5' cap is to prevent break down of mature mRNA molecules before translation, the cap also aids binding of the ribosome to the mRNA to start translation [ 8 ] and enables mRNA to be differentiated from other RNAs in the cell. [ 1 ] In contrast, the 3' Poly(A) tail is added to the 3' end of the mRNA molecule and is composed of 100-200 adenine bases. [ 8 ] These distinct mRNA modifications enable the cell to detect that the full mRNA message is intact if both the 5' cap and 3' tail are present. [ 1 ]
This modified pre-mRNA molecule then undergoes the process of RNA splicing. Genes are composed of a series of introns and exons , introns are nucleotide sequences which do not encode a protein while, exons are nucleotide sequences that directly encode a protein. Introns and exons are present in both the underlying DNA sequence and the pre-mRNA molecule, therefore, to produce a mature mRNA molecule encoding a protein, splicing must occur. [ 6 ] During splicing, the intervening introns are removed from the pre-mRNA molecule by a multi-protein complex known as a spliceosome (composed of over 150 proteins and RNA). [ 9 ] This mature mRNA molecule is then exported into the cytoplasm through nuclear pores in the envelope of the nucleus. [ citation needed ]
During translation, ribosomes synthesize polypeptide chains from mRNA template molecules. In eukaryotes, translation occurs in the cytoplasm of the cell, where the ribosomes are located either free floating or attached to the endoplasmic reticulum . In prokaryotes, which lack a nucleus, the processes of both transcription and translation occur in the cytoplasm. [ 10 ]
Ribosomes are complex molecular machines , made of a mixture of protein and ribosomal RNA , arranged into two subunits (a large and a small subunit), which surround the mRNA molecule. The ribosome reads the mRNA molecule in a 5'-3' direction and uses it as a template to determine the order of amino acids in the polypeptide chain. [ 11 ] To translate the mRNA molecule, the ribosome uses small molecules, known as transfer RNAs (tRNA), to deliver the correct amino acids to the ribosome. Each tRNA is composed of 70-80 nucleotides and adopts a characteristic cloverleaf structure due to the formation of hydrogen bonds between the nucleotides within the molecule. There are around 60 different types of tRNAs, each tRNA binds to a specific sequence of three nucleotides (known as a codon ) within the mRNA molecule and delivers a specific amino acid. [ 12 ]
The ribosome initially attaches to the mRNA at the start codon (AUG) and begins to translate the molecule. The mRNA nucleotide sequence is read in triplets ; three adjacent nucleotides in the mRNA molecule correspond to a single codon. Each tRNA has an exposed sequence of three nucleotides, known as the anticodon, which are complementary in sequence to a specific codon that may be present in mRNA. For example, the first codon encountered is the start codon composed of the nucleotides AUG. The correct tRNA with the anticodon (complementary 3 nucleotide sequence UAC) binds to the mRNA using the ribosome. This tRNA delivers the correct amino acid corresponding to the mRNA codon, in the case of the start codon, this is the amino acid methionine. The next codon (adjacent to the start codon) is then bound by the correct tRNA with complementary anticodon, delivering the next amino acid to ribosome. The ribosome then uses its peptidyl transferase enzymatic activity to catalyze the formation of the covalent peptide bond between the two adjacent amino acids. [ 6 ]
The ribosome then moves along the mRNA molecule to the third codon. The ribosome then releases the first tRNA molecule, as only two tRNA molecules can be brought together by a single ribosome at one time. The next complementary tRNA with the correct anticodon complementary to the third codon is selected, delivering the next amino acid to the ribosome which is covalently joined to the growing polypeptide chain. This process continues with the ribosome moving along the mRNA molecule adding up to 15 amino acids per second to the polypeptide chain. Behind the first ribosome, up to 50 additional ribosomes can bind to the mRNA molecule forming a polysome , this enables simultaneous synthesis of multiple identical polypeptide chains. [ 6 ] Termination of the growing polypeptide chain occurs when the ribosome encounters a stop codon (UAA, UAG, or UGA) in the mRNA molecule. When this occurs, no tRNA can recognise it and a release factor induces the release of the complete polypeptide chain from the ribosome. [ 12 ] Dr. Har Gobind Khorana , a scientist originating from India, decoded the RNA sequences for about 20 amino acids. [ citation needed ] He was awarded the Nobel Prize in 1968, along with two other scientists, for his work.
Once synthesis of the polypeptide chain is complete, the polypeptide chain folds to adopt a specific structure which enables the protein to carry out its functions. The basic form of protein structure is known as the primary structure , which is simply the polypeptide chain i.e. a sequence of covalently bonded amino acids. The primary structure of a protein is encoded by a gene. Therefore, any changes to the sequence of the gene can alter the primary structure of the protein and all subsequent levels of protein structure, ultimately changing the overall structure and function. [ citation needed ]
The primary structure of a protein (the polypeptide chain) can then fold or coil to form the secondary structure of the protein. The most common types of secondary structure are known as an alpha helix or beta sheet , these are small structures produced by hydrogen bonds forming within the polypeptide chain. This secondary structure then folds to produce the tertiary structure of the protein. The tertiary structure is the proteins overall 3D structure which is made of different secondary structures folding together. In the tertiary structure, key protein features e.g. the active site, are folded and formed enabling the protein to function. Finally, some proteins may adopt a complex quaternary structure . Most proteins are made of a single polypeptide chain, however, some proteins are composed of multiple polypeptide chains (known as subunits) which fold and interact to form the quaternary structure. Hence, the overall protein is a multi-subunit complex composed of multiple folded, polypeptide chain subunits e.g. haemoglobin . [ 13 ]
There are events that follow protein biosynthesis such as proteolysis [ 14 ] and protein-folding. Proteolysis refers to the cleavage of proteins by proteases and the breakdown of proteins into amino acids by the action of enzymes.
When protein folding into the mature, functional 3D state is complete, it is not necessarily the end of the protein maturation pathway. A folded protein can still undergo further processing through post-translational modifications. There are over 200 known types of post-translational modification, these modifications can alter protein activity, the ability of the protein to interact with other proteins and where the protein is found within the cell e.g. in the cell nucleus or cytoplasm. [ 15 ] Through post-translational modifications, the diversity of proteins encoded by the genome is expanded by 2 to 3 orders of magnitude . [ 16 ]
There are four key classes of post-translational modification: [ 3 ]
Cleavage of proteins is an irreversible post-translational modification carried out by enzymes known as proteases . These proteases are often highly specific and cause hydrolysis of a limited number of peptide bonds within the target protein. The resulting shortened protein has an altered polypeptide chain with different amino acids at the start and end of the chain. This post-translational modification often alters the proteins function, the protein can be inactivated or activated by the cleavage and can display new biological activities. [ 17 ]
Following translation, small chemical groups can be added onto amino acids within the mature protein structure. [ 18 ] Examples of processes which add chemical groups to the target protein include methylation, acetylation and phosphorylation . [ 19 ]
Methylation is the reversible addition of a methyl group onto an amino acid catalyzed by methyltransferase enzymes. Methylation occurs on at least 9 of the 20 common amino acids, however, it mainly occurs on the amino acids lysine and arginine . One example of a protein which is commonly methylated is a histone . Histones are proteins found in the nucleus of the cell. DNA is tightly wrapped round histones and held in place by other proteins and interactions between negative charges in the DNA and positive charges on the histone. A highly specific pattern of amino acid methylation on the histone proteins is used to determine which regions of DNA are tightly wound and unable to be transcribed and which regions are loosely wound and able to be transcribed. [ 20 ]
Histone-based regulation of DNA transcription is also modified by acetylation. Acetylation is the reversible covalent addition of an acetyl group onto a lysine amino acid by the enzyme acetyltransferase . The acetyl group is removed from a donor molecule known as acetyl coenzyme A and transferred onto the target protein. [ 21 ] Histones undergo acetylation on their lysine residues by enzymes known as histone acetyltransferase . The effect of acetylation is to weaken the charge interactions between the histone and DNA, thereby making more genes in the DNA accessible for transcription. [ 22 ]
The final, prevalent post-translational chemical group modification is phosphorylation. Phosphorylation is the reversible, covalent addition of a phosphate group to specific amino acids ( serine , threonine and tyrosine ) within the protein. The phosphate group is removed from the donor molecule ATP by a protein kinase and transferred onto the hydroxyl group of the target amino acid, this produces adenosine diphosphate as a byproduct. This process can be reversed and the phosphate group removed by the enzyme protein phosphatase . Phosphorylation can create a binding site on the phosphorylated protein which enables it to interact with other proteins and generate large, multi-protein complexes. Alternatively, phosphorylation can change the level of protein activity by altering the ability of the protein to bind its substrate. [ 1 ]
Post-translational modifications can incorporate more complex, large molecules into the folded protein structure. One common example of this is glycosylation , the addition of a polysaccharide molecule, which is widely considered to be most common post-translational modification. [ 16 ]
In glycosylation, a polysaccharide molecule (known as a glycan ) is covalently added to the target protein by glycosyltransferases enzymes and modified by glycosidases in the endoplasmic reticulum and Golgi apparatus . Glycosylation can have a critical role in determining the final, folded 3D structure of the target protein. In some cases glycosylation is necessary for correct folding. N-linked glycosylation promotes protein folding by increasing solubility and mediates the protein binding to protein chaperones . Chaperones are proteins responsible for folding and maintaining the structure of other proteins. [ 1 ]
There are broadly two types of glycosylation, N-linked glycosylation and O-linked glycosylation . N-linked glycosylation starts in the endoplasmic reticulum with the addition of a precursor glycan. The precursor glycan is modified in the Golgi apparatus to produce complex glycan bound covalently to the nitrogen in an asparagine amino acid. In contrast, O-linked glycosylation is the sequential covalent addition of individual sugars onto the oxygen in the amino acids serine and threonine within the mature protein structure. [ 1 ]
Many proteins produced within the cell are secreted outside the cell to function as extracellular proteins. Extracellular proteins are exposed to a wide variety of conditions. To stabilize the 3D protein structure, covalent bonds are formed either within the protein or between the different polypeptide chains in the quaternary structure. The most prevalent type is a disulfide bond (also known as a disulfide bridge). A disulfide bond is formed between two cysteine amino acids using their side chain chemical groups containing a Sulphur atom, these chemical groups are known as thiol functional groups. Disulfide bonds act to stabilize the pre-existing structure of the protein. Disulfide bonds are formed in an oxidation reaction between two thiol groups and therefore, need an oxidizing environment to react. As a result, disulfide bonds are typically formed in the oxidizing environment of the endoplasmic reticulum catalyzed by enzymes called protein disulfide isomerases. Disulfide bonds are rarely formed in the cytoplasm as it is a reducing environment. [ 1 ]
Many diseases are caused by mutations in genes, due to the direct connection between the DNA nucleotide sequence and the amino acid sequence of the encoded protein. Changes to the primary structure of the protein can result in the protein mis-folding or malfunctioning. Mutations within a single gene have been identified as a cause of multiple diseases, including sickle cell disease , known as single gene disorders. [ citation needed ]
Sickle cell disease is a group of diseases caused by a mutation in a subunit of hemoglobin, a protein found in red blood cells responsible for transporting oxygen. The most dangerous of the sickle cell diseases is known as sickle cell anemia. Sickle cell anemia is the most common homozygous recessive single gene disorder , meaning the affected individual must carry a mutation in both copies of the affected gene (one inherited from each parent) to experience the disease. Hemoglobin has a complex quaternary structure and is composed of four polypeptide subunits – two A subunits and two B subunits. [ 23 ] Patients with sickle cell anemia have a missense or substitution mutation in the gene encoding the hemoglobin B subunit polypeptide chain. A missense mutation means the nucleotide mutation alters the overall codon triplet such that a different amino acid is paired with the new codon. In the case of sickle cell anemia, the most common missense mutation is a single nucleotide mutation from thymine to adenine in the hemoglobin B subunit gene. [ 24 ] This changes codon 6 from encoding the amino acid glutamic acid to encoding valine. [ 23 ]
This change in the primary structure of the hemoglobin B subunit polypeptide chain alters the functionality of the hemoglobin multi-subunit complex in low oxygen conditions. When red blood cells unload oxygen into the tissues of the body, the mutated haemoglobin protein starts to stick together to form a semi-solid structure within the red blood cell. This distorts the shape of the red blood cell, resulting in the characteristic "sickle" shape, and reduces cell flexibility. This rigid, distorted red blood cell can accumulate in blood vessels creating a blockage. The blockage prevents blood flow to tissues and can lead to tissue death which causes great pain to the individual. [ 25 ]
Cancers form as a result of gene mutations as well as improper protein translation. In addition to cancer cells proliferating abnormally, they suppress the expression of anti-apoptotic or pro-apoptotic genes or proteins. Most cancer cells see a mutation in the signaling protein Ras, which functions as an on/off signal transductor in cells. In cancer cells, the RAS protein becomes persistently active, thus promoting the proliferation of the cell due to the absence of any regulation. [ 26 ] Additionally, most cancer cells carry two mutant copies of the regulator gene p53, which acts as a gatekeeper for damaged genes and initiates apoptosis in malignant cells. In its absence, the cell cannot initiate apoptosis or signal for other cells to destroy it. [ 27 ]
As the tumor cells proliferate, they either remain confined to one area and are called benign, or become malignant cells that migrate to other areas of the body. Oftentimes, these malignant cells secrete proteases that break apart the extracellular matrix of tissues. This then allows the cancer to enter its terminal stage called Metastasis, in which the cells enter the bloodstream or the lymphatic system to travel to a new part of the body. [ 26 ] | https://en.wikipedia.org/wiki/Protein_biosynthesis |
In molecular biology , protein catabolism is the breakdown of proteins into smaller peptides and ultimately into amino acids . Protein catabolism is a key function of digestion process. Protein catabolism often begins with pepsin , which converts proteins into polypeptides. These polypeptides are then further degraded. In humans, the pancreatic proteases include trypsin , chymotrypsin , and other enzymes. In the intestine, the small peptides are broken down into amino acids that can be absorbed into the bloodstream. These absorbed amino acids can then undergo amino acid catabolism , where they are utilized as an energy source or as precursors to new proteins. [ 1 ]
The amino acids produced by catabolism may be directly recycled to form new proteins, converted into different amino acids, or can undergo amino acid catabolism to be converted to other compounds via the Krebs cycle . [ 2 ]
Protein catabolism produces amino acids that are used to form other proteins or oxidized to meet the energy needs of the cell. The amino acids that are produced by protein catabolism can then be further catabolized in amino acid catabolism. Among the several degradative processes for amino acids are Deamination (removal of an amino group), transamination (transfer of amino group), decarboxylation (removal of carboxyl group), and dehydrogenation (removal of hydrogen). Degradation of amino acids can function as part of a salvage pathway, whereby parts of degraded amino acids are used to create new amino acids, or as part of a metabolic pathway whereby the amino acid is broken down to release or recapture chemical energy. For example, the chemical energy that is released by oxidization in a dehydrogenation reaction can be used to reduce NAD + to NADH , which can then be fed directly into the Krebs/Citric Acid (TCA) Cycle . [ 2 ]
Protein degradation differs from protein catabolism. Proteins are produced and destroyed routinely as part of the normal operations of the cell. Transcription factors , proteins that help regulate protein synthesis, are targets of such degradations. Their degradation is not a significant contributor to the energy needs of the cell. [ 3 ] The addition of ubiquitin (ubiquitylation) marks a protein for degradation via the proteasome . [ 4 ]
Oxidative deamination is the first step to breaking down the amino acids so that they can be converted to sugars. The process begins by removing the amino group of the amino acids. The amino group becomes ammonium as it is lost and later undergoes the urea cycle to become urea, in the liver. It is then released into the blood stream, where it is transferred to the kidneys, which will secrete the urea as urine. [ 5 ] [ 6 ] The remaining portion of the amino acid becomes oxidized, resulting in an α- keto acid . The alpha-keto acid will then proceed into the TCA cycle, in order to produce energy. The acid can also enter glycolysis , where it will be eventually converted into pyruvate . The pyruvate is then converted into acetyl-CoA so that it can enter the TCA cycle and convert the original pyruvate molecules into ATP , or usable energy for the organism. [ 7 ]
Transamination leads to the same result as deamination: the remaining acid will undergo either glycolysis or the TCA cycle to produce energy that the organism's body will use for various purposes. This process transfers the amino group instead of losing the amino group to be converted into ammonium. The amino group is transferred to α-ketoglutarate , so that it can be converted to glutamate . Then glutamate transfers the amino group to oxaloacetate . This transfer is so that the oxaloacetate can be converted to aspartate or other amino acids. Eventually, this product will also proceed into oxidative deamination to once again produce alpha-ketoglutarate, an alpha-keto acid that will undergo the TCA cycle, and ammonium, which will eventually undergo the urea cycle. [ 8 ]
Transaminases are enzymes that help catalyze the reactions that take place in transamination. They help catalyze the reaction at the point when the amino group is transferred from the original amino acid, like glutamate to α-ketoglutarate, and hold onto it to transfer it to another α-ketoacid. [ 8 ]
Some key factors that determine overall rate include protein half-life, pH, and temperature.
Protein half-life helps determine the overall rate as this designates the first step in protein catabolism. Depending on whether this step is short or long will influence the rest of the metabolic process. One key component in determining the protein half-life is based on the N-end rule . This states that the amino acid present at the N-terminus of a protein helps determine the protein's half-life. [ 9 ] | https://en.wikipedia.org/wiki/Protein_catabolism |
Protein chemical shift prediction is a branch of biomolecular nuclear magnetic resonance spectroscopy that aims to accurately calculate protein chemical shifts from protein coordinates. Protein chemical shift prediction was first attempted in the late 1960s using semi-empirical methods applied to protein structures solved by X-ray crystallography . [ 1 ] Since that time protein chemical shift prediction has evolved to employ much more sophisticated approaches including quantum mechanics , machine learning and empirically derived chemical shift hypersurfaces. [ 1 ] The most recently developed methods exhibit remarkable precision and accuracy.
NMR chemical shifts are often called the mileposts of nuclear magnetic resonance spectroscopy . Chemists have used chemical shifts for more than 50 years as highly reproducible, easily measured parameters to map out the covalent structure of small organic molecules. Indeed, the sensitivity of NMR chemical shifts to the type and character of neighbouring atoms , combined with their reasonably predictable tendencies has made them invaluable for both deciphering and describing the structure of thousands of newly synthesized or newly isolated compounds [ 1 ] [ 2 ] [ 3 ] [ 4 ] The same sensitivity to a variety of important protein structural features has made protein chemical shifts equally valuable to protein chemists and biomolecular NMR spectroscopists. [ 4 ] In particular, protein chemical shifts are sensitive not only to substituent or covalent atom effects (such as electronegativity , redox states or ring currents ) but they are also sensitive to backbone torsion angles (i.e. secondary structure), hydrogen bonding, local atomic motions and solvent accessibility.
Predicted or estimated protein chemical shifts can be used to assist with the chemical shift assignment process. This is especially true if a similar (or identical) protein structure has been solved by X-ray crystallography. In this case, the three-dimensional structure can be used to estimate what the NMR chemical shifts should be and thereby simplify the process of assigning the experimentally observed chemical shifts. Predicted/estimated protein chemical shifts can also be used to identify incorrect or mis-assignments, to correct mis-referenced or incorrectly referenced chemical shifts, to optimize protein structures via chemical shift refinement and to identify the relative contributions of different electronic or geometric effects to nucleus-specific shifts. [ 1 ] Protein chemical shifts can also be used to identify secondary structures, to estimate backbone torsion angles, to determine the location of aromatic rings , to assess cysteine oxidation states, to estimate solvent exposure and to measure backbone flexibility. [ 4 ]
Significant progress in chemical shift prediction has been made through continuous improvements in our understanding of the key physico-chemical factors contributing to chemical shift changes. These improvements have also been helped along through significant computational advancements [ 5 ] [ 6 ] [ 7 ] [ 8 ] and the rapid expansion of biomolecular chemical shift databases [ 9 ] . [ 10 ] Over the past four decades, at least three different methods for calculating or predicting protein chemical shifts have emerged. The first is based on using sequence/structure alignment against protein chemical shift databases, the second is based on directly calculating shifts from atomic coordinates, and the third is based on using a combination of the two approaches. [ 1 ] [ 4 ]
By early 2000, several research groups realized that protein chemical shifts could be more efficiently and accurately calculated by combining different methods together as shown in Figure 1. This led to the development of several programs and web servers that rapidly calculate protein chemical shifts when provided with protein coordinate data. [ 1 ] These “hybrid” programs, along with some of their features and URLs, are listed below in Table 1.
http://www.wishartlab.com
This table (Figure 2) lists the correlation coefficients between the experimentally observed backbone chemical shifts and the calculated/predicted backbone shifts for different chemical shift predictors using an identical test set of 61 test proteins.
Different methods have different levels of coverage and rates of calculation. Some methods only calculate or predict chemical shifts for backbone atoms (6 atom types). Some calculate chemical shifts for backbone and certain side chain atoms (C and N only) and still others are able to calculate shifts for all atoms (40 atom types). For chemical shift refinement there is a need for rapid calculation as thousands of structures are generated during a molecular dynamics or simulated annealing run and their chemical shifts must be calculated equally rapidly.
All the computational speed tests for SPARTA, SPARTA+, SHIFTS, CamShift, SHIFTX and SHIFTX2 were performed on the same computer using the same set of proteins. The calculation speed reported for PROSHIFT is based on the response rate of its web server. [ 4 ] | https://en.wikipedia.org/wiki/Protein_chemical_shift_prediction |
Protein chemical shift re-referencing is a post-assignment process of adjusting the assigned NMR chemical shifts to match IUPAC and BMRB recommended standards in protein chemical shift referencing . In NMR chemical shifts are normally referenced to an internal standard that is dissolved in the NMR sample. These internal standards include tetramethylsilane (TMS), 4,4-dimethyl-4-silapentane-1-sulfonic acid (DSS) and trimethylsilyl propionate (TSP). For protein NMR spectroscopy the recommended standard is DSS , which is insensitive to pH variations (unlike TSP). Furthermore, the DSS 1H signal may be used to indirectly reference 13C and 15N shifts using a simple ratio calculation [1]. Unfortunately, many biomolecular NMR spectroscopy labs use non-standard methods for determining the 1H , 13C or 15N “zero-point” chemical shift position. This lack of standardization makes it difficult to compare chemical shifts for the same protein between different laboratories. It also makes it difficult to use chemical shifts to properly identify or assign secondary structures or to improve their 3D structures via chemical shift refinement. Chemical shift re-referencing offers a means to correct these referencing errors and to standardize the reporting of protein chemical shifts across laboratories.
Incorrect chemical shift referencing is a particularly acute problem in biomolecular NMR. [ 1 ] It has been estimated that up to 20% of 13C and up to 35% of 15N shift assignments are improperly referenced. [ 2 ] [ 3 ] [ 4 ] Given that the structural and dynamic information contained within chemical shifts is often quite subtle, it is critical that protein chemical shifts be properly referenced so that these subtle differences can be detected. Fundamentally, the problem with chemical shift referencing comes from the fact that chemical shifts are relative frequency measurements rather than absolute frequency measurements. Because of the historic problems with chemical shift referencing, chemical shifts are perhaps the most precisely measurable but the least accurately measured parameters in all of NMR spectroscopy . [ 5 ] [ 3 ]
Because of the magnitude and severity of the problems with chemical shift referencing in biomolecular NMR, a number of computer programs have been developed to help mitigate the problem (see Table 1 for a summary). The first program to comprehensively tackle chemical shift mis-referencing in biomolecular NMR was SHIFTCOR. [ 2 ]
Table 1. Summary and comparison of different chemical shift re-referencing and mis-assignment detection programs. [ 5 ]
SHIFTCOR is an automated protein chemical shift correction program that uses statistical methods to compare and correct predicted NMR chemical shifts (derived from the 3D structure of the protein) relative to an input set of experimentally measured chemical shifts. SHIFTCOR uses several simple statistical approaches and pre-determined cut-off values to identify and correct potential referencing, assignment and typographical errors . SHIFTCOR identifies potential chemical shift referencing problems by comparing the difference between the average value of each set of observed backbone (1Hα, 13Cα, 13Cβ, 13CO, 15N and 1HN) shifts and their corresponding predicted chemical shifts. The difference between these two averages results in a nucleus-specific chemical shift offset or reference correction (i.e. one for 1H , one for 13C and one for 15N). In order to ensure that certain extreme outliers do not unduly bias these average offset values, the average of the observed shifts is only calculated after excluding potential mis-assignments or typographical errors . [ 2 ]
SHIFTCOR generates and reports chemical shift offsets or differences for each nucleus. The results contain the chemical shift analyses (including lists of potential mis-assignments, the estimated referencing errors, the estimated error in the calculated reference offset (95% confidence interval), the applied or suggested reference offset, correlation coefficients, RMSD values) and the corrected BMRB formatted chemical shift file (see Figure 1 for details). [ 2 ]
SHIFTCOR uses the chemical shift calculation program SHIFTX [ 12 ] to predict 1Hα, 13Cα,15N shifts based on the 3D structure coordinates of the protein being analyzed. By comparing the predicted shifts to the observed shifts, SHIFTCOR is able to accurately identify chemical shift reference offsets as well as potential mis-assignments. A key limitation to the SHIFTCOR approach is that requires that the 3D structure for the target protein be available to assess the chemical shift reference offsets. Given that chemical shift assignments are typically made before the structure is determined, it was soon realized that structure-independent approaches were required to develop. [ 5 ]
Several methods have been developed that make use of the estimated (via 1H or 13C shifts) or predicted (via sequence) secondary structure content of the protein being analyzed. These programs include PSSI, [ 10 ] CheckShift, [ 6 ] [ 7 ] LACS, [ 4 ] [ 9 ] and PANAV. [ 11 ] Both PANAV < [1] > and CheckShift are also available as web servers.
The PSSI and PANAV programs use the secondary structure determined by 1H shifts (which are almost never mis-referenced) to adjust the target protein’s 13C and 15N shifts to match the 1H-derived secondary structure. LACS uses the difference between secondary 13Cα and 13Cβ shifts plotted against secondary 13Cα shifts or secondary 13Cβ shifts to determine reference offsets. A more recent version of LACS has been adapted to identify 15N chemical shift mis-referencing. [ 4 ] This new version of LACS exploits the well-known relationship between secondary 15N shifts and the secondary 13Cα and 13Cβ shifts of the preceding residue. [ 3 ] In contrast to LACS and PANAV/PSSI, CheckShift uses secondary structure predicted from high-performance secondary structure prediction programs such as PSIPRED [ 13 ] to iteratively adjust 13C and 15N chemical shifts so that their secondary shifts match the predicted secondary structure. These programs have all been shown to accurately identify mis-referenced and properly re-reference protein chemical shifts deposited in the BMRB,. [ 7 ] [ 11 ] Note that both LACS and CheckShift are programmed to always predict the same offset for 13Cα and 13Cβ shifts, whereas PSSI and PANAV do not make this assumption. As a general rule, PANAV and PSSI typically exhibit a smaller spread (or standard deviation ) in calculated reference offsets, indicating that these programs are slightly more precise than either LACS or CheckShift. Neither LACS nor CheckShift are able to handle proteins that have the extremely large (above 40 ppm) reference offsets, whereas PANAV and PSSI seem to be able to deal with these kinds of anomalous proteins. [ 11 ]
In a recent study, [ 11 ] a chemical shift re-referencing program (PANAV) was run on a total of 2421 BMRB entries that had a sufficient proportion of (>80%) of assigned chemical shifts to perform a robust chemical shift reference correction. A total of 243 entries were found with 13Cα shifts offset by more than 1.0 ppm, 238 entries with 13Cβ shifts offset of more than 1.0 ppm, 200 entries with 13C’ shifts offset of more than 1.0 ppm and 137 entries with 15N shifts offset by more than 1.5 ppm. From this study, 19.7% of the entries in the BMRB appear to be mis-referenced. Evidently, chemical shift referencing continues to be a significant, and as yet unresolved problem for the biomolecular NMR community. [ 5 ] [ 11 ] | https://en.wikipedia.org/wiki/Protein_chemical_shift_re-referencing |
Protein crystallization is the process of formation of a regular array of individual protein molecules stabilized by crystal contacts. If the crystal is sufficiently ordered, it will diffract . Some proteins naturally form crystalline arrays, like aquaporin in the lens of the eye. [ 1 ] [ 2 ]
In the process of protein crystallization, proteins are dissolved in an aqueous environment and sample solution until they reach the supersaturated state . [ 3 ] Different methods are used to reach that state such as vapor diffusion, microbatch, microdialysis, and free-interface diffusion. Developing protein crystals is a difficult process influenced by many factors, including pH, temperature, ionic strength in the crystallization solution, and even gravity. [ 3 ] Once formed, these crystals can be used in structural biology to study the molecular structure of the protein, particularly for various industrial or medical purposes. [ 4 ] [ 5 ]
For over 150 years, scientists from all around the world have known about the crystallization of protein molecules. [ 6 ]
In 1840, Friedrich Ludwig Hünefeld accidentally discovered the formation of crystalline material in samples of earthworm blood held under two glass slides and occasionally observed small plate-like crystals in desiccated swine or human blood samples. These crystals were named as 'haemoglobin', by Felix Hoppe-Seyler in 1864. The seminal findings of Hünefeld inspired many scientists in the future. [ 7 ]
In 1851, Otto Funke described the process of producing human haemoglobin crystals by diluting red blood cells with solvents, such as pure water, alcohol or ether, followed by slow evaporation of the solvent from the protein solution. In 1871, William T. Preyer, Professor at University of Jena, published a book entitled Die Blutkrystalle (The Crystals of Blood), reviewing the features of haemoglobin crystals from around 50 species of mammals, birds, reptiles and fishes. [ 7 ]
In 1909, the physiologist Edward T. Reichert, together with the mineralogist Amos P. Brown, published a treatise on the preparation, physiology and geometrical characterization of haemoglobin crystals from several hundreds animals, including extinct species such as the Tasmanian wolf. [ 7 ] Increasing protein crystals were found.
In 1934, John Desmond Bernal and his student Dorothy Hodgkin discovered that protein crystals surrounded by their mother liquor gave better diffraction patterns than dried crystals. Using pepsin , they were the first to discern the diffraction pattern of a wet, globular protein. Prior to Bernal and Hodgkin, protein crystallography had only been performed in dry conditions with inconsistent and unreliable results. This is the first X‐ray diffraction pattern of a protein crystal. [ 8 ]
In 1958, the structure of myoglobin (a red protein containing heme), determined by X-ray crystallography, was first reported by John Kendrew . [ 9 ] Kendrew shared the 1962 Nobel Prize in Chemistry with Max Perutz for this discovery. [ 4 ]
Now, based on the protein crystals, the structures of them play a significant role in biochemistry and translational medicine.
Protein crystallization is governed by the same physics that governs the formation of inorganic crystals. For crystallization to occur spontaneously, the crystal state must be favored thermodynamically. This is described by the Gibbs free energy (∆G), defined as ∆G = ∆H- T∆S, which captures how the enthalpy change of a process, ∆H, trades off with the corresponding change in entropy , ∆S. [ 10 ] Entropy, roughly, describes the disorder of a system. Highly ordered states, such as protein crystals, are disfavored thermodynamically compared to more disordered states, such as solutions of proteins in solvent, because the transition to a more ordered state would decrease the total entropy of the system (negative ∆S). For crystals to form spontaneously, the ∆G of crystal formation must be negative. In other words, the entropic penalty must be paid by a corresponding decrease in the total energy of the system (∆H). Familiar inorganic crystals such as sodium chloride spontaneously form at ambient conditions because the crystal state decreases the total energy of the system. However, crystallization of some proteins under ambient conditions would both decrease the entropy (negative ∆S) and increase the total energy (positive ∆H) of the system, and thus does not occur spontaneously. To achieve crystallization of such proteins conditions are modified to make crystal formation energetically favorable. This is often accomplished by creation of a supersaturated solution of the sample. [ 3 ]
Crystal formation requires two steps: nucleation and growth . [ 3 ] Nucleation is the initiation step for crystallization. [ 3 ] At the nucleation phase, protein molecules in solution come together as aggregates to form a stable solid nucleus. [ 3 ] As the nucleus forms, the crystal grows bigger and bigger by molecules attaching to this stable nucleus. [ 3 ] The nucleation step is critical for crystal formation since it is the first-order phase transition of samples moving from having a high degree of freedom to obtaining an ordered state (aqueous to solid). [ 3 ] For the nucleation step to succeed, the manipulation of crystallization parameters is essential. The approach behind getting a protein to crystallize is to yield a lower solubility of the targeted protein in solution. [ 3 ] Once the solubility limit is exceeded and crystals are present, crystallization is accomplished. [ 3 ]
Vapor diffusion is the most commonly employed method of protein crystallization. In this method, droplets containing purified protein, buffer , and precipitant are allowed to equilibrate with a larger reservoir containing similar buffers and precipitants in higher concentrations. Initially, the droplet of protein solution contains comparatively low precipitant and protein concentrations, but as the drop and reservoir equilibrate, the precipitant and protein concentrations increase in the drop. If the appropriate crystallization solutions are used for a given protein, crystal growth occurs in the drop. [ 11 ] [ 12 ] This method is used because it allows for gentle and gradual changes in concentration of protein and precipitant concentration, which aid in the growth of large and well-ordered crystals.
Vapor diffusion can be performed in either hanging-drop or sitting-drop format. Hanging-drop apparatus involve a drop of protein solution placed on an inverted cover slip, which is then suspended above the reservoir. Sitting-drop crystallization apparatus place the drop on a pedestal that is separated from the reservoir. Both of these methods require sealing of the environment so that equilibration between the drop and reservoir can occur. [ 11 ] [ 13 ]
A microbatch usually involves immersing a very small volume of protein droplets in oil (as little as 1 μL). The reason that oil is required is because such low volume of protein solution is used and therefore evaporation must be inhibited to carry out the experiment aqueously. Although there are various oils that can be used, the two most common sealing agent are paraffin oils (described by Chayen et al.) and silicon oils (described by D’Arcy). There are also other methods for microbatching that do not use a liquid sealing agent and instead require a scientist to quickly place a film or some tape on a welled plate after placing the drop in the well.
Besides the very limited amounts of sample needed, this method also has as a further advantage that the samples are protected from airborne contamination, as they are never exposed to the air during the experiment.
Microdialysis takes advantage of a semi-permeable membrane , across which small molecules and ions can pass, while proteins and large polymers cannot cross. By establishing a gradient of solute concentration across the membrane and allowing the system to progress toward equilibrium, the system can slowly move toward supersaturation, at which point protein crystals may form.
Microdialysis can produce crystals by salting out , employing high concentrations of salt or other small membrane-permeable compounds that decrease the solubility of the protein. Very occasionally, some proteins can be crystallized by dialysis salting in, by dialyzing against pure water, removing solutes, driving self-association and crystallization.
This technique brings together protein and precipitation solutions without premixing them, but instead, injecting them through either sides of a channel, allowing equilibrium through diffusion. The two solutions come into contact in a reagent chamber, both at their maximum concentrations, initiating spontaneous nucleation. As the system comes into equilibrium, the level of supersaturation decreases, favouring crystal growth. [ 14 ]
The basic driving force for protein crystallization is to optimize the number of bonds one can form with another protein through intermolecular interactions. [ 3 ] These interactions depend on electron densities of molecules and the protein side chains that change as a function of pH . [ 10 ] The tertiary and quaternary structure of proteins are determined by intermolecular interactions between the amino acids’ side groups, in which the hydrophilic groups are usually facing outwards to the solution to form a hydration shell to the solvent (water). [ 10 ] As the pH changes, the charge on these polar side group also change with respect to the solution pH and the protein's pKa . Hence, the choice of pH is essential either to promote the formation of crystals where the bonding between molecules to each other is more favorable than with water molecules. [ 10 ] pH is one of the most powerful manipulations that one can assign for the optimal crystallization condition.
Temperature is another interesting parameter to discuss since protein solubility is a function of temperature. [ 15 ] In protein crystallization, manipulation of temperature to yield successful crystals is one common strategy. Unlike pH, temperature of different components of the crystallography experiments could impact the final results such as temperature of buffer preparation, [ 16 ] temperature of the actual crystallization experiment, etc.
Chemical additives are small chemical compounds that are added to the crystallization process to increase the yield of crystals. [ 17 ] The role of small molecules in protein crystallization had not been well thought of in the early days since they were thought of as contaminants in most case. [ 17 ] Smaller molecules crystallize better than macromolecules such as proteins, therefore, the use of chemical additives had been limited prior to the study by McPherson. However, this is a powerful aspect of the experimental parameters for crystallization that is important for biochemists and crystallographers to further investigate and apply. [ 17 ]
High through-put methods exist to help streamline the large number of experiments required to explore the various conditions that are necessary for successful crystal growth. There are numerous commercial kits available for order which apply preassembled ingredients in systems guaranteed to produce successful crystallization. Using such a kit, a scientist avoids the hassle of purifying a protein and determining the appropriate crystallization conditions. [ 18 ]
Liquid-handling robots can be used to set up and automate large number of crystallization experiments simultaneously. What would otherwise be slow and potentially error-prone process carried out by a human can be accomplished efficiently and accurately with an automated system. Robotic crystallization systems use the same components described above, but carry out each step of the procedure quickly and with a large number of replicates. Each experiment utilizes tiny amounts of solution, and the advantage of the smaller size is two-fold: the smaller sample sizes not only cut-down on expenditure of purified protein, but smaller amounts of solution lead to quicker crystallizations. Each experiment is monitored by a camera which detects crystal growth. [ 12 ]
Proteins can be engineered to improve the chance of successful protein crystallization by using techniques like Surface Entropy Reduction [ 19 ] or engineering in crystal contacts. [ 20 ] Frequently, problematic cysteine residues can be replaced by alanine to avoid disulfide -mediated aggregation, and residues such as lysine, glutamate, and glutamine can be changed to alanine to reduce intrinsic protein flexibility, which can hinder crystallization..
Macromolecular structures can be determined from protein crystal using a variety of methods, including X-ray diffraction / X-ray crystallography , cryogenic electron microscopy (CryoEM) (including electron crystallography and microcrystal electron diffraction (MicroED) ), small-angle X-ray scattering , and neutron diffraction . See also Structural biology .
Crystallization of proteins can also be useful in the formulation of proteins for pharmaceutical purposes. [ 21 ] | https://en.wikipedia.org/wiki/Protein_crystallization |
Protein engineering is the process of developing useful or valuable proteins through the design and production of unnatural polypeptides , often by altering amino acid sequences found in nature. [ 1 ] It is a young discipline, with much research taking place into the understanding of protein folding and recognition for protein design principles. It has been used to improve the function of many enzymes for industrial catalysis. [ 2 ] It is also a product and services market, with an estimated value of $168 billion by 2017. [ 3 ]
There are two general strategies for protein engineering: rational protein design and directed evolution . These methods are not mutually exclusive; researchers will often apply both. In the future, more detailed knowledge of protein structure and function , and advances in high-throughput screening , may greatly expand the abilities of protein engineering. Eventually, even unnatural amino acids may be included, via newer methods, such as expanded genetic code , that allow encoding novel amino acids in genetic code.
The applications in numerous fields, including medicine and industrial bioprocessing, are vast and numerous.
In rational protein design, a scientist uses detailed knowledge of the structure and function of a protein to make desired changes. In general, this has the advantage of being inexpensive and technically easy, since site-directed mutagenesis methods are well-developed. However, its major drawback is that detailed structural knowledge of a protein is often unavailable, and, even when available, it can be very difficult to predict the effects of various mutations since structural information most often provide a static picture of a protein structure. However, programs such as Folding@home and Foldit have utilized crowdsourcing techniques in order to gain insight into the folding motifs of proteins. [ 4 ]
Computational protein design algorithms seek to identify novel amino acid sequences that are low in energy when folded to the pre-specified target structure. While the sequence-conformation space that needs to be searched is large, the most challenging requirement for computational protein design is a fast, yet accurate, energy function that can distinguish optimal sequences from similar suboptimal ones.
Without structural information about a protein, sequence analysis is often useful in elucidating information about the protein. These techniques involve alignment of target protein sequences with other related protein sequences. This alignment can show which amino acids are conserved between species and are important for the function of the protein. These analyses can help to identify hot spot amino acids that can serve as the target sites for mutations. Multiple sequence alignment utilizes data bases such as PREFAB, SABMARK, OXBENCH, IRMBASE, and BALIBASE in order to cross reference target protein sequences with known sequences. Multiple sequence alignment techniques are listed below. [ 5 ] [ page needed ]
This method begins by performing pair wise alignment of sequences using k-tuple or Needleman–Wunsch methods. These methods calculate a matrix that depicts the pair wise similarity among the sequence pairs. Similarity scores are then transformed into distance scores that are used to produce a guide tree using the neighbor joining method. This guide tree is then employed to yield a multiple sequence alignment. [ 5 ] [ page needed ]
This method is capable of aligning up to 190,000 sequences by utilizing the k-tuple method. Next sequences are clustered using the mBed and k -means methods. A guide tree is then constructed using the UPGMA method that is used by the HH align package. This guide tree is used to generate multiple sequence alignments. [ 5 ] [ page needed ]
This method utilizes fast Fourier transform (FFT) that converts amino acid sequences into a sequence composed of volume and polarity values for each amino acid residue. This new sequence is used to find homologous regions. [ 5 ] [ page needed ]
This method utilizes the Wu-Manber approximate string matching algorithm to generate multiple sequence alignments. [ 5 ] [ page needed ]
This method utilizes Kmer and Kimura distances to generate multiple sequence alignments. [ 5 ] [ page needed ]
This method utilizes tree based consistency objective functions for alignment evolution. This method has been shown to be 5–10% more accurate than Clustal W. [ 5 ] [ page needed ]
Coevolutionary analysis is also known as correlated mutation, covariation, or co-substitution. This type of rational design involves reciprocal evolutionary changes at evolutionarily interacting loci. Generally this method begins with the generation of a curated multiple sequence alignments for the target sequence. This alignment is then subjected to manual refinement that involves removal of highly gapped sequences, as well as sequences with low sequence identity. This step increases the quality of the alignment. Next, the manually processed alignment is utilized for further coevolutionary measurements using distinct correlated mutation algorithms. These algorithms result in a coevolution scoring matrix. This matrix is filtered by applying various significance tests to extract significant coevolution values and wipe out background noise. Coevolutionary measurements are further evaluated to assess their performance and stringency. Finally, the results from this coevolutionary analysis are validated experimentally. [ 5 ] [ page needed ]
De novo generation of protein benefits from knowledge of existing protein structures. This knowledge of existing protein structure assists with the prediction of new protein structures. Methods for protein structure prediction fall under one of the four following classes: ab initio, fragment based methods, homology modeling, and protein threading. [ 5 ] [ page needed ]
These methods involve free modeling without using any structural information about the template. Ab initio methods are aimed at prediction of the native structures of proteins corresponding to the global minimum of its free energy. some examples of ab initio methods are AMBER, GROMOS, GROMACS, CHARMM, OPLS, and ENCEPP12. General steps for ab initio methods begin with the geometric representation of the protein of interest. Next, a potential energy function model for the protein is developed. This model can be created using either molecular mechanics potentials or protein structure derived potential functions. Following the development of a potential model, energy search techniques including molecular dynamic simulations, Monte Carlo simulations and genetic algorithms are applied to the protein. [ 5 ] [ page needed ]
These methods use database information regarding structures to match homologous structures to the created protein sequences. These homologous structures are assembled to give compact structures using scoring and optimization procedures, with the goal of achieving the lowest potential energy score. Webservers for fragment information are I-TASSER, ROSETTA, ROSETTA @ home, FRAGFOLD, CABS fold, PROFESY, CREF, QUARK, UNDERTAKER, HMM, and ANGLOR. [ 5 ] : 72
These methods are based upon the homology of proteins. These methods are also known as comparative modeling. The first step in homology modeling is generally the identification of template sequences of known structure which are homologous to the query sequence. Next the query sequence is aligned to the template sequence. Following the alignment, the structurally conserved regions are modeled using the template structure. This is followed by the modeling of side chains and loops that are distinct from the template. Finally the modeled structure undergoes refinement and assessment of quality. Servers that are available for homology modeling data are listed here: SWISS MODEL, MODELLER, ReformAlign, PyMOD, TIP-STRUCTFAST, COMPASS, 3d-PSSM, SAMT02, SAMT99, HHPRED, FAGUE, 3D-JIGSAW, META-PP, ROSETTA, and I-TASSER. [ 5 ] [ page needed ]
Protein threading can be used when a reliable homologue for the query sequence cannot be found. This method begins by obtaining a query sequence and a library of template structures. Next, the query sequence is threaded over known template structures. These candidate models are scored using scoring functions. These are scored based upon potential energy models of both query and template sequence. The match with the lowest potential energy model is then selected. Methods and servers for retrieving threading data and performing calculations are listed here: GenTHREADER, pGenTHREADER, pDomTHREADER, ORFEUS, PROSPECT, BioShell-Threading, FFASO3, RaptorX, HHPred, LOOPP server, Sparks-X, SEGMER, THREADER2, ESYPRED3D, LIBRA, TOPITS, RAPTOR, COTH, MUSTER. [ 5 ] [ page needed ]
For more information on rational design see site-directed mutagenesis .
Multivalent binding can be used to increase the binding specificity and affinity through avidity effects. Having multiple binding domains in a single biomolecule or complex increases the likelihood of other interactions to occur via individual binding events. Avidity or effective affinity can be much higher than the sum of the individual affinities providing a cost and time-effective tool for targeted binding. [ 6 ]
Multivalent proteins are relatively easy to produce by post-translational modifications or multiplying the protein-coding DNA sequence. The main advantage of multivalent and multispecific proteins is that they can increase the effective affinity for a target of a known protein. In the case of an inhomogeneous target using a combination of proteins resulting in multispecific binding can increase specificity, which has high applicability in protein therapeutics.
The most common example for multivalent binding are the antibodies, and there is extensive research for bispecific antibodies. Applications of bispecific antibodies cover a broad spectrum that includes diagnosis, imaging, prophylaxis, and therapy. [ 7 ] [ 8 ]
In directed evolution, random mutagenesis , e.g. by error-prone PCR or sequence saturation mutagenesis , is applied to a protein, and a selection regime is used to select variants having desired traits. Further rounds of mutation and selection are then applied. This method mimics natural evolution and, in general, produces superior results to rational design. An added process, termed DNA shuffling , mixes and matches pieces of successful variants to produce better results. Such processes mimic the recombination that occurs naturally during sexual reproduction . Advantages of directed evolution are that it requires no prior structural knowledge of a protein, nor is it necessary to be able to predict what effect a given mutation will have. Indeed, the results of directed evolution experiments are often surprising in that desired changes are often caused by mutations that were not expected to have some effect. The drawback is that they require high-throughput screening , which is not feasible for all proteins. Large amounts of recombinant DNA must be mutated and the products screened for desired traits. The large number of variants often requires expensive robotic equipment to automate the process. Further, not all desired activities can be screened for easily.
Natural Darwinian evolution can be effectively imitated in the lab toward tailoring protein properties for diverse applications, including catalysis. Many experimental technologies exist to produce large and diverse protein libraries and for screening or selecting folded, functional variants. Folded proteins arise surprisingly frequently in random sequence space, an occurrence exploitable in evolving selective binders and catalysts. While more conservative than direct selection from deep sequence space, redesign of existing proteins by random mutagenesis and selection/screening is a particularly robust method for optimizing or altering extant properties. It also represents an excellent starting point for achieving more ambitious engineering goals. Allying experimental evolution with modern computational methods is likely the broadest, most fruitful strategy for generating functional macromolecules unknown to nature. [ 9 ]
The main challenges of designing high quality mutant libraries have shown significant progress in the recent past. This progress has been in the form of better descriptions of the effects of mutational loads on protein traits. Also computational approaches have shown large advances in the innumerably large sequence space to more manageable screenable sizes, thus creating smart libraries of mutants. Library size has also been reduced to more screenable sizes by the identification of key beneficial residues using algorithms for systematic recombination. Finally a significant step forward toward efficient reengineering of enzymes has been made with the development of more accurate statistical models and algorithms quantifying and predicting coupled mutational effects on protein functions. [ 10 ]
Generally, directed evolution may be summarized as an iterative two step process which involves generation of protein mutant libraries, and high throughput screening processes to select for variants with improved traits. This technique does not require prior knowledge of the protein structure and function relationship. Directed evolution utilizes random or focused mutagenesis to generate libraries of mutant proteins. Random mutations can be introduced using either error prone PCR, or site saturation mutagenesis. Mutants may also be generated using recombination of multiple homologous genes. Nature has evolved a limited number of beneficial sequences. Directed evolution makes it possible to identify undiscovered protein sequences which have novel functions. This ability is contingent on the proteins ability to tolerant amino acid residue substitutions without compromising folding or stability. [ 5 ] [ page needed ]
Directed evolution methods can be broadly categorized into two strategies, asexual and sexual methods.
Asexual methods do not generate any cross links between parental genes. Single genes are used to create mutant libraries using various mutagenic techniques. These asexual methods can produce either random or focused mutagenesis.
Random mutagenic methods produce mutations at random throughout the gene of interest. Random mutagenesis can introduce the following types of mutations: transitions, transversions, insertions, deletions, inversion, missense, and nonsense. Examples of methods for producing random mutagenesis are below.
Error prone PCR utilizes the fact that Taq DNA polymerase lacks 3' to 5' exonuclease activity. This results in an error rate of 0.001–0.002% per nucleotide per replication. This method begins with choosing the gene, or the area within a gene, one wishes to mutate. Next, the extent of error required is calculated based upon the type and extent of activity one wishes to generate. This extent of error determines the error prone PCR strategy to be employed. Following PCR, the genes are cloned into a plasmid and introduced to competent cell systems. These cells are then screened for desired traits. Plasmids are then isolated for colonies which show improved traits, and are then used as templates the next round of mutagenesis. Error prone PCR shows biases for certain mutations relative to others. Such as biases for transitions over transversions. [ 5 ] [ page needed ]
Rates of error in PCR can be increased in the following ways: [ 5 ] [ page needed ]
Also see polymerase chain reaction for more information.
This PCR method is based upon rolling circle amplification, which is modeled from the method that bacteria use to amplify circular DNA. This method results in linear DNA duplexes. These fragments contain tandem repeats of circular DNA called concatamers, which can be transformed into bacterial strains. Mutations are introduced by first cloning the target sequence into an appropriate plasmid. Next, the amplification process begins using random hexamer primers and Φ29 DNA polymerase under error prone rolling circle amplification conditions. Additional conditions to produce error prone rolling circle amplification are 1.5 pM of template DNA, 1.5 mM MnCl 2 and a 24 hour reaction time. MnCl 2 is added into the reaction mixture to promote random point mutations in the DNA strands. Mutation rates can be increased by increasing the concentration of MnCl 2 , or by decreasing concentration of the template DNA. Error prone rolling circle amplification is advantageous relative to error prone PCR because of its use of universal random hexamer primers, rather than specific primers. Also the reaction products of this amplification do not need to be treated with ligases or endonucleases. This reaction is isothermal. [ 5 ] [ page needed ]
Chemical mutagenesis involves the use of chemical agents to introduce mutations into genetic sequences. Examples of chemical mutagens follow.
Sodium bisulfate is effective at mutating G/C rich genomic sequences. This is because sodium bisulfate catalyses deamination of unmethylated cytosine to uracil. [ 5 ] [ page needed ]
Ethyl methane sulfonate alkylates guanidine residues. This alteration causes errors during DNA replication. [ 5 ] [ page needed ]
Nitrous acid causes transversion by de-amination of adenine and cytosine. [ 5 ] [ page needed ]
The dual approach to random chemical mutagenesis is an iterative two step process. First it involves the in vivo chemical mutagenesis of the gene of interest via EMS. Next, the treated gene is isolated and cloning into an untreated expression vector in order to prevent mutations in the plasmid backbone. [ 5 ] [ page needed ] This technique preserves the plasmids genetic properties. [ 5 ] [ page needed ]
This method has been used to create targeted in vivo mutagenesis in yeast. This method involves the fusion of a 3-methyladenine DNA glycosylase to tetR DNA-binding domain. This has been shown to increase mutation rates by over 800 time in regions of the genome containing tetO sites. [ 5 ] [ page needed ]
This method involves alteration in length of the sequence via simultaneous deletion and insertion of chunks of bases of arbitrary length. This method has been shown to produce proteins with new functionalities via introduction of new restriction sites, specific codons, four base codons for non-natural amino acids. [ 5 ] [ page needed ]
Recently many methods for transposon based random mutagenesis have been reported. This methods include, but are not limited to the following: PERMUTE-random circular permutation, random protein truncation, random nucleotide triplet substitution, random domain/tag/multiple amino acid insertion, codon scanning mutagenesis, and multicodon scanning mutagenesis. These aforementioned techniques all require the design of mini-Mu transposons. Thermo scientific manufactures kits for the design of these transposons. [ 5 ] [ page needed ]
These methods involve altering gene length via insertion and deletion mutations. An example is the tandem repeat insertion (TRINS) method. This technique results in the generation of tandem repeats of random fragments of the target gene via rolling circle amplification and concurrent incorporation of these repeats into the target gene. [ 5 ] [ page needed ]
Mutator strains are bacterial cell lines which are deficient in one or more DNA repair mechanisms. An example of a mutator strand is the E. coli XL1-RED. [ 5 ] [ page needed ] This subordinate strain of E. coli is deficient in the MutS, MutD, MutT DNA repair pathways. Use of mutator strains is useful at introducing many types of mutation; however, these strains show progressive sickness of culture because of the accumulation of mutations in the strains own genome. [ 5 ] [ page needed ]
Focused mutagenic methods produce mutations at predetermined amino acid residues. These techniques require and understanding of the sequence-function relationship for the protein of interest. Understanding of this relationship allows for the identification of residues which are important in stability, stereoselectivity, and catalytic efficiency. [ 5 ] [ page needed ] Examples of methods that produce focused mutagenesis are below.
Site saturation mutagenesis is a PCR based method used to target amino acids with significant roles in protein function. The two most common techniques for performing this are whole plasmid single PCR, and overlap extension PCR.
Whole plasmid single PCR is also referred to as site directed mutagenesis (SDM). SDM products are subjected to Dpn endonuclease digestion. This digestion results in cleavage of only the parental strand, because the parental strand contains a GmATC which is methylated at N6 of adenine. SDM does not work well for large plasmids of over ten kilobases. Also, this method is only capable of replacing two nucleotides at a time. [ 5 ] [ page needed ]
Overlap extension PCR requires the use of two pairs of primers. One primer in each set contains a mutation. A first round of PCR using these primer sets is performed and two double stranded DNA duplexes are formed. A second round of PCR is then performed in which these duplexes are denatured and annealed with the primer sets again to produce heteroduplexes, in which each strand has a mutation. Any gaps in these newly formed heteroduplexes are filled with DNA polymerases and further amplified. [ 5 ] [ page needed ]
Sequence saturation mutagenesis results in the randomization of the target sequence at every nucleotide position. This method begins with the generation of variable length DNA fragments tailed with universal bases via the use of template transferases at the 3' termini. Next, these fragments are extended to full length using a single stranded template. The universal bases are replaced with a random standard base, causing mutations. There are several modified versions of this method such as SeSAM-Tv-II, SeSAM-Tv+, and SeSAM-III. [ 5 ] [ page needed ]
This site saturation mutagenesis method involves two separate PCR reaction. The first of which uses only forward primers, while the second reaction uses only reverse primers. This avoids the formation of primer dimer formation. [ 5 ] [ page needed ]
This site saturation mutagenic technique begins with one mutagenic oligonucleotide and one universal flanking primer. These two reactants are used for an initial PCR cycle. Products from this first PCR cycle are used as mega primers for the next PCR. [ 5 ] [ page needed ]
This site saturation mutagenic method is based on overlap extension PCR. It is used to introduce mutations at any site in a circular plasmid. [ 5 ] [ page needed ]
This method utilizes user defined site directed mutagenesis at single or multiple sites simultaneously. OSCARR is an acronym for one pot simple methodology for cassette randomization and recombination . This randomization and recombination results in randomization of desired fragments of a protein. Omnichange is a sequence independent, multisite saturation mutagenesis which can saturate up to five independent codons on a gene.
This method removes redundant codons and stop codons.
This is a PCR based method. Cassette mutagenesis begins with the synthesis of a DNA cassette containing the gene of interest, which is flanked on either side by restriction sites. The endonuclease which cleaves these restriction sites also cleaves sites in the target plasmid. The DNA cassette and the target plasmid are both treated with endonucleases to cleave these restriction sites and create sticky ends. Next the products from this cleavage are ligated together, resulting in the insertion of the gene into the target plasmid. An alternative form of cassette mutagenesis called combinatorial cassette mutagenesis is used to identify the functions of individual amino acid residues in the protein of interest. Recursive ensemble mutagenesis then utilizes information from previous combinatorial cassette mutagenesis. Codon cassette mutagenesis allows you to insert or replace a single codon at a particular site in double stranded DNA. [ 5 ] [ page needed ]
Sexual methods of directed evolution involve in vitro recombination which mimic natural in vivo recombination. Generally these techniques require high sequence homology between parental sequences. These techniques are often used to recombine two different parental genes, and these methods do create cross overs between these genes. [ 5 ] [ page needed ]
Homologous recombination can be categorized as either in vivo or in vitro. In vitro homologous recombination mimics natural in vivo recombination. These in vitro recombination methods require high sequence homology between parental sequences. These techniques exploit the natural diversity in parental genes by recombining them to yield chimeric genes. The resulting chimera show a blend of parental characteristics. [ 5 ] [ page needed ]
This in vitro technique was one of the first techniques in the era of recombination. It begins with the digestion of homologous parental genes into small fragments by DNase1. These small fragments are then purified from undigested parental genes. Purified fragments are then reassembled using primer-less PCR. This PCR involves homologous fragments from different parental genes priming for each other, resulting in chimeric DNA. The chimeric DNA of parental size is then amplified using end terminal primers in regular PCR. [ 5 ] [ page needed ]
This in vitro homologous recombination method begins with the synthesis of many short gene fragments exhibiting point mutations using random sequence primers. These fragments are reassembled to full length parental genes using primer-less PCR. These reassembled sequences are then amplified using PCR and subjected to further selection processes. This method is advantageous relative to DNA shuffling because there is no use of DNase1, thus there is no bias for recombination next to a pyrimidine nucleotide. This method is also advantageous due to its use of synthetic random primers which are uniform in length, and lack biases. Finally this method is independent of the length of DNA template sequence, and requires a small amount of parental DNA. [ 5 ] [ page needed ]
This method generates chimeric genes directly from metagenomic samples. It begins with isolation of the desired gene by functional screening from metagenomic DNA sample. Next, specific primers are designed and used to amplify the homologous genes from different environmental samples. Finally, chimeric libraries are generated to retrieve the desired functional clones by shuffling these amplified homologous genes. [ 5 ] [ page needed ]
This in vitro method is based on template switching to generate chimeric genes. This PCR based method begins with an initial denaturation of the template, followed by annealing of primers and a short extension time. All subsequent cycle generate annealing between the short fragments generated in previous cycles and different parts of the template. These short fragments and the templates anneal together based on sequence complementarity. This process of fragments annealing template DNA is known as template switching. These annealed fragments will then serve as primers for further extension. This method is carried out until the parental length chimeric gene sequence is obtained. Execution of this method only requires flanking primers to begin. There is also no need for Dnase1 enzyme. [ 5 ] [ page needed ]
This method has been shown to generate chimeric gene libraries with an average of 14 crossovers per chimeric gene. It begins by aligning fragments from a parental top strand onto the bottom strand of a uracil containing template from a homologous gene. 5' and 3' overhang flaps are cleaved and gaps are filled by the exonuclease and endonuclease activities of Pfu and taq DNA polymerases. The uracil containing template is then removed from the heteroduplex by treatment with a uracil DNA glcosylase, followed by further amplification using PCR. This method is advantageous because it generates chimeras with relatively high crossover frequency. However it is somewhat limited due to the complexity and the need for generation of single stranded DNA and uracil containing single stranded template DNA. [ 5 ] [ page needed ]
Shuffling of synthetic degenerate oligonucleotides adds flexibility to shuffling methods, since oligonucleotides containing optimal codons and beneficial mutations can be included. [ 5 ] [ page needed ]
Cloning performed in yeast involves PCR dependent reassembly of fragmented expression vectors. These reassembled vectors are then introduced to, and cloned in yeast. Using yeast to clone the vector avoids toxicity and counter-selection that would be introduced by ligation and propagation in E. coli. [ 5 ] [ page needed ]
This method introduces mutations into specific regions of genes while leaving other parts intact by utilizing the high frequency of homologous recombination in yeast. [ 5 ] [ page needed ]
This method utilizes a bacteriophage with a modified life cycle to transfer evolving genes from host to host. The phage's life cycle is designed in such a way that the transfer is correlated with the activity of interest from the enzyme. This method is advantageous because it requires minimal human intervention for the continuous evolution of the gene. [ 5 ]
These methods are based upon the fact that proteins can exhibit similar structural identity while lacking sequence homology.
Exon shuffling is the combination of exons from different proteins by recombination events occurring at introns. Orthologous exon shuffling involves combining exons from orthologous genes from different species. Orthologous domain shuffling involves shuffling of entire protein domains from orthologous genes from different species. Paralogous exon shuffling involves shuffling of exon from different genes from the same species. Paralogous domain shuffling involves shuffling of entire protein domains from paralogous proteins from the same species. Functional homolog shuffling involves shuffling of non-homologous domains which are functional related. All of these processes being with amplification of the desired exons from different genes using chimeric synthetic oligonucleotides. This amplification products are then reassembled into full length genes using primer-less PCR. During these PCR cycles the fragments act as templates and primers. This results in chimeric full length genes, which are then subjected to screening. [ 5 ] [ page needed ]
Fragments of parental genes are created using controlled digestion by exonuclease III. These fragments are blunted using endonuclease, and are ligated to produce hybrid genes. THIOITCHY is a modified ITCHY technique which utilized nucleotide triphosphate analogs such as α-phosphothioate dNTPs. Incorporation of these nucleotides blocks digestion by exonuclease III. This inhibition of digestion by exonuclease III is called spiking. Spiking can be accomplished by first truncating genes with exonuclease to create fragments with short single stranded overhangs. These fragments then serve as templates for amplification by DNA polymerase in the presence of small amounts of phosphothioate dNTPs. These resulting fragments are then ligated together to form full length genes. Alternatively the intact parental genes can be amplified by PCR in the presence of normal dNTPs and phosphothioate dNTPs. These full length amplification products are then subjected to digestion by an exonuclease. Digestion will continue until the exonuclease encounters an α-pdNTP, resulting in fragments of different length. These fragments are then ligated together to generate chimeric genes. [ 5 ] [ page needed ]
This method generates libraries of hybrid genes inhibiting multiple crossovers by combining DNA shuffling and ITCHY. This method begins with the construction of two independent ITCHY libraries. The first with gene A on the N-terminus. And the other having gene B on the N-terminus. These hybrid gene fragments are separated using either restriction enzyme digestion or PCR with terminus primers via agarose gel electrophoresis. These isolated fragments are then mixed together and further digested using DNase1. Digested fragments are then reassembled by primerless PCR with template switching. [ 5 ] [ page needed ]
This method generates libraries of hybrid genes by template switching of uni-directionally growing polynucleotides in the presence of single stranded DNA fragments as templates for chimeras. This method begins with the preparation of single stranded DNA fragments by reverse transcription from target mRNA. Gene specific primers are then annealed to the single stranded DNA. These genes are then extended during a PCR cycle. This cycle is followed by template switching and annealing of the short fragments obtained from the earlier primer extension to other single stranded DNA fragments. This process is repeated until full length single stranded DNA is obtained. [ 5 ] [ page needed ]
This method generates recombination between genes with little to no sequence homology. These chimeras are fused via a linker sequence containing several restriction sites. This construct is then digested using DNase1. Fragments are made are made blunt ended using S1 nuclease. These blunt end fragments are put together into a circular sequence by ligation. This circular construct is then linearized using restriction enzymes for which the restriction sites are present in the linker region. This results in a library of chimeric genes in which contribution of genes to 5' and 3' end will be reversed as compared to the starting construct. [ 5 ] [ page needed ]
This method results in a library of genes with multiple crossovers from several parental genes. This method does not require sequence identity among the parental genes. This does require one or two conserved amino acids at every crossover position. It begins with alignment of parental sequences and identification of consensus regions which serve as crossover sites. This is followed by the incorporation of specific tags containing restriction sites followed by the removal of the tags by digestion with Bac1, resulting in genes with cohesive ends. These gene fragments are mixed and ligated in an appropriate order to form chimeric libraries. [ 5 ] [ page needed ]
This method begins with alignment of homologous genes, followed by identification of regions of polymorphism. Next the top strand of the gene is divided into small degenerate oligonucleotides. The bottom strand is also digested into oligonucleotides to serve as scaffolds. These fragments are combined in solution are top strand oligonucleotides are assembled onto bottom strand oligonucleotides. Gaps between these fragments are filled with polymerase and ligated. [ 5 ] [ page needed ]
This method involves the shuffling of plural DNA fragments without homology, in a single PCR. This results in the reconstruction of complete proteins by assembly of modules encoding different structural units. [ 5 ] [ page needed ]
This method begins with the amplification of gene fragments which need to be recombined, using uracil dNTPs. This amplification solution also contains primers, PfuTurbo, and Cx Hotstart DNA polymerase. Amplified products are next incubated with USER enzyme. This enzyme catalyzes the removal of uracil residues from DNA creating single base pair gaps. The USER enzyme treated fragments are mixed and ligated using T4 DNA ligase and subjected to Dpn1 digestion to remove the template DNA. These resulting dingle stranded fragments are subjected to amplification using PCR, and are transformed into E. coli. [ 5 ] [ page needed ]
This method allows you to recombine at least 9 different fragments in an acceptor vector by using type 2 restriction enzyme which cuts outside of the restriction sites. It begins with sub cloning of fragments in separate vectors to create Bsa1 flanking sequences on both sides. These vectors are then cleaved using type II restriction enzyme Bsa1, which generates four nucleotide single strand overhangs. Fragments with complementary overhangs are hybridized and ligated using T4 DNA ligase. Finally these constructs are then transformed into E. coli cells, which are screened for expression levels. [ 5 ] [ page needed ]
This method can be used to recombine structural elements or entire protein domains. This method is based on phosphorothioate chemistry which allows the specific cleavage of phosphorothiodiester bonds. The first step in the process begins with amplification of fragments that need to be recombined along with the vector backbone. This amplification is accomplished using primers with phosphorothiolated nucleotides at 5' ends. Amplified PCR products are cleaved in an ethanol-iodine solution at high temperatures. Next these fragments are hybridized at room temperature and transformed into E. coli which repair any nicks. [ 5 ] [ page needed ]
This system is based upon a natural site specific recombination system in E. coli. This system is called the integron system, and produces natural gene shuffling. This method was used to construct and optimize a functional tryptophan biosynthetic operon in trp-deficient E. coli by delivering individual recombination cassettes or trpA-E genes along with regulatory elements with the integron system. [ 5 ] [ page needed ]
This method generates single stranded DNA strands, which encompass a single block sequence either at the 5' or 3' end, complementary sequences in a stem loop region, and a D branch region serving as a primer binding site for PCR. Equivalent amounts of both 5' and 3' half strands are mixed and formed a hybrid due to the complementarity in the stem region. Hybrids with free phosphorylated 5' end in 3' half strands are then ligated with free 3' ends in 5' half strands using T4 DNA ligase in the presence of 0.1 mM ATP. Ligated products are then amplified by two types of PCR to generate pre 5' half and pre 3' half PCR products. These PCR product are converted to single strands via avidin-biotin binding to the 5' end of the primes containing stem sequences that were biotin labeled. Next, biotinylated 5' half strands and non-biotinylated 3' half strands are used as 5' and 3' half strands for the next Y-ligation cycle. [ 5 ] [ page needed ]
Semi-rational design uses information about a proteins sequence, structure and function, in tandem with predictive algorithms. Together these are used to identify target amino acid residues which are most likely to influence protein function. Mutations of these key amino acid residues create libraries of mutant proteins that are more likely to have enhanced properties. [ 11 ]
Advances in semi-rational enzyme engineering and de novo enzyme design provide researchers with powerful and effective new strategies to manipulate biocatalysts. Integration of sequence and structure based approaches in library design has proven to be a great guide for enzyme redesign. Generally, current computational de novo and redesign methods do not compare to evolved variants in catalytic performance. Although experimental optimization may be produced using directed evolution, further improvements in the accuracy of structure predictions and greater catalytic ability will be achieved with improvements in design algorithms. Further functional enhancements may be included in future simulations by integrating protein dynamics. [ 11 ]
Biochemical and biophysical studies, along with fine-tuning of predictive frameworks will be useful to experimentally evaluate the functional significance of individual design features. Better understanding of these functional contributions will then give feedback for the improvement of future designs. [ 11 ]
Directed evolution will likely not be replaced as the method of choice for protein engineering, although computational protein design has fundamentally changed the way protein engineering can manipulate bio-macromolecules. Smaller, more focused and functionally-rich libraries may be generated by using in methods which incorporate predictive frameworks for hypothesis-driven protein engineering. New design strategies and technical advances have begun a departure from traditional protocols, such as directed evolution, which represents the most effective strategy for identifying top-performing candidates in focused libraries. Whole-gene library synthesis is replacing shuffling and mutagenesis protocols for library preparation. Also highly specific low throughput screening assays are increasingly applied in place of monumental screening and selection efforts of millions of candidates. Together, these developments are poised to take protein engineering beyond directed evolution and towards practical, more efficient strategies for tailoring biocatalysts. [ 11 ]
Once a protein has undergone directed evolution, ration design or semi-ration design, the libraries of mutant proteins must be screened to determine which mutants show enhanced properties. Phage display methods are one option for screening proteins. This method involves the fusion of genes encoding the variant polypeptides with phage coat protein genes. Protein variants expressed on phage surfaces are selected by binding with immobilized targets in vitro. Phages with selected protein variants are then amplified in bacteria, followed by the identification of positive clones by enzyme linked immunosorbent assay. These selected phages are then subjected to DNA sequencing. [ 5 ] [ page needed ]
Cell surface display systems can also be utilized to screen mutant polypeptide libraries. The library mutant genes are incorporated into expression vectors which are then transformed into appropriate host cells. These host cells are subjected to further high throughput screening methods to identify the cells with desired phenotypes. [ 5 ] [ page needed ]
Cell free display systems have been developed to exploit in vitro protein translation or cell free translation. These methods include mRNA display, ribosome display, covalent and non covalent DNA display, and in vitro compartmentalization. [ 5 ] : 53
Enzyme engineering is the application of modifying an enzyme's structure (and, thus, its function) or modifying the catalytic activity of isolated enzymes to produce new metabolites, to allow new (catalyzed) pathways for reactions to occur, [ 12 ] or to convert from certain compounds into others ( biotransformation ). These products are useful as chemicals, pharmaceuticals, fuel, food, or agricultural additives.
An enzyme reactor [ 13 ] consists of a vessel containing a reactional medium that is used to perform a desired conversion by enzymatic means. Enzymes used in this process are free in the solution. Also Microorganisms are one of important origin for genuine enzymes . [ 14 ]
Computing methods have been used to design a protein with a novel fold, such as Top7 , [ 15 ] and sensors for unnatural molecules. [ 16 ] The engineering of fusion proteins has yielded rilonacept , a pharmaceutical that has secured Food and Drug Administration (FDA) approval for treating cryopyrin-associated periodic syndrome .
Another computing method, IPRO, successfully engineered the switching of cofactor specificity of Candida boidinii xylose reductase. [ 17 ] Iterative Protein Redesign and Optimization (IPRO) redesigns proteins to increase or give specificity to native or novel substrates and cofactors . This is done by repeatedly randomly perturbing the structure of the proteins around specified design positions, identifying the lowest energy combination of rotamers , and determining whether the new design has a lower binding energy than prior ones. The iterative nature of this process allows IPRO to make additive mutations to a protein sequence that collectively improve the specificity toward desired substrates and/or cofactors. [ 17 ]
Computation-aided design has also been used to engineer complex properties of a highly ordered nano-protein assembly. [ 18 ] A protein cage, E. coli bacterioferritin (EcBfr), which naturally shows structural instability and an incomplete self-assembly behavior by populating two oligomerization states, is the model protein in this study. Through computational analysis and comparison to its homologs , it has been found that this protein has a smaller-than-average dimeric interface on its two-fold symmetry axis due mainly to the existence of an interfacial water pocket centered on two water-bridged asparagine residues. To investigate the possibility of engineering EcBfr for modified structural stability, a semi-empirical computational method is used to virtually explore the energy differences of the 480 possible mutants at the dimeric interface relative to the wild type EcBfr. This computational study also converges on the water-bridged asparagines . Replacing these two asparagines with hydrophobic amino acids results in proteins that fold into alpha-helical monomers and assemble into cages as evidenced by circular dichroism and transmission electron microscopy. Both thermal and chemical denaturation confirm that, all redesigned proteins, in agreement with the calculations, possess increased stability. One of the three mutations shifts the population in favor of the higher order oligomerization state in solution as shown by both size exclusion chromatography and native gel electrophoresis. [ 18 ]
A in silico method, PoreDesigner, [ 19 ] was developed to redesign bacterial channel protein (OmpF) to reduce its 1 nm pore size to any desired sub-nm dimension. Transport experiments on the narrowest designed pores revealed complete salt rejection when assembled in biomimetic block-polymer matrices. | https://en.wikipedia.org/wiki/Protein_engineering |
A protein family is a group of evolutionarily related proteins . In many cases, a protein family has a corresponding gene family , in which each gene encodes a corresponding protein with a 1:1 relationship. The term "protein family" should not be confused with family as it is used in taxonomy.
Proteins in a family descend from a common ancestor and typically have similar three-dimensional structures , functions, and significant sequence similarity . [ 1 ] [ 2 ] Sequence similarity (usually amino-acid sequence) is one of the most common indicators of homology, or common evolutionary ancestry. [ 3 ] [ 4 ] Some frameworks for evaluating the significance of similarity between sequences use sequence alignment methods. Proteins that do not share a common ancestor are unlikely to show statistically significant sequence similarity, making sequence alignment a powerful tool for identifying the members of protein families. [ 3 ] [ 4 ] Families are sometimes grouped together into larger clades called superfamilies based on structural similarity, even if there is no identifiable sequence homology.
Currently, over 60,000 protein families have been defined, [ 5 ] although ambiguity in the definition of "protein family" leads different researchers to highly varying numbers.
The term protein family has broad usage and can be applied to large groups of proteins with barely detectable sequence similarity as well as narrow groups of proteins with near identical sequence, function, and structure. To distinguish between these cases, a hierarchical terminology is in use. At the highest level of classification are protein superfamilies , which group distantly related proteins, often based on their structural similarity. [ 6 ] [ 7 ] [ 8 ] [ 9 ] Next are protein families, which refer to proteins with a shared evolutionary origin exhibited by significant sequence similarity . [ 2 ] [ 10 ] Subfamilies can be defined within families to denote closely related proteins that have similar or identical functions. [ 11 ] For example, a superfamily like the PA clan of proteases has less sequence conservation than the C04 family within it.
Protein families were first recognised when most proteins that were structurally understood were small, single-domain proteins such as myoglobin , hemoglobin , and cytochrome c . Since then, many proteins have been found with multiple independent structural and functional units called domains . Due to evolutionary shuffling, different domains in a protein have evolved independently. This has led to a focus on families of protein domains. Several online resources are devoted to identifying and cataloging these domains. [ 12 ] [ 13 ]
Different regions of a protein have differing functional constraints. For example, the active site of an enzyme requires certain amino-acid residues to be precisely oriented. A protein–protein binding interface may consist of a large surface with constraints on the hydrophobicity or polarity of the amino-acid residues. Functionally constrained regions of proteins evolve more slowly than unconstrained regions such as surface loops, giving rise to blocks of conserved sequence when the sequences of a protein family are compared (see multiple sequence alignment ). These blocks are most commonly referred to as motifs, although many other terms are used (blocks, signatures, fingerprints, etc.). Several online resources are devoted to identifying and cataloging protein motifs. [ 14 ]
According to current consensus, protein families arise in two ways. First, the separation of a parent species into two genetically isolated descendant species allows a gene/protein to independently accumulate variations ( mutations ) in these two lineages. This results in a family of orthologous proteins, usually with conserved sequence motifs. Second, a gene duplication may create a second copy of a gene (termed a paralog ). Because the original gene is still able to perform its function, the duplicated gene is free to diverge and may acquire new functions (by random mutation).
Certain gene/protein families, especially in eukaryotes , undergo extreme expansions and contractions in the course of evolution, sometimes in concert with whole genome duplications . Expansions are less likely, and losses more likely, for intrinsically disordered proteins and for protein domains whose hydrophobic amino acids are further from the optimal degree of dispersion along the primary sequence. [ 15 ] This expansion and contraction of protein families is one of the salient features of genome evolution , but its importance and ramifications are currently unclear.
As the total number of sequenced proteins increases and interest expands in proteome analysis, an effort is ongoing to organize proteins into families and to describe their component domains and motifs. Reliable identification of protein families is critical to phylogenetic analysis, functional annotation, and the exploration of the diversity of protein function in a given phylogenetic branch. The Enzyme Function Initiative uses protein families and superfamilies as the basis for development of a sequence/structure-based strategy for large scale functional assignment of enzymes of unknown function. [ 16 ] The algorithmic means for establishing protein families on a large scale are based on a notion of similarity.
Many biological databases catalog protein families and allow users to match query sequences to known families. These include:
Similarly, many database-searching algorithms exist, for example: | https://en.wikipedia.org/wiki/Protein_family |
In electrochemistry , protein film voltammetry (or protein film electrochemistry , or direct electrochemistry of proteins ) is a technique for examining the behavior of proteins immobilized (either adsorbed or covalently attached) on an electrode . The technique is applicable to proteins and enzymes that engage in electron transfer reactions and it is part of the methods available to study enzyme kinetics . [ citation needed ]
Provided that it makes suitable contact with the electrode surface (electron transfer between the electrode and the protein is direct) and provided that it is not denatured , the protein can be fruitfully interrogated by monitoring current as a function of electrode potential and other experimental parameters.
Various electrode materials can be used. [ 1 ] Special electrode designs are required to address membrane-bound proteins. [ 2 ]
Small redox proteins such as cytochromes and ferredoxins can be investigated on condition that their electroactive coverage (the amount of protein undergoing direct electron transfer) is large enough (in practice, greater than a fraction of pmol/cm 2 ).
Electrochemical data obtained with small proteins can be used to measure the redox potentials of the protein's redox sites, [ 3 ] the rate of electron transfer between the protein and the electrode, [ 4 ] or the rates of chemical reactions (such as protonations) that are coupled to electron transfer. [ 5 ]
In a cyclic voltammetry experiment carried out with an adsorbed redox protein, the oxidation and reduction of each redox site shows as a pair of positive and negative peaks. Since all the sample is oxidised or reduced during the potential sweep, the peak current and peak area should be proportional to scan rate (observing that the peak current is proportional to scan rate proves that the redox species that gives the peak is actually immobilised). [ 3 ] The same is true for experiments performed with non-biological redox molecules adsorbed onto electrodes. The theory was mainly developed by the French electrochemist Etienne Laviron in the 1980s [ 4 ] , [ 6 ] , . [ 7 ]
Since both this faradaic current (which results from the oxidation/reduction of the adsorbed molecule) and the capacitive current (which results from electrode charging) increase in proportion to scan rate, the peaks should remain visible when the scan rate is increased. In contrast, when the redox analyte is in solution and diffuses to/from the electrode, the peak current is proportional to the square root of the scan rate (see: Randles–Sevcik equation ).
Irrespective of scan rate, the area under the peak (in units of AV) is equal to n F A Γ ν {\displaystyle nFA\Gamma \nu } , where n {\displaystyle n} is the number of electrons exchanged in the oxidation/reduction of the center, A {\displaystyle A} is the electrode surface and Γ {\displaystyle \Gamma } is the electroactive coverage (in units of mol/cm 2 ). [ 3 ] The latter can therefore be deduced from the area under the peak after subtraction of the capacitive current.
At slow scan rates there should be no separation between the oxidative and reductive peaks.
If the reaction is a simple electron transfer reaction, the peaks should remain symmetrical at fast scan rates. A peak separation is observed when the scan rate ν ≫ R T k 0 / F {\displaystyle \nu \gg RTk^{0}/F} , where k 0 {\displaystyle k_{0}} is the exchange electron transfer rate constant in Butler Volmer theory . Laviron equation [ 4 ] , [ 8 ] , [ 9 ] predicts that at fast scan rates, the peaks separate in proportion to log ( ν / k 0 ) {\displaystyle \log(\nu /k^{0})} . The larger ν {\displaystyle \nu } or the smaller k 0 {\displaystyle k^{0}} , the larger the peak separation. The peak potentials are E p = E 0 ± R T α F ln α F ν k 0 R T {\displaystyle E_{p}=E^{0}\pm {\frac {RT}{\alpha F}}\ln {\frac {\alpha F\nu }{k^{0}RT}}} , [ 4 ] as shown by lines in fig 2B ( α {\displaystyle \alpha } is the charge transfer coefficient ). Examining the experimental change in peak position against scan rate therefore informs on the rate of interfacial electron transfer k 0 {\displaystyle k^{0}} .
Coupled reactions are reactions whose rate or equilibrium constant is not the same for the oxidized and reduced forms of the species that is being investigated. For example, reduction should favour protonation ( p K a o x < p K a r e d {\displaystyle pK_{a}^{\rm {ox}}<pK_{a}^{\rm {red}}} ): the protonation reaction is coupled to the reduction at p K a o x < p H < p K a r e d {\displaystyle pK_{a}^{\rm {ox}}<pH<pK_{a}^{\rm {red}}} . The binding of a small molecule (other than the proton) may also be coupled to a redox reaction.
Two cases must be considered depending on whether the coupled reaction is slow or fast (meaning that the time scale of the coupled reaction is larger or smaller than the voltammetric time scale [ 10 ] τ = R T / F ν {\displaystyle \tau =RT/F\nu } ).
In studies of enzymes , the current results from the catalytic oxydation or reduction of the enzyme's substrate .
The electroactive coverage of large redox enzymes (such as laccase , hydrogenase etc.) is often too low to detect any signal in the absence of substrate, but the electrochemical signal is amplified by catalysis: indeed, the catalytic current is proportional to turnover rate times electroactive coverage. The effect of varying the electrode potential, the pH or the concentration of substrates and inhibitors etc. can be examined to learn about various steps in the catalytic mechanism. [ 8 ]
For an enzyme immobilised on an electrode, the value of the current at a certain potential equates ± n F A Γ × T O F {\displaystyle \pm nFA\Gamma \times {\rm {TOF}}} , where n {\displaystyle n} is the number of electrons exchanged in the catalytic reaction, A {\displaystyle A} is the electrode surface, Γ {\displaystyle \Gamma } is the electroactive coverage, and TOF is the turnover frequency (or "turnover number") , that is, the number of substrate molecules transformed per second and per molecule of adsorbed enzyme).The latter can be deduced from the absolute value of the current only on condition that A Γ {\displaystyle A\Gamma } is known, which is rarely the case. However, information is obtained by analysing the relative change in current that results from changing the experimental conditions.
The factors that may influence the TOF are (i) the mass transport of substrate towards the electrode where the enzyme is immobilised ( diffusion and convection ), (ii) the rate of electron transfer between the electrode and the enzyme (interfacial electron transfer), and (iii) the "intrinsic" activity of the enzyme, all of which may depend on electrode potential.
The enzyme is often immobilized on a rotating disk working electrode (RDE) that is spun quickly to prevent the depletion of the substrate near the electrode. In that case, mass transport of substrate towards the electrode where the enzyme is adsorbed may not be influential.
Under very oxidising or very reducing conditions, the steady-state catalytic current sometimes tends to a limiting value (a plateau) which (still provided there is no mass transport limitation) relates to the activity of the fully oxidised or fully reduced enzyme, respectively. If interfacial electron transfer is slow and if there is a distribution of electron transfer rates (resulting from a distribution of orientations of the enzymes molecules on the electrode), the current keeps increasing linearly with potential instead of reaching a plateau; in that case the limiting slope is proportional to the turnover rate of the fully oxidised or fully reduced enzyme. [ 8 ]
The change in steady-state current against potential is often complex (e.g. not merely sigmoidal). [ 12 ]
Another level of complexity comes from the existence of slow redox-driven reactions that may change the activity of the enzyme and make the response depart from steady-state. [ 13 ] Here, slow means that the time scale of the (in)activation is similar to the voltammetric time scale [ 10 ] τ = R T / F ν {\displaystyle \tau =RT/F\nu } . If a RDE is used, these slow (in)activations are detected by a hysteresis in the catalytic voltammogram that is not due to mass-transport. The hysteresis may disappear at very fast scan rates (if the inactivation has no time to proceed) or at very slow scan rates (if the (in)activation reaction reaches a steady-state). [ 14 ]
Conventional voltammetry offers a limited picture of the enzyme-electrode interface and on the structure of the species involved in the reaction. Complementing standard electrochemistry with other methods can provide a more complete picture of catalysis. [ 15 ] [ 16 ] [ 17 ] | https://en.wikipedia.org/wiki/Protein_film_voltammetry |
Protein folding is the physical process by which a protein , after synthesis by a ribosome as a linear chain of amino acids , changes from an unstable random coil into a more ordered three-dimensional structure . This structure permits the protein to become biologically functional or active. [ 1 ]
The folding of many proteins begins even during the translation of the polypeptide chain. The amino acids interact with each other to produce a well-defined three-dimensional structure, known as the protein's native state . This structure is determined by the amino-acid sequence or primary structure . [ 2 ]
The correct three-dimensional structure is essential to function, although some parts of functional proteins may remain unfolded , [ 3 ] indicating that protein dynamics are important. Failure to fold into a native structure generally produces inactive proteins, but in some instances, misfolded proteins have modified or toxic functionality. Several neurodegenerative and other diseases are believed to result from the accumulation of amyloid fibrils formed by misfolded proteins, the infectious varieties of which are known as prions . [ 4 ] Many allergies are caused by the incorrect folding of some proteins because the immune system does not produce the antibodies for certain protein structures. [ 5 ]
Denaturation of proteins is a process of transition from a folded to an unfolded state . It happens in cooking , burns , proteinopathies , and other contexts. Residual structure present, if any, in the supposedly unfolded state may form a folding initiation site and guide the subsequent folding reactions. [ 6 ]
The duration of the folding process varies dramatically depending on the protein of interest. When studied outside the cell , the slowest folding proteins require many minutes or hours to fold, primarily due to proline isomerization , and must pass through a number of intermediate states, like checkpoints, before the process is complete. [ 7 ] On the other hand, very small single- domain proteins with lengths of up to a hundred amino acids typically fold in a single step. [ 8 ] Time scales of milliseconds are the norm, and the fastest known protein folding reactions are complete within a few microseconds. [ 9 ] The folding time scale of a protein depends on its size, contact order , and circuit topology . [ 10 ]
Understanding and simulating the protein folding process has been an important challenge for computational biology since the late 1960s.
The primary structure of a protein, its linear amino-acid sequence, determines its native conformation. [ 11 ] The specific amino acid residues and their position in the polypeptide chain are the determining factors for which portions of the protein fold closely together and form its three-dimensional conformation. The amino acid composition is not as important as the sequence. [ 12 ] The essential fact of folding, however, remains that the amino acid sequence of each protein contains the information that specifies both the native structure and the pathway to attain that state. This is not to say that nearly identical amino acid sequences always fold similarly. [ 13 ] Conformations differ based on environmental factors as well; similar proteins fold differently based on where they are found.
Formation of a secondary structure is the first step in the folding process that a protein takes to assume its native structure. Characteristic of secondary structure are the structures known as alpha helices and beta sheets that fold rapidly because they are stabilized by intramolecular hydrogen bonds , as was first characterized by Linus Pauling . Formation of intramolecular hydrogen bonds provides another important contribution to protein stability. [ 14 ] α-helices are formed by hydrogen bonding of the backbone to form a spiral shape (refer to figure on the right). [ 12 ] The β pleated sheet is a structure that forms with the backbone bending over itself to form the hydrogen bonds (as displayed in the figure to the left). The hydrogen bonds are between the amide hydrogen and carbonyl oxygen of the peptide bond . There exists anti-parallel β pleated sheets and parallel β pleated sheets where the stability of the hydrogen bonds is stronger in the anti-parallel β sheet as it hydrogen bonds with the ideal 180 degree angle compared to the slanted hydrogen bonds formed by parallel sheets. [ 12 ]
The α-Helices and β-Sheets are commonly amphipathic, meaning they have a hydrophilic and a hydrophobic portion. This ability helps in forming tertiary structure of a protein in which folding occurs so that the hydrophilic sides are facing the aqueous environment surrounding the protein and the hydrophobic sides are facing the hydrophobic core of the protein. [ 15 ] Secondary structure hierarchically gives way to tertiary structure formation. Once the protein's tertiary structure is formed and stabilized by the hydrophobic interactions, there may also be covalent bonding in the form of disulfide bridges formed between two cysteine residues. These non-covalent and covalent contacts take a specific topological arrangement in a native structure of a protein. Tertiary structure of a protein involves a single polypeptide chain; however, additional interactions of folded polypeptide chains give rise to quaternary structure formation. [ 16 ]
Tertiary structure may give way to the formation of quaternary structure in some proteins, which usually involves the "assembly" or "coassembly" of subunits that have already folded; in other words, multiple polypeptide chains could interact to form a fully functional quaternary protein. [ 12 ]
Folding is a spontaneous process that is mainly guided by hydrophobic interactions, formation of intramolecular hydrogen bonds , van der Waals forces , and it is opposed by conformational entropy . [ 17 ] The folding time scale of an isolated protein depends on its size, contact order , and circuit topology . Inside cells, the process of folding often begins co-translationally , so that the N-terminus of the protein begins to fold while the C-terminal portion of the protein is still being synthesized by the ribosome ; however, a protein molecule may fold spontaneously during or after biosynthesis . [ 18 ] While these macromolecules may be regarded as " folding themselves ", the process also depends on the solvent ( water or lipid bilayer ), [ 19 ] the concentration of salts , the pH , the temperature , the possible presence of cofactors and of molecular chaperones .
Proteins will have limitations on their folding abilities by the restricted bending angles or conformations that are possible. These allowable angles of protein folding are described with a two-dimensional plot known as the Ramachandran plot , depicted with psi and phi angles of allowable rotation. [ 20 ]
Protein folding must be thermodynamically favorable within a cell in order for it to be a spontaneous reaction. Since it is known that protein folding is a spontaneous reaction, then it must assume a negative Gibbs free energy value. Gibbs free energy in protein folding is directly related to enthalpy and entropy . [ 12 ] For a negative delta G to arise and for protein folding to become thermodynamically favorable, then either enthalpy, entropy, or both terms must be favorable.
Minimizing the number of hydrophobic side-chains exposed to water is an important driving force behind the folding process. [ 21 ] The hydrophobic effect is the phenomenon in which the hydrophobic chains of a protein collapse into the core of the protein (away from the hydrophilic environment). [ 12 ] In an aqueous environment, the water molecules tend to aggregate around the hydrophobic regions or side chains of the protein, creating water shells of ordered water molecules. [ 22 ] An ordering of water molecules around a hydrophobic region increases order in a system and therefore contributes a negative change in entropy (less entropy in the system). The water molecules are fixed in these water cages which drives the hydrophobic collapse , or the inward folding of the hydrophobic groups. The hydrophobic collapse introduces entropy back to the system via the breaking of the water cages which frees the ordered water molecules. [ 12 ] The multitude of hydrophobic groups interacting within the core of the globular folded protein contributes a significant amount to protein stability after folding, because of the vastly accumulated van der Waals forces (specifically London Dispersion forces ). [ 12 ] The hydrophobic effect exists as a driving force in thermodynamics only if there is the presence of an aqueous medium with an amphiphilic molecule containing a large hydrophobic region. [ 23 ] The strength of hydrogen bonds depends on their environment; thus, H-bonds enveloped in a hydrophobic core contribute more than H-bonds exposed to the aqueous environment to the stability of the native state. [ 24 ]
In proteins with globular folds, hydrophobic amino acids tend to be interspersed along the primary sequence, rather than randomly distributed or clustered together. [ 25 ] [ 26 ] However, proteins that have recently been born de novo , which tend to be intrinsically disordered , [ 27 ] [ 28 ] show the opposite pattern of hydrophobic amino acid clustering along the primary sequence. [ 29 ]
Molecular chaperones are a class of proteins that aid in the correct folding of other proteins in vivo . Chaperones exist in all cellular compartments and interact with the polypeptide chain in order to allow the native three-dimensional conformation of the protein to form; however, chaperones themselves are not included in the final structure of the protein they are assisting in. [ 30 ] Chaperones may assist in folding even when the nascent polypeptide is being synthesized by the ribosome. [ 31 ] Molecular chaperones operate by binding to stabilize an otherwise unstable structure of a protein in its folding pathway, but chaperones do not contain the necessary information to know the correct native structure of the protein they are aiding; rather, chaperones work by preventing incorrect folding conformations. [ 31 ] In this way, chaperones do not actually increase the rate of individual steps involved in the folding pathway toward the native structure; instead, they work by reducing possible unwanted aggregations of the polypeptide chain that might otherwise slow down the search for the proper intermediate and they provide a more efficient pathway for the polypeptide chain to assume the correct conformations. [ 30 ] Chaperones are not to be confused with folding catalyst proteins, which catalyze chemical reactions responsible for slow steps in folding pathways. Examples of folding catalysts are protein disulfide isomerases and peptidyl-prolyl isomerases that may be involved in formation of disulfide bonds or interconversion between cis and trans stereoisomers of peptide group. [ 31 ] Chaperones are shown to be critical in the process of protein folding in vivo because they provide the protein with the aid needed to assume its proper alignments and conformations efficiently enough to become "biologically relevant". [ 32 ] This means that the polypeptide chain could theoretically fold into its native structure without the aid of chaperones, as demonstrated by protein folding experiments conducted in vitro ; [ 32 ] however, this process proves to be too inefficient or too slow to exist in biological systems; therefore, chaperones are necessary for protein folding in vivo. Along with its role in aiding native structure formation, chaperones are shown to be involved in various roles such as protein transport, degradation, and even allow denatured proteins exposed to certain external denaturant factors an opportunity to refold into their correct native structures. [ 33 ]
A fully denatured protein lacks both tertiary and secondary structure, and exists as a so-called random coil . Under certain conditions some proteins can refold; however, in many cases, denaturation is irreversible. [ 34 ] Cells sometimes protect their proteins against the denaturing influence of heat with enzymes known as heat shock proteins (a type of chaperone), which assist other proteins both in folding and in remaining folded. Heat shock proteins have been found in all species examined, from bacteria to humans, suggesting that they evolved very early and have an important function. Some proteins never fold in cells at all except with the assistance of chaperones which either isolate individual proteins so that their folding is not interrupted by interactions with other proteins or help to unfold misfolded proteins, allowing them to refold into the correct native structure. [ 35 ] This function is crucial to prevent the risk of precipitation into insoluble amorphous aggregates. The external factors involved in protein denaturation or disruption of the native state include temperature, external fields (electric, magnetic), [ 36 ] molecular crowding, [ 37 ] and even the limitation of space (i.e. confinement), which can have a big influence on the folding of proteins. [ 38 ] High concentrations of solutes , extremes of pH , mechanical forces, and the presence of chemical denaturants can contribute to protein denaturation, as well. These individual factors are categorized together as stresses. Chaperones are shown to exist in increasing concentrations during times of cellular stress and help the proper folding of emerging proteins as well as denatured or misfolded ones. [ 30 ]
Under some conditions proteins will not fold into their biochemically functional forms. Temperatures above or below the range that cells tend to live in will cause thermally unstable proteins to unfold or denature (this is why boiling makes an egg white turn opaque). Protein thermal stability is far from constant, however; for example, hyperthermophilic bacteria have been found that grow at temperatures as high as 122 °C, [ 39 ] which of course requires that their full complement of vital proteins and protein assemblies be stable at that temperature or above.
The bacterium E. coli is the host for bacteriophage T4 , and the phage encoded gp31 protein ( P17313 ) appears to be structurally and functionally homologous to E. coli chaperone protein GroES and able to substitute for it in the assembly of bacteriophage T4 virus particles during infection. [ 40 ] Like GroES, gp31 forms a stable complex with GroEL chaperonin that is absolutely necessary for the folding and assembly in vivo of the bacteriophage T4 major capsid protein gp23. [ 40 ]
Some proteins have multiple native structures, and change their fold based on some external factors. For example, the KaiB protein switches fold throughout the day , acting as a clock for cyanobacteria. It has been estimated that around 0.5–4% of PDB ( Protein Data Bank ) proteins switch folds. [ 41 ]
A protein is considered to be misfolded if it cannot achieve its normal native state. This can be due to mutations in the amino acid sequence or a disruption of the normal folding process by external factors. [ 42 ] The misfolded protein typically contains β-sheets that are organized in a supramolecular arrangement known as a cross-β structure. These β-sheet-rich assemblies are very stable, very insoluble, and generally resistant to proteolysis. [ 43 ] The structural stability of these fibrillar assemblies is caused by extensive interactions between the protein monomers, formed by backbone hydrogen bonds between their β-strands. [ 43 ] The misfolding of proteins can trigger the further misfolding and accumulation of other proteins into aggregates or oligomers. The increased levels of aggregated proteins in the cell leads to formation of amyloid -like structures which can cause degenerative disorders and cell death. [ 42 ] The amyloids are fibrillary structures that contain intermolecular hydrogen bonds which are highly insoluble and made from converted protein aggregates. [ 42 ] Therefore, the proteasome pathway may not be efficient enough to degrade the misfolded proteins prior to aggregation. Misfolded proteins can interact with one another and form structured aggregates and gain toxicity through intermolecular interactions. [ 42 ]
Aggregated proteins are associated with prion -related illnesses such as Creutzfeldt–Jakob disease , bovine spongiform encephalopathy (mad cow disease), amyloid-related illnesses such as Alzheimer's disease and familial amyloid cardiomyopathy or polyneuropathy , [ 44 ] as well as intracellular aggregation diseases such as Huntington's and Parkinson's disease . [ 4 ] [ 45 ] These age onset degenerative diseases are associated with the aggregation of misfolded proteins into insoluble, extracellular aggregates and/or intracellular inclusions including cross-β amyloid fibrils . It is not completely clear whether the aggregates are the cause or merely a reflection of the loss of protein homeostasis, the balance between synthesis, folding, aggregation and protein turnover. Recently the European Medicines Agency approved the use of Tafamidis or Vyndaqel (a kinetic stabilizer of tetrameric transthyretin) for the treatment of transthyretin amyloid diseases. This suggests that the process of amyloid fibril formation (and not the fibrils themselves) causes the degeneration of post-mitotic tissue in human amyloid diseases. [ 46 ] Misfolding and excessive degradation instead of folding and function leads to a number of proteopathy diseases such as antitrypsin -associated emphysema , cystic fibrosis and the lysosomal storage diseases , where loss of function is the origin of the disorder. While protein replacement therapy has historically been used to correct the latter disorders, an emerging approach is to use pharmaceutical chaperones to fold mutated proteins to render them functional.
While inferences about protein folding can be made through mutation studies , typically, experimental techniques for studying protein folding rely on the gradual unfolding or folding of proteins and observing conformational changes using standard non-crystallographic techniques.
X-ray crystallography is one of the more efficient and important methods for attempting to decipher the three dimensional configuration of a folded protein. [ 47 ] To be able to conduct X-ray crystallography, the protein under investigation must be located inside a crystal lattice. To place a protein inside a crystal lattice, one must have a suitable solvent for crystallization, obtain a pure protein at supersaturated levels in solution, and precipitate the crystals in solution. [ 48 ] Once a protein is crystallized, X-ray beams can be concentrated through the crystal lattice which would diffract the beams or shoot them outwards in various directions. These exiting beams are correlated to the specific three-dimensional configuration of the protein enclosed within. The X-rays specifically interact with the electron clouds surrounding the individual atoms within the protein crystal lattice and produce a discernible diffraction pattern. [ 15 ] Only by relating the electron density clouds with the amplitude of the X-rays can this pattern be read and lead to assumptions of the phases or phase angles involved that complicate this method. [ 49 ] Without the relation established through a mathematical basis known as Fourier transform , the " phase problem " would render predicting the diffraction patterns very difficult. [ 15 ] Emerging methods like multiple isomorphous replacement use the presence of a heavy metal ion to diffract the X-rays into a more predictable manner, reducing the number of variables involved and resolving the phase problem. [ 47 ]
Fluorescence spectroscopy is a highly sensitive method for studying the folding state of proteins. Three amino acids, phenylalanine (Phe), tyrosine (Tyr) and tryptophan (Trp), have intrinsic fluorescence properties, but only Tyr and Trp are used experimentally because their quantum yields are high enough to give good fluorescence signals. Both Trp and Tyr are excited by a wavelength of 280 nm, whereas only Trp is excited by a wavelength of 295 nm. Because of their aromatic character, Trp and Tyr residues are often found fully or partially buried in the hydrophobic core of proteins, at the interface between two protein domains, or at the interface between subunits of oligomeric proteins. In this apolar environment, they have high quantum yields and therefore high fluorescence intensities. Upon disruption of the protein's tertiary or quaternary structure, these side chains become more exposed to the hydrophilic environment of the solvent, and their quantum yields decrease, leading to low fluorescence intensities. For Trp residues, the wavelength of their maximal fluorescence emission also depend on their environment.
Fluorescence spectroscopy can be used to characterize the equilibrium unfolding of proteins by measuring the variation in the intensity of fluorescence emission or in the wavelength of maximal emission as functions of a denaturant value. [ 50 ] [ 51 ] The denaturant can be a chemical molecule (urea, guanidinium hydrochloride), temperature, pH, pressure, etc. The equilibrium between the different but discrete protein states, i.e. native state, intermediate states, unfolded state, depends on the denaturant value; therefore, the global fluorescence signal of their equilibrium mixture also depends on this value. One thus obtains a profile relating the global protein signal to the denaturant value. The profile of equilibrium unfolding may enable one to detect and identify intermediates of unfolding. [ 52 ] [ 53 ] General equations have been developed by Hugues Bedouelle to obtain the thermodynamic parameters that characterize the unfolding equilibria for homomeric or heteromeric proteins, up to trimers and potentially tetramers, from such profiles. [ 50 ] Fluorescence spectroscopy can be combined with fast-mixing devices such as stopped flow , to measure protein folding kinetics, [ 54 ] generate a chevron plot and derive a Phi value analysis .
Circular dichroism is one of the most general and basic tools to study protein folding. Circular dichroism spectroscopy measures the absorption of circularly polarized light . In proteins, structures such as alpha helices and beta sheets are chiral, and thus absorb such light. The absorption of this light acts as a marker of the degree of foldedness of the protein ensemble. This technique has been used to measure equilibrium unfolding of the protein by measuring the change in this absorption as a function of denaturant concentration or temperature . A denaturant melt measures the free energy of unfolding as well as the protein's m value, or denaturant dependence. A temperature melt measures the denaturation temperature (Tm) of the protein. [ 50 ] As for fluorescence spectroscopy, circular-dichroism spectroscopy can be combined with fast-mixing devices such as stopped flow to measure protein folding kinetics and to generate chevron plots .
The more recent developments of vibrational circular dichroism (VCD) techniques for proteins, currently involving Fourier transform (FT) instruments, provide powerful means for determining protein conformations in solution even for very large protein molecules. Such VCD studies of proteins can be combined with X-ray diffraction data for protein crystals, FT-IR data for protein solutions in heavy water (D 2 O), or quantum computations .
Protein nuclear magnetic resonance (NMR) is able to collect protein structural data by inducing a magnet field through samples of concentrated protein. In NMR, depending on the chemical environment, certain nuclei will absorb specific radio-frequencies. [ 55 ] [ 56 ] Because protein structural changes operate on a time scale from ns to ms, NMR is especially equipped to study intermediate structures in timescales of ps to s. [ 57 ] Some of the main techniques for studying proteins structure and non-folding protein structural changes include COSY , TOCSY , HSQC , time relaxation (T1 & T2), and NOE . [ 55 ] NOE is especially useful because magnetization transfers can be observed between spatially proximal hydrogens are observed. [ 55 ] Different NMR experiments have varying degrees of timescale sensitivity that are appropriate for different protein structural changes. NOE can pick up bond vibrations or side chain rotations, however, NOE is too sensitive to pick up protein folding because it occurs at larger timescale. [ 57 ]
Because protein folding takes place in about 50 to 3000 s −1 CPMG Relaxation dispersion and chemical exchange saturation transfer have become some of the primary techniques for NMR analysis of folding. [ 56 ] In addition, both techniques are used to uncover excited intermediate states in the protein folding landscape. [ 58 ] To do this, CPMG Relaxation dispersion takes advantage of the spin echo phenomenon. This technique exposes the target nuclei to a 90 pulse followed by one or more 180 pulses. [ 59 ] As the nuclei refocus, a broad distribution indicates the target nuclei is involved in an intermediate excited state. By looking at Relaxation dispersion plots the data collect information on the thermodynamics and kinetics between the excited and ground. [ 59 ] [ 58 ] Saturation Transfer measures changes in signal from the ground state as excited states become perturbed. It uses weak radio frequency irradiation to saturate the excited state of a particular nuclei which transfers its saturation to the ground state. [ 56 ] This signal is amplified by decreasing the magnetization (and the signal) of the ground state. [ 56 ] [ 58 ]
The main limitations in NMR is that its resolution decreases with proteins that are larger than 25 kDa and is not as detailed as X-ray crystallography . [ 56 ] Additionally, protein NMR analysis is quite difficult and can propose multiple solutions from the same NMR spectrum. [ 55 ]
In a study focused on the folding of an amyotrophic lateral sclerosis involved protein SOD1 , excited intermediates were studied with relaxation dispersion and Saturation transfer. [ 60 ] SOD1 had been previously tied to many disease causing mutants which were assumed to be involved in protein aggregation, however the mechanism was still unknown. By using Relaxation Dispersion and Saturation Transfer experiments many excited intermediate states were uncovered misfolding in the SOD1 mutants. [ 60 ]
Dual polarisation interferometry is a surface-based technique for measuring the optical properties of molecular layers. When used to characterize protein folding, it measures the conformation by determining the overall size of a monolayer of the protein and its density in real time at sub-Angstrom resolution, [ 61 ] although real-time measurement of the kinetics of protein folding are limited to processes that occur slower than ~10 Hz. Similar to circular dichroism , the stimulus for folding can be a denaturant or temperature .
The study of protein folding has been greatly advanced in recent years by the development of fast, time-resolved techniques. Experimenters rapidly trigger the folding of a sample of unfolded protein and observe the resulting dynamics . Fast techniques in use include neutron scattering , [ 62 ] ultrafast mixing of solutions, photochemical methods, and laser temperature jump spectroscopy . Among the many scientists who have contributed to the development of these techniques are Jeremy Cook, Heinrich Roder, Terry Oas, Harry Gray , Martin Gruebele , Brian Dyer, William Eaton, Sheena Radford , Chris Dobson , Alan Fersht , Bengt Nölting and Lars Konermann.
Proteolysis is routinely used to probe the fraction unfolded under a wide range of solution conditions (e.g. fast parallel proteolysis (FASTpp) . [ 63 ] [ 64 ]
Single molecule techniques such as optical tweezers and AFM have been used to understand protein folding mechanisms of isolated proteins as well as proteins with chaperones. [ 65 ] Optical tweezers have been used to stretch single protein molecules from their C- and N-termini and unfold them to allow study of the subsequent refolding. [ 66 ] The technique allows one to measure folding rates at single-molecule level; for example, optical tweezers have been recently applied to study folding and unfolding of proteins involved in blood coagulation. von Willebrand factor (vWF) is a protein with an essential role in blood clot formation process. It discovered – using single molecule optical tweezers measurement – that calcium-bound vWF acts as a shear force sensor in the blood. Shear force leads to unfolding of the A2 domain of vWF, whose refolding rate is dramatically enhanced in the presence of calcium. [ 67 ] Recently, it was also shown that the simple src SH3 domain accesses multiple unfolding pathways under force. [ 68 ]
Biotin painting enables condition-specific cellular snapshots of (un)folded proteins. Biotin 'painting' shows a bias towards predicted Intrinsically disordered proteins . [ 69 ]
Computational studies of protein folding includes three main aspects related to the prediction of protein stability, kinetics, and structure. A 2013 review summarizes the available computational methods for protein folding. [ 70 ]
In 1969, Cyrus Levinthal noted that, because of the very large number of degrees of freedom in an unfolded polypeptide chain, the molecule has an astronomical number of possible conformations. An estimate of 3 300 or 10 143 was made in one of his papers. [ 71 ] Levinthal's paradox is a thought experiment based on the observation that if a protein were folded by sequential sampling of all possible conformations, it would take an astronomical amount of time to do so, even if the conformations were sampled at a rapid rate (on the nanosecond or picosecond scale). [ 72 ] Based upon the observation that proteins fold much faster than this, Levinthal then proposed that a random conformational search does not occur, and the protein must, therefore, fold through a series of meta-stable intermediate states .
The configuration space of a protein during folding can be visualized as an energy landscape . According to Joseph Bryngelson and Peter Wolynes , proteins follow the principle of minimal frustration , meaning that naturally evolved proteins have optimized their folding energy landscapes, [ 73 ] and that nature has chosen amino acid sequences so that the folded state of the protein is sufficiently stable. In addition, the acquisition of the folded state had to become a sufficiently fast process. Even though nature has reduced the level of frustration in proteins, some degree of it remains up to now as can be observed in the presence of local minima in the energy landscape of proteins.
A consequence of these evolutionarily selected sequences is that proteins are generally thought to have globally "funneled energy landscapes" (a term coined by José Onuchic ) [ 74 ] that are largely directed toward the native state. This " folding funnel " landscape allows the protein to fold to the native state through any of a large number of pathways and intermediates, rather than being restricted to a single mechanism. The theory is supported by both computational simulations of model proteins and experimental studies, [ 73 ] and it has been used to improve methods for protein structure prediction and design . [ 73 ] The description of protein folding by the leveling free-energy landscape is also consistent with the 2nd law of thermodynamics. [ 75 ] Physically, thinking of landscapes in terms of visualizable potential or total energy surfaces simply with maxima, saddle points, minima, and funnels, rather like geographic landscapes, is perhaps a little misleading. The relevant description is really a high-dimensional phase space in which manifolds might take a variety of more complicated topological forms. [ 76 ]
The unfolded polypeptide chain begins at the top of the funnel where it may assume the largest number of unfolded variations and is in its highest energy state. Energy landscapes such as these indicate that there are a large number of initial possibilities, but only a single native state is possible; however, it does not reveal the numerous folding pathways that are possible. A different molecule of the same exact protein may be able to follow marginally different folding pathways, seeking different lower energy intermediates, as long as the same native structure is reached. [ 77 ] Different pathways may have different frequencies of utilization depending on the thermodynamic favorability of each pathway. This means that if one pathway is found to be more thermodynamically favorable than another, it is likely to be used more frequently in the pursuit of the native structure. [ 77 ] As the protein begins to fold and assume its various conformations, it always seeks a more thermodynamically favorable structure than before and thus continues through the energy funnel. Formation of secondary structures is a strong indication of increased stability within the protein, and only one combination of secondary structures assumed by the polypeptide backbone will have the lowest energy and therefore be present in the native state of the protein. [ 77 ] Among the first structures to form once the polypeptide begins to fold are alpha helices and beta turns, where alpha helices can form in as little as 100 nanoseconds and beta turns in 1 microsecond. [ 30 ]
There exists a saddle point in the energy funnel landscape where the transition state for a particular protein is found. [ 30 ] The transition state in the energy funnel diagram is the conformation that must be assumed by every molecule of that protein if the protein wishes to finally assume the native structure. No protein may assume the native structure without first passing through the transition state. [ 30 ] The transition state can be referred to as a variant or premature form of the native state rather than just another intermediary step. [ 78 ] The folding of the transition state is shown to be rate-determining, and even though it exists in a higher energy state than the native fold, it greatly resembles the native structure. Within the transition state, there exists a nucleus around which the protein is able to fold, formed by a process referred to as "nucleation condensation" where the structure begins to collapse onto the nucleus. [ 78 ]
De novo or ab initio techniques for computational protein structure prediction can be used for simulating various aspects of protein folding. Molecular dynamics (MD) was used in simulations of protein folding and dynamics in silico . [ 79 ] First equilibrium folding simulations were done using implicit solvent model and umbrella sampling . [ 80 ] Because of computational cost, ab initio MD folding simulations with explicit water are limited to peptides and small proteins. [ 81 ] [ 82 ] MD simulations of larger proteins remain restricted to dynamics of the experimental structure or its high-temperature unfolding. Long-time folding processes (beyond about 1 millisecond), like folding of larger proteins (>150 residues) can be accessed using coarse-grained models . [ 83 ] [ 84 ] [ 85 ]
Several large-scale computational projects, such as Rosetta@home , [ 86 ] Folding@home [ 87 ] and Foldit , [ 88 ] target protein folding.
Long continuous-trajectory simulations have been performed on Anton , a massively parallel supercomputer designed and built around custom ASICs and interconnects by D. E. Shaw Research . The longest published result of a simulation performed using Anton as of 2011 was a 2.936 millisecond simulation of NTL9 at 355 K. [ 89 ] Such simulations are currently able to unfold and refold small proteins (<150 amino acids residues) in equilibrium and predict how mutations affect folding kinetics and stability. [ 90 ]
In 2020 a team of researchers that used AlphaFold , an artificial intelligence (AI) protein structure prediction program developed by DeepMind placed first in CASP , a long-standing structure prediction contest. [ 91 ] The team achieved a level of accuracy much higher than any other group. [ 92 ] It scored above 90% for around two-thirds of the proteins in CASP's global distance test (GDT) , a test that measures the degree of similarity between the structure predicted by a computational program, and the empirical structure determined experimentally in a lab. A score of 100 is considered a complete match, within the distance cutoff used for calculating GDT. [ 93 ]
AlphaFold's protein structure prediction results at CASP were described as "transformational" and "astounding". [ 94 ] [ 95 ] Some researchers noted that the accuracy is not high enough for a third of its predictions, and that it does not reveal the physical mechanism of protein folding for the protein folding problem to be considered solved. [ 96 ] Nevertheless, it is considered a significant achievement in computational biology [ 93 ] and great progress towards a decades-old grand challenge of biology, predicting the structure of proteins. [ 94 ] | https://en.wikipedia.org/wiki/Protein_folding |
Protein footprinting is a term used to refer to a method of biochemical analysis that investigates protein structure , assembly, and interactions within a larger macromolecular assembly . It was originally coined in reference to the use of limited proteolysis to investigate contact sites within a monoclonal antibody - protein antigen complex [ 1 ] and a year later to examine the protection from hydroxyl radical cleavage conferred by a protein bound to DNA within a DNA-protein complex. [ 2 ] In DNA footprinting the protein is envisioned to make an imprint (or footprint ) at a particular point of interaction. [ 3 ] This latter method was adapted through the direct treatment of proteins and their complexes with hydroxyl radicals [ 4 ] [ 5 ] and can be generally denoted RP-MS (for Radical Probe - Mass Spectrometry) [ 6 ] akin to the designation used for Hydrogen-deuterium exchange Mass Spectrometry (denoted HD-MS or HX-MS).
Time-resolved hydroxyl radical protein footprinting (HRPF) employing mass spectrometry analysis was originated and developed in the late 1990s in synchrotron radiolysis studies. [ 7 ] [ 8 ] The same year, these authors (Maleknia et al.) reported on the use of an electrical discharge source to effect the oxidation of proteins on millisecond timescales as proteins pass from the electrosprayed solution into the mass spectrometer. [ 9 ] Years later in 2005, researchers Hambly and Gross introduced a method for protein oxidation on the microsecond timescale using laser flash photolysis of hydrogen peroxide to generate hydroxyl radicals. [ 10 ] This method, fast photochemical oxidation of proteins (FPOP), claimed to footprint proteins faster than they change their fold [ 11 ] though this timeframe has been challenged given hydrogen peroxide, not present in the original studies, and secondary radicals, react alone in situ over tens of milliseconds. [ 12 ] The combined approaches have since been used to determine protein structures, [ 13 ] protein folding, protein dynamics, and protein–protein interactions. [ 14 ]
Unlike nucleic acids, proteins oxidize rather than cleave on these timescales. Analysis of the products by mass spectrometry reveals that proteins are oxidized in a limited manner (some 10–30% of total protein) at a number of amino acid side chains across the proteins. The rate or level of oxidation at the reactive amino acid side chains (Met, Cys, Trp, Tyr, Phe, His, Pro and Leu) provides a measure of their accessibility to the bulk solvent. The mechanisms of side chain oxidation were explored by performing the radiolysis reactions in 18 O-labeled water .
A critical feature of these experiments is the need to expose proteins to hydroxyl radicals for limited timescales on the order of 1–50 ms inducing 10-30% oxidation of total protein. A further requirement is to generate hydroxyl radicals from the bulk solvent (i.e. water) (equations 1 and 2) not hydrogen peroxide which can remain to oxidize proteins even without other stimuli. [ 15 ]
Hydroxyl radicals can be produced in solution by an electrical discharge within a conventional atmospheric pressure electrospray ionization (ESI) source. When a high voltage difference (~8 keV) is held between an electrospray needle and a sampling orifice to the mass analyzer, radicals can be produced in solution at the electrospray needle tip. This method was the first employed to apply protein footprinting to the study of a protein complex. [ 16 ]
The exposure of proteins to a "white" X-ray beam of synchrotron light or an electrical discharge for tens of milliseconds provides sufficient oxidative modification to the surface amino acid side chains without damage to the protein structure. These products can be easily detected and quantified by mass spectrometry. By adjusting the time for radiolysis or which protein ions spend in the discharge source, a time-resolved approach is possible which is valuable for the study of protein dynamics.
A computer program (PROXIMO) has also been written to help model protein complexes using data from the RP-MS/Protein footprinting approach. [ 17 ] RP-MS/Protein footprinting studies of protein complexes can also employ computational approaches to assist with this modeling. [ 18 ]
The application of ion mobility mass spectrometry has conclusively demonstrated that the conditions employed in RP-MS/Protein footprinting experiments do not alter the structure of proteins. [ 19 ]
Other studies have extended the method to study early onset protein damage given the radical basis of the method and the significance of oxygen based radicals in the pathogenesis of many diseases including neurological disorders and even blindness. [ 20 ] | https://en.wikipedia.org/wiki/Protein_footprinting |
Protein backbone fragment libraries have been used successfully in a variety of structural biology applications, including homology modeling , [ 1 ] de novo structure prediction , [ 2 ] [ 3 ] [ 4 ] and structure determination . [ 5 ] By reducing the complexity of the search space, these fragment libraries enable more rapid search of conformational space , leading to more efficient and accurate models.
Proteins can adopt an exponential number of states when modeled discretely. Typically, a protein's conformations are represented as sets of dihedral angles , bond lengths , and bond angles between all connected atoms. The most common simplification is to assume ideal bond lengths and bond angles. However, this still leaves the phi-psi angles of the backbone, and up to four dihedral angles for each side chain , leading to a worst case complexity of k 6* n possible states of the protein, where n is the number of residues and k is the number of discrete states modeled for each dihedral angle. In order to reduce the conformational space, one can use protein fragment libraries rather than explicitly model every phi-psi angle.
Fragments are short segments of the peptide backbone, typically from 5 to 15 residues long, and do not include the side chains. They may specify the location of just the C-alpha atoms if it is a reduced atom representation, or all the backbone heavy atoms (N, C-alpha, C carbonyl, O). Note that side chains are typically not modeled using the fragment library approach. To model discrete states of a side chain, one could use a rotamer library approach. [ 6 ]
This approach operates under the assumption that local interactions play a large role in stabilizing the overall protein conformation. In any short sequence, the molecular forces constrain the structure, leading to only a small number of possible conformations, which can be modeled by fragments. Indeed, according to Levinthal's paradox , a protein could not possibly sample all possible conformations within a biologically reasonable amount of time. Locally stabilized structures would reduce the search space and allow proteins to fold on the order of milliseconds.
Libraries of these fragments are constructed from an analysis of the Protein Data Bank (PDB). First, a representative subset of the PDB is chosen which should cover a diverse array of structures, preferably at a good resolution. Then, for each structure, every set of n consecutive residues is taken as a sample fragment. The samples are then clustered into k groups, based upon how similar they are to each other in spatial configuration, using algorithms such as k -means clustering . The parameters n and k are chosen according to the application (see discussion on complexity below). The centroids of the clusters are then taken to represent the fragment. Further optimization can be performed to ensure that the centroid possesses ideal bond geometry, as it was derived by averaging other geometries. [ 7 ]
Because the fragments are derived from structures that exist in nature, the segment of backbone they represent will have realistic bonding geometries. This helps avoid having to explore the full space of conformation angles, much of which would lead to unrealistic geometries.
The clustering above can be performed without regard to the identities of the residues, or it can be residue-specific. [ 2 ] That is, for any given input sequence of amino acids, a clustering can be derived using only samples found in the PDB with the same sequence in the k -mer fragment. This requires more computational work than deriving a sequence-independent fragment library but can potentially produce more accurate models. Conversely, a larger sample set is required, and one may not achieve full coverage.
In homology modeling , a common application of fragment libraries is to model the loops of the structure. Typically, the alpha helices and beta sheets are threaded against a template structure, but the loops in between are not specified and need to be predicted. Finding the loop with the optimal configuration is NP-hard . To reduce the conformational space that needs to be explored, one can model the loop as a series of overlapping fragments. The space can then be sampled, or if the space is now small enough, exhaustively enumerated.
One approach for exhaustive enumeration goes as follows. [ 1 ] Loop construction begins by aligning all possible fragments to overlap with the three residues at the N terminus of the loop (the anchor point). Then all possible choices for a second fragment are aligned to (all possible choices of) the first fragment, ensuring that the last three residues of the first fragment overlap with the first three residues of the second fragment. This ensures that the fragment chain forms realistic angles both within the fragment and between fragments. This is then repeated until a loop with the correct length of residues is constructed.
The loop must both begin at the anchor on the N side and end at the anchor on the C side. Each loop must therefore be tested to see if its last few residues overlap with the C terminal anchor. Very few of these exponential numbers of candidate loops will close the loop. After filtering out loops that don't close, one must then determine which loop has the optimal configuration, as determined by having the lowest energy using some molecular mechanics force field.
The complexity of the state space is still exponential in the number of residues, even after using fragment libraries. However, the degree of the exponent is reduced. For a library of F -mer fragments, with L fragments in the library, and to model a chain of N residues overlapping each fragment by 3, there will be L [ N /( F -3)]+1 possible chains. [ 7 ] This is much less than the K N possibilities if explicitly modeling the phi-psi angles as K possible combinations, as the complexity grows at a degree smaller than N .
The complexity increases in L , the size of the fragment library. However, libraries with more fragments will capture a greater diversity of fragment structures, so there is a trade off in the accuracy of the model vs the speed of exploring the search space. This choice governs what K is used when performing the clustering.
Additionally, for any fixed L , the diversity of structures capable of being modeled decreases as the length of the fragments increases. Shorter fragments are more capable of covering the diverse array of structures found in the PDB than longer ones. Recently, it was shown that libraries of up to length 15 are capable of modeling 91% of the fragments in the PDB to within 2.0 angstroms. [ 8 ] | https://en.wikipedia.org/wiki/Protein_fragment_library |
Protein function prediction methods are techniques that bioinformatics researchers use to assign biological or biochemical roles to proteins . These proteins are usually ones that are poorly studied or predicted based on genomic sequence data. These predictions are often driven by data-intensive computational procedures. Information may come from nucleic acid sequence homology , gene expression profiles, protein domain structures, text mining of publications, phylogenetic profiles, phenotypic profiles, and protein-protein interaction. Protein function is a broad term: the roles of proteins range from catalysis of biochemical reactions to transport to signal transduction , and a single protein may play a role in multiple processes or cellular pathways. [ 1 ]
Generally, function can be thought of as, "anything that happens to or through a protein". [ 1 ] The Gene Ontology Consortium provides a useful classification of functions, based on a dictionary of well-defined terms divided into three main categories of molecular function, biological process and cellular component . [ 2 ] Researchers can query this database with a protein name or accession number to retrieve associated Gene Ontology (GO) terms or annotations based on computational or experimental evidence.
While techniques such as microarray analysis, RNA interference , and the yeast two-hybrid system can be used to experimentally demonstrate the function of a protein, advances in sequencing technologies have made the rate at which proteins can be experimentally characterized much slower than the rate at which new sequences become available. [ 3 ] Thus, the annotation of new sequences is mostly by prediction through computational methods, as these types of annotation can often be done quickly and for many genes or proteins at once. The first such methods inferred function based on homologous proteins with known functions ( homology-based function prediction ). The development of context-based and structure based methods have expanded what information can be predicted, and a combination of methods can now be used to get a picture of complete cellular pathways based on sequence data. [ 3 ] The importance and prevalence of computational prediction of gene function is underlined by an analysis of 'evidence codes' used by the GO database: as of 2010, 98% of annotations were listed under the code IEA (inferred from electronic annotation) while only 0.6% were based on experimental evidence. [ 4 ]
Proteins of similar sequence are usually homologous [ 5 ] and thus have a similar function. Hence proteins in a newly sequenced genome are routinely annotated using the sequences of similar proteins in related genomes.
However, closely related proteins do not always share the same function. [ 6 ] For example, the yeast Gal1 and Gal3 proteins are paralogs (73% identity and 92% similarity) that have evolved very different functions with Gal1 being a galactokinase and Gal3 being a transcriptional inducer. [ 7 ]
There is no hard sequence-similarity threshold for "safe" function prediction; many proteins of barely detectable sequence similarity have the same function while others (such as Gal1 and Gal3) are highly similar but have evolved different functions. As a rule of thumb, sequences that are more than 30-40% identical are usually considered as having the same or a very similar function.
For enzymes , predictions of specific functions are especially difficult, as they only need a few key residues in their active site , hence very different sequences can have very similar activities. By contrast, even with sequence identity of 70% or greater, 10% of any pair of enzymes have different substrates; and differences in the actual enzymatic reactions are not uncommon near 50% sequence identity. [ 8 ] [ 9 ]
The development of protein domain databases such as Pfam (Protein Families Database) [ 10 ] allow us to find known domains within a query sequence, providing evidence for likely functions. The dcGO website [ 11 ] contains annotations to both the individual domains and supra-domains (i.e., combinations of two or more successive domains), thus via dcGO Predictor allowing for the function predictions in a more realistic manner. Within protein domains , shorter signatures known as ' motifs ' are associated with particular functions, [ 12 ] and motif databases such as PROSITE ('database of protein domains, families and functional sites') can be searched using a query sequence. [ 13 ] Motifs can, for example, be used to predict subcellular localization of a protein (where in the cell the protein is sent after synthesis). Short signal peptides direct certain proteins to a particular location such as the mitochondria, and various tools exist for the prediction of these signals in a protein sequence. [ 14 ] For example, SignalP , which has been updated several times as methods are improved. [ 15 ] Thus, aspects of a protein's function can be predicted without comparison to other full-length homologous protein sequences.
Because 3D protein structure is generally more well conserved than protein sequence, structural similarity is a good indicator of similar function in two or more proteins. [ 6 ] [ 12 ] Many programs have been developed to screen a known protein structure against the Protein Data Bank [ 16 ] and report similar structures (for example, FATCAT (Flexible structure AlignmenT by Chaining AFPs (Aligned Fragment Pairs) with Twists), [ 17 ] CE (combinatorial extension) [ 18 ] ) and DeepAlign (protein structure alignment beyond spatial proximity). [ 19 ] Similarly, the main protein databases, such as UniProt , have built-in tools to search any given protein sequences against structure databases, and link to related proteins of known structure.
To deal with the situation that many protein sequences have no solved structures, some function prediction servers such as RaptorX are also developed that can first predict the 3D model of a sequence and then use structure-based method to predict functions based upon the predicted 3D model. In many cases instead of the whole protein structure, the 3D structure of a particular motif representing an active site or binding site can be targeted. [ 12 ] [ 20 ] [ 21 ] [ 22 ] [ 23 ] The Structurally Aligned Local Sites of Activity (SALSA) [ 21 ] method, developed by Mary Jo Ondrechen and students, utilizes computed chemical properties of the individual amino acids to identify local biochemically active sites. Databases such as Catalytic Site Atlas [ 24 ] have been developed that can be searched using novel protein sequences to predict specific functional sites.
One of the challenges involved in protein function prediction is discovery of the active site. This is complicated by certain active sites not being formed – essentially existing – until the protein undergoes conformational changes brought on by the binding of small molecules. Most protein structures have been determined by X-ray crystallography which requires a purified protein crystal . As a result, existing structural models are generally of a purified protein and as such lack the conformational changes that are created when the protein interacts with small molecules. [ 26 ]
Computational solvent mapping utilizes probes (small organic molecules) that are computationally 'moved' over the surface of the protein searching for sites where they tend to cluster. Multiple different probes are generally applied with the goal being to obtain a large number of different protein-probe conformations. The generated clusters are then ranked based on the cluster's average free energy. After computationally mapping multiple probes, the site of the protein where relatively large numbers of clusters form typically corresponds to an active site on the protein. [ 26 ]
This technique is a computational adaptation of 'wet lab' work from 1996. It was discovered that ascertaining the structure of a protein while it is suspended in different solvents and then superimposing those structures on one another produces data where the organic solvent molecules (that the proteins were suspended in) typically cluster at the protein's active site. This work was carried out as a response to realizing that water molecules are visible in the electron density maps produced by X-ray crystallography . The water molecules are interacting with the protein and tend to cluster at the protein's polar regions. This led to the idea of immersing the purified protein crystal in other solvents (e.g. ethanol , isopropanol , etc.) to determine where these molecules cluster on the protein. The solvents can be chosen based on what they approximate, that is, what molecule this protein may interact with (e.g. ethanol can probe for interactions with the amino acid serine , isopropanol a probe for threonine , etc.). It is vital that the protein crystal maintains its tertiary structure in each solvent. This process is repeated for multiple solvents and then this data can be used to try to determine potential active sites on the protein. [ 27 ] Ten years later this technique was developed into an algorithm by Clodfelter et al.
Many of the newer methods for protein function prediction are not based on comparison of sequence or structure as above, but on some type of correlation between novel genes/proteins and those that already have annotations. Several methods have been developed to predict gene function on the local genomic or phylogenomic context and structure of genes:
Phylogenetic profiling is based on the observation that two or more proteins with the same pattern of presence or absence in many different genomes most likely have a functional link. [ 12 ] [ 28 ] Whereas homology-based methods can often be used to identify molecular functions of a protein, context-based approaches can be used to predict cellular function, or the biological process in which a protein acts. [ 3 ] [ 28 ] For example, proteins involved in the same metabolic pathway are likely to be present in a genome together or are absent altogether, suggesting that these genes work together in a functional context.
Operons are clusters of genes that are transcribed together. Based on co-transcription data but also based on the fact that the order of genes in operons is often conserved across many bacteria, indicates that they act together. [ 29 ]
Gene fusion occurs when two or more genes encode two or more proteins in one organism and have, through evolution, combined to become a single gene in another organism (or vice versa for gene fission ). [ 3 ] [ 30 ] This concept has been used, for example, to search all E. coli protein sequences for homology in other genomes and find over 6000 pairs of sequences with shared homology to single proteins in another genome, indicating potential interaction between each of the pairs. [ 30 ] Because the two sequences in each protein pair are non-homologous, these interactions could not be predicted using homology-based methods.
In prokaryotes , clusters of genes that are physically close together in the genome often conserve together through evolution, and tend to encode proteins that interact or are part of the same operon . [ 3 ] Thus, chromosomal proximity also called the gene neighbour method [ 31 ] can be used to predict functional similarity between proteins, at least in prokaryotes. Chromosomal proximity has also been seen to apply for some pathways in selected eukaryotic genomes, including Homo sapiens , [ 32 ] and with further development gene neighbor methods may be valuable for studying protein interactions in eukaryotes. [ 28 ]
Genes involved in similar functions are also often co-transcribed, so that an unannotated protein can often be predicted to have a related function to proteins with which it co-expresses. [ 12 ] The guilt by association algorithms developed based on this approach can be used to analyze large amounts of sequence data and identify genes with expression patterns similar to those of known genes. [ 33 ] [ 34 ] Often, a guilt by association study compares a group of candidate genes (unknown function) to a target group (for example, a group of genes known to be associated with a particular disease), and rank the candidate genes by their likelihood of belonging to the target group based on the data. [ 35 ] Based on recent studies, however, it has been suggested that some problems exist with this type of analysis. For example, because many proteins are multifunctional, the genes encoding them may belong to several target groups. It is argued that such genes are more likely to be identified in guilt by association studies, and thus predictions are not specific. [ 35 ]
With the accumulation of RNA-seq data that are capable of estimating expression profiles for alternatively spliced isoforms, machine learning algorithms have also been developed for predicting and differentiating functions at the isoform level. [ 36 ] This represents an emerging research area in function prediction, which integrates large-scale, heterogeneous genomic data to infer functions at the isoform level. [ 37 ]
Guilt by association type algorithms may be used to produce a functional association network for a given target group of genes or proteins. [ 38 ] These networks serve as a representation of the evidence for shared/similar function within a group of genes, where nodes represent genes/proteins and are linked to each other by edges representing evidence of shared function. [ 39 ]
Several networks based on different data sources can be combined into a composite network, which can then be used by a prediction algorithm to annotate candidate genes or proteins. [ 40 ] For example, the developers of the bioPIXIE system used a wide variety of Saccharomyces cerevisiae (yeast) genomic data to produce a composite functional network for that species. [ 41 ] This resource allows the visualization of known networks representing biological processes, as well as the prediction of novel components of those networks. Many algorithms have been developed to predict function based on the integration of several data sources (e.g. genomic, proteomic, protein interaction, etc.), and testing on previously annotated genes indicates a high level of accuracy. [ 39 ] [ 42 ] Disadvantages of some function prediction algorithms have included a lack of accessibility, and the time required for analysis. Faster, more accurate algorithms such as GeneMANIA (multiple association network integration algorithm) have however been developed in recent years [ 40 ] and are publicly available on the web, indicating the future direction of function prediction.
STRING : web tool that integrates various data sources for function prediction. [ 43 ]
VisANT : Visual analysis of networks and integrative visual data-mining. [ 44 ]
Mantis : A consensus-driven function prediction tool that dynamically integrates multiple reference databases. [ 45 ] | https://en.wikipedia.org/wiki/Protein_function_prediction |
In cell biology , protein kinase A ( PKA ) is a family of serine-threonine kinases [ 1 ] whose activity is dependent on cellular levels of cyclic AMP (cAMP). PKA is also known as cAMP-dependent protein kinase ( EC 2.7.11.11 ). PKA has several functions in the cell, including regulation of glycogen , sugar , and lipid metabolism . It should not be confused with 5'- AMP-activated protein kinase ( AMP-activated protein kinase ).
Protein kinase A, more precisely known as adenosine 3',5'-monophosphate (cyclic AMP)-dependent protein kinase, abbreviated to PKA, was discovered by chemists Edmond H. Fischer and Edwin G. Krebs in 1968. They won the Nobel Prize in Physiology or Medicine in 1992 for their work on phosphorylation and dephosphorylation and how it relates to PKA activity. [ 2 ]
PKA is one of the most widely researched protein kinases , in part because of its uniqueness; out of 540 different protein kinase genes that make up the human kinome , only one other protein kinase, casein kinase 2 , is known to exist in a physiological tetrameric complex, meaning it consists of four subunits. [ 1 ]
The diversity of mammalian PKA subunits was realized after Dr. Stan McKnight and others identified four possible catalytic subunit genes and four regulatory subunit genes. In 1991, Susan Taylor and colleagues crystallized the PKA Cα subunit, which revealed the bi-lobe structure of the protein kinase core for the very first time, providing a blueprint for all the other protein kinases in a genome (the kinome). [ 3 ]
When inactive, the PKA apoenzyme exists as a tetramer which consists of two regulatory subunits and two catalytic subunits. The catalytic subunit contains the active site, a series of canonical residues found in protein kinases that bind and hydrolyse ATP , and a domain to bind the regulatory subunit. The regulatory subunit has domains to bind to cyclic AMP, a domain that interacts with catalytic subunit, and an auto inhibitory domain. There are two major forms of regulatory subunit; RI and RII. [ 4 ]
Mammalian cells have at least two types of PKAs: type I is mainly in the cytosol , whereas type II is bound via its regulatory subunits and special anchoring proteins, described in the anchorage section , to the plasma membrane , nuclear membrane , mitochondrial outer membrane , and microtubules . In both types, once the catalytic subunits are freed and active, they can migrate into the nucleus (where they can phosphorylate transcription regulatory proteins), while the regulatory subunits remain in the cytoplasm. [ 5 ]
The following human genes encode PKA subunits:
PKA is also commonly known as cAMP-dependent protein kinase, because it has traditionally been thought to be activated through release of the catalytic subunits when levels of the second messenger called cyclic adenosine monophosphate , or cAMP, rise in response to a variety of signals. However, recent studies evaluating the intact holoenzyme complexes, including regulatory AKAP-bound signalling complexes, have suggested that the local sub cellular activation of the catalytic activity of PKA might proceed without physical separation of the regulatory and catalytic components, especially at physiological concentrations of cAMP. [ 6 ] [ 7 ] In contrast, experimentally induced supra physiological concentrations of cAMP, meaning higher than normally observed in cells, are able to cause separation of the holoenzymes, and release of the catalytic subunits. [ 6 ]
Extracellular hormones, such as glucagon and epinephrine , begin an intracellular signalling cascade that triggers protein kinase A activation by first binding to a G protein–coupled receptor (GPCR) on the target cell. When a GPCR is activated by its extracellular ligand, a conformational change is induced in the receptor that is transmitted to an attached intracellular heterotrimeric G protein complex by protein domain dynamics . The Gs alpha subunit of the stimulated G protein complex exchanges GDP for GTP in a reaction catalyzed by the GPCR and is released from the complex. The activated Gs alpha subunit binds to and activates an enzyme called adenylyl cyclase , which, in turn, catalyzes the conversion of ATP into cAMP, directly increasing the cAMP level. Four cAMP molecules are able to bind to the two regulatory subunits. This is done by two cAMP molecules binding to each of the two cAMP binding sites (CNB-B and CNB-A) which induces a conformational change in the regulatory subunits of PKA, causing the subunits to detach and unleash the two, now activated, catalytic subunits. [ 8 ]
Once released from inhibitory regulatory subunit, the catalytic subunits can go on to phosphorylate a number of other proteins in the minimal substrate context Arg-Arg-X-Ser/Thr., [ 9 ] although they are still subject to other layers of regulation, including modulation by the heat stable pseudosubstrate inhibitor of PKA, termed PKI. [ 7 ] [ 10 ]
Below is a list of the steps involved in PKA activation:
The liberated catalytic subunits can then catalyze the transfer of ATP terminal phosphates to protein substrates at serine , or threonine residues . This phosphorylation usually results in a change in activity of the substrate. Since PKAs are present in a variety of cells and act on different substrates, PKA regulation and cAMP regulation are involved in many different pathways.
The mechanisms of further effects may be divided into direct protein phosphorylation and protein synthesis:
The Serine/Threonine residue of the substrate peptide is orientated in such a way that the hydroxyl group faces the gamma phosphate group of the bound ATP molecule. Both the substrate, ATP, and two Mg2+ ions form intensive contacts with the catalytic subunit of PKA. In the active conformation, the C helix packs against the N-terminal lobe and the Aspartate residue of the conserved DFG motif chelates the Mg2+ ions, assisting in positioning the ATP substrate. The triphosphate group of ATP points out of the adenosine pocket for the transfer of gamma-phosphate to the Serine/Threonine of the peptide substrate. There are several conserved residues, include Glutamate (E) 91 and Lysine (K) 72, that mediate the positioning of alpha- and beta-phosphate groups. The hydroxyl group of the peptide substrate's Serine/Threonine attacks the gamma phosphate group at the phosphorus via an SN2 nucleophilic reaction, which results in the transfer of the terminal phosphate to the peptide substrate and cleavage of the phosphodiester bond between the beta-phosphate and the gamma-phosphate groups. PKA acts as a model for understanding protein kinase biology, with the position of the conserved residues helping to distinguish the active protein kinase and inactive pseudokinase members of the human kinome.
Downregulation of protein kinase A occurs by a feedback mechanism and uses a number of cAMP hydrolyzing phosphodiesterase (PDE) enzymes, which belong to the substrates activated by PKA. Phosphodiesterase quickly converts cAMP to AMP, thus reducing the amount of cAMP that can activate protein kinase A. PKA is also regulated by a complex series of phosphorylation events, which can include modification by autophosphorylation and phosphorylation by regulatory kinases, such as PDK1. [ 7 ]
Thus, PKA is controlled, in part, by the levels of cAMP . Also, the catalytic subunit itself can be down-regulated by phosphorylation.
The regulatory subunit dimer of PKA is important for localizing the kinase inside the cell. The dimerization and docking (D/D) domain of the dimer binds to the A-kinase binding (AKB) domain of A-kinase anchor protein (AKAP). The AKAPs localize PKA to various locations (e.g., plasma membrane, mitochondria, etc.) within the cell.
AKAPs bind many other signaling proteins, creating a very efficient signaling hub at a certain location within the cell. For example, an AKAP located near the nucleus of a heart muscle cell would bind both PKA and phosphodiesterase (hydrolyzes cAMP), which allows the cell to limit the productivity of PKA, since the catalytic subunit is activated once cAMP binds to the regulatory subunits.
PKA phosphorylates proteins that have the motif Arginine-Arginine-X-Serine exposed, in turn (de)activating the proteins. Many possible substrates of PKA exist; a list of such substrates is available and maintained by the NIH . [ 11 ]
As protein expression varies from cell type to cell type, the proteins that are available for phosphorylation will depend upon the cell in which PKA is present. Thus, the effects of PKA activation vary with cell type :
Epinephrine and glucagon affect the activity of protein kinase A by changing the levels of cAMP in a cell via the G-protein mechanism, using adenylate cyclase . Protein kinase A acts to phosphorylate many enzymes important in metabolism. For example, protein kinase A phosphorylates acetyl-CoA carboxylase and pyruvate dehydrogenase . Such covalent modification has an inhibitory effect on these enzymes, thus inhibiting lipogenesis and promoting net gluconeogenesis . Insulin, on the other hand, decreases the level of phosphorylation of these enzymes, which instead promotes lipogenesis. Recall that gluconeogenesis does not occur in myocytes.
PKA helps transfer/translate the dopamine signal into cells in the nucleus accumbens , which mediates reward, motivation, and task salience . The vast majority of reward perception involves neuronal activation in the nucleus accumbens, some examples of which include sex, recreational drugs, and food. Protein Kinase A signal transduction pathway helps in modulation of ethanol consumption and its sedative effects. A mouse study reports that mice with genetically reduced cAMP-PKA signalling results into less consumption of ethanol and are more sensitive to its sedative effects. [ 18 ]
PKA is directed to specific sub-cellular locations after tethering to AKAPs . Ryanodine receptor (RyR) co-localizes with the muscle AKAP and RyR phosphorylation and efflux of Ca 2+ is increased by localization of PKA at RyR by AKAPs. [ 19 ]
In a cascade mediated by a GPCR known as β 1 adrenoceptor , activated by catecholamines (notably norepinephrine ), PKA gets activated and phosphorylates numerous targets, namely: L-type calcium channels , phospholamban , troponin I , myosin binding protein C , and potassium channels . This increases inotropy as well as lusitropy , increasing contraction force as well as enabling the muscles to relax faster. [ 20 ] [ 21 ]
PKA has always been considered important in formation of a memory . In the fruit fly , reductions in expression activity of DCO (PKA catalytic subunit encoding gene) can cause severe learning disabilities, middle term memory and short term memory. Long term memory is dependent on the CREB transcription factor, regulated by PKA. A study done on drosophila reported that an increase in PKA activity can affect short term memory. However, a decrease in PKA activity by 24% inhibited learning abilities and a decrease by 16% affected both learning ability and memory retention. Formation of a normal memory is highly sensitive to PKA levels. [ 22 ] | https://en.wikipedia.org/wiki/Protein_kinase_A |
Protein splicing is an intramolecular reaction of a particular protein in which an internal protein segment (called an intein ) is removed from a precursor protein with a ligation of C-terminal and N-terminal external proteins (called exteins ) on both sides. The splicing junction of the precursor protein is mainly a cysteine or a serine , which are amino acids containing a nucleophilic side chain . The protein splicing reactions which are known now do not require exogenous cofactors or energy sources such as adenosine triphosphate (ATP) or guanosine triphosphate (GTP). Normally, splicing is associated only with pre-mRNA splicing . This precursor protein contains three segments—an N-extein followed by the intein followed by a C-extein . After splicing has taken place, the resulting protein contains the N-extein linked to the C-extein; this splicing product is also termed an extein.
The first intein was discovered in 1988 through sequence comparison between the Neurospora crassa [ 1 ] and carrot [ 2 ] vacuolar ATPase (without intein) and the homologous gene in yeast (with intein) that was first described as a putative calcium ion transporter . [ 3 ] In 1990 Hirata et al. [ 4 ] demonstrated that the extra sequence in the yeast gene was transcribed into mRNA and removed itself from the host protein only after translation. Since then, inteins have been found in all three domains of life (eukaryotes, bacteria, and archaea) and in viruses .
Protein splicing was unanticipated and its mechanisms were discovered by two groups (Anraku [ 5 ] and Stevens [ 6 ] ) in 1990. They both discovered a Saccharomyces cerevisiae VMA1 in a precursor of a vacuolar H + -ATPase enzyme. The amino acid sequence of the N- and C-termini corresponded to 70% DNA sequence of that of a vacuolar H + -ATPase from other organisms, while the amino acid sequence of the central position corresponded to 30% of the total DNA sequence of the yeast HO nuclease .
Many genes have unrelated intein-coding segments inserted at different positions. For these and other reasons, inteins (or more properly, the gene segments coding for inteins) are sometimes called selfish genetic elements , but it may be more accurate to call them parasitic . According to the gene centered view of evolution, most genes are "selfish" only insofar as to compete with other genes or alleles but usually they fulfill a function for the organisms, whereas "parasitic genetic elements", at least initially, do not make a positive contribution to the fitness of the organism. [ 7 ] [ 8 ]
As of December 2019, the UniProtKB database contains 188 entries manually annotated as inteins, ranging from just tens of amino acid residues to thousands. [ 9 ] The first intein was found encoded within the VMA gene of Saccharomyces cerevisiae . They were later found in fungi ( ascomycetes , basidiomycetes , zygomycetes and chytrids ) and in diverse proteins as well. A protein distantly related to known inteins containing protein, but closely related to metazoan hedgehog proteins , has been described to have the intein sequence from Glomeromycota . Many of the newly described inteins contain homing endonucleases and some of these are apparently active. [ 10 ] The abundance of intein in fungi indicates lateral transfer of intein-containing genes. While in eubacteria and archaea, there are 289 and 182 currently known inteins. Not surprisingly, most intein in eubacteria and archaea are found to be inserted into nucleic acid metabolic protein, like fungi. [ 10 ]
Inteins vary greatly, but many of the same intein-containing proteins are found in a number of species. For example, pre-mRNA processing factor 8 ( Prp8 ) protein, instrumental in the spliceosome , has seven different intein insertion sites across eukaryotic species. [ 11 ] Intein-containing Prp8 is most commonly found in fungi, but is also seen in Amoebozoa , Chlorophyta , Capsaspora , and Choanoflagellida . Many mycobacteria contain inteins within DnaB (bacterial replicative helicase), RecA (bacterial DNA recombinase), and SufB ( FeS cluster assembly protein). [ 12 ] [ 13 ] There is remarkable variety within the structure and number of DnaB inteins, both within the mycobacterium genus and beyond. Interestingly, intein-containing DnaB is also found in the chloroplasts of algae. [ 14 ] Intein-containing proteins found in archaea include RadA (RecA homolog), RFC, PolB, RNR. [ 15 ] Many of the same intein-containing proteins (or their homologs) are found in two or even all three domains of life. Inteins are also seen in the proteomes encoded by bacteriophages and eukaryotic viruses. Viruses may have been involved as vectors of intein distribution across the wide variety of intein containing organisms. [ 15 ]
The process for class 1 inteins begins with an N-O or N-S shift when the side chain of the first residue (a serine , threonine , or cysteine ) of the intein portion of the precursor protein nucleophilically attacks the peptide bond of the residue immediately upstream (that is, the final residue of the N-extein) to form a linear ester (or thioester ) intermediate. A transesterification occurs when the side chain of the first residue of the C-extein attacks the newly formed (thio)ester to free the N-terminal end of the intein. This forms a branched intermediate in which the N-extein and C-extein are attached, albeit not through a peptide bond. The last residue of the intein is always an asparagine (Asn), and the amide nitrogen atom of this side chain cleaves apart the peptide bond between the intein and the C-extein, resulting in a free intein segment with a terminal cyclic imide . Finally, the free amino group of the C-extein now attacks the (thio)ester linking the N- and C-exteins together. An O-N or S-N shift produces a peptide bond and the functional, ligated protein. [ 16 ]
Class 2 inteins have no nucleophilic first side chain, only an alanine. Instead, the reaction starts directly with a nucleophilic displacement, with the first residue of the C-extein atticking the peptide carboxyl on the final residue of the N-extein. The rest proceeds as usual, starting with Asn turning into a cyclic imide. [ 17 ]
Class 3 inteins have no nucleophilic first side chain, only an alanine, yet they have an internal noncontiguous "WCT" motif. The internal C (cysteine) residue attacks the peptide carboxyl on the final residue of the N-extein (nucleophilic displacement). Transesterification occurs when the first residue of the C-extein attacks the newly formed thioester. The rest proceeds as usual. [ 18 ]
The mechanism for the splicing effect is a naturally occurring analogy to the technique for chemically generating medium-sized proteins called native chemical ligation .
An intein is a segment of a protein that is able to excise itself and join the remaining portions (the exteins ) with a peptide bond during protein splicing. [ 19 ] Inteins have also been called protein introns , by analogy with (RNA) introns .
The first part of an intein name is based on the scientific name of the organism in which it is found, and the second part is based on the name of the corresponding gene or extein. For example, the intein found in Thermoplasma acidophilum and associated with Vacuolar ATPase subunit A (VMA) is called "Tac VMA".
Normally, as in this example, just three letters suffice to specify the organism, but there are variations. For example, additional letters may be added to indicate a strain. If more than one intein is encoded in the corresponding gene, the inteins are given a numerical suffix starting from 5 ′ to 3 ′ or in order of their identification (for example, "Msm dnaB-1").
The segment of the gene that encodes the intein is usually given the same name as the intein, but to avoid confusion the name of the intein proper is usually capitalized ( e.g. , Pfu RIR1-1), whereas the name of the corresponding gene segment is italicized ( e.g. , Pfu rir1-1 ). A different disambiguating convention is to place a lowercase "i" after the source protein name, e.g. "Msm DnaBi1". [ 20 ]
Inteins can be classified on many criteria.
Inteins can contain a homing endonuclease gene (HEG) domain in addition to the splicing domains. This domain is responsible for the spread of the intein by cleaving DNA at an intein-free allele on the homologous chromosome , triggering the DNA double-stranded break repair (DSBR) system, which then repairs the break, thus copying the intein-coding DNA into a previously intein-free site. [ 17 ] The HEG domain is not necessary for intein splicing, and so it can be lost, forming a minimal , or mini , intein . Several studies have demonstrated the modular nature of inteins by adding or removing HEG domains and determining the activity of the new construct. [ citation needed ]
Sometimes, the intein of the precursor protein comes from two genes. In this case, the intein is said to be a split intein . For example, in cyanobacteria , DnaE , the catalytic subunit α of DNA polymerase III , is encoded by two separate genes, dnaE-n and dnaE-c . The dnaE-n product consists of an N-extein sequence followed by a 123-AA intein sequence, whereas the dnaE-c product consists of a 36-AA intein sequence followed by a C-extein sequence. [ 21 ]
Inteins are very efficient at protein splicing, and they have accordingly found an important role in biotechnology . There are more than 200 inteins identified to date; sizes range from 100–800 AAs . Inteins have been engineered for particular applications such as protein semisynthesis [ 22 ] and the selective labeling of protein segments, which is useful for NMR studies of large proteins. [ 23 ]
Pharmaceutical inhibition of intein excision may be a useful tool for drug development ; the protein that contains the intein will not carry out its normal function if the intein does not excise, since its structure will be disrupted.
It has been suggested that inteins could prove useful for achieving allotopic expression of certain highly hydrophobic proteins normally encoded by the mitochondrial genome, for example in gene therapy . [ 24 ] The hydrophobicity of these proteins is an obstacle to their import into mitochondria. Therefore, the insertion of a non-hydrophobic intein may allow this import to proceed. Excision of the intein after import would then restore the protein to wild-type .
Affinity tags have been widely used to purify recombinant proteins, as they allow the accumulation of recombinant protein with little impurities. However, the affinity tag must be removed by proteases in the final purification step. The extra proteolysis step raises the problems of protease specificity in removing affinity tags from recombinant protein, and the removal of the digestion product. This problem can be avoided by fusing an affinity tag to self-cleavable inteins in a controlled environment. The first generation of expression vectors of this kind used modified Saccharomyces cerevisiae VMA (Sce VMA) intein. Chong et al. [ 25 ] used a chitin binding domain (CBD) from Bacillus circulans as an affinity tag, and fused this tag with a modified Sce VMA intein. The modified intein undergoes a self-cleavage reaction at its N-terminal peptide linkage with 1,4-dithiothreitol (DTT), β-mercaptoethanol (β-ME), or cystine at low temperatures over a broad pH range. After expressing the recombinant protein, the cell homogenate is passed through the column containing chitin . This allows the CBD of the chimeric protein to bind to the column. Furthermore, when the temperature is lowered and the molecules described above pass through the column, the chimeric protein undergoes self-splicing and only the target protein is eluted. This novel technique eliminates the need for a proteolysis step, and modified Sce VMA stays in column attached to chitin through CBD. [ 25 ]
Recently inteins have been used to purify proteins based on self aggregating peptides. Elastin-like polypeptides (ELPs) are a useful tool in biotechnology. Fused with target protein, they tend to form aggregates inside the cells. [ 26 ] This eliminates the chromatographic step needed in protein purification. The ELP tags have been used in the fusion protein of intein, so that the aggregates can be isolated without chromatography (by centrifugation) and then intein and tag can be cleaved in controlled manner to release the target protein into solution. This protein isolation can be done using continuous media flow, yielding high amounts of protein, making this process more economically efficient than conventional methods. [ 26 ] Another group of researchers used smaller self aggregating tags to isolate target protein. Small amphipathic peptides 18A and ELK16 (figure 5) were used to form self cleaving aggregating protein. [ 27 ]
Over the last twenty years, there has been increasing interest in leveraging inteins for antimicrobial applications. [ 12 ] Intein splicing is found exclusively in unicellular organisms, with a particularly high abundance in pathogenic microorganisms. [ 28 ] Furthermore, inteins are commonly found within housekeeping proteins and/or proteins involved in the survival of the organism within a human host. Post-translational intein removal is necessary for the protein to properly fold and function. For example, Gaëlle Huet et al. demonstrated that in Mycobacterium tuberculosis , unspliced SufB prevents the formation of the SufBCD complex, a component of the SUF machinery. [ 29 ] As such, the inhibition of intein splicing may serve as a powerful platform for the development of antimicrobials.
Current research on intein splicing inhibitors has focused on developing antimycobacterials ( M. tb. has three intein-containing proteins), as well as agents active against pathogenic fungi Cryptococcus and Aspergillus. [ 13 ] Cisplatin and similar platinum-containing compounds inhibit splicing of the M. tb. RecA intein through coordinating to catalytic residues. [ 30 ] Divalent cations, such as copper (II) and zinc (II) ions, function similarly to reversibly inhibit splicing. [ 12 ] However, neither of these methods are currently suitable for an effective and safe antibiotic. The fungal Prp8 intein is also inhibited by divalent cations and cisplatin through interfering with the catalytic Cys1 residue. [ 12 ] In 2021, Li et al. showed that small molecule inhibitors of Prp8 intein splicing were selective and effective at slowing the growth of C. neoformans and C. gattii , providing exciting evidence for the antimicrobial potential of intein splicing inhibitors. [ 31 ] | https://en.wikipedia.org/wiki/Protein_ligation |
The Lowry protein assay is a biochemical assay for determining the total level of protein in a solution . The total protein concentration is exhibited by a color change of the sample solution in proportion to protein concentration, which can then be measured using colorimetric techniques . It is named for the biochemist Oliver H. Lowry who developed the reagent in the 1940s. His 1951 paper describing the technique is the most-highly cited paper ever in the scientific literature, cited over 300,000 times. [ 1 ] [ 2 ] [ 3 ]
The method combines the reactions of copper ions with the peptide bonds under alkaline conditions (the Biuret test ) with the oxidation of aromatic protein residues. The Lowry method is based on the reaction of Cu + , produced by the oxidation of peptide bonds, with Folin–Ciocalteu reagent (a mixture of phosphotungstic acid and phosphomolybdic acid in the Folin–Ciocalteu reaction). The reaction mechanism is not well understood, but involves reduction of the Folin–Ciocalteu reagent and oxidation of aromatic residues (mainly tryptophan , also tyrosine ).
Proper caution must be taken when dealing with the Folin's reagent, which is only active in acidic conditions. Although this is true, the reduction reaction, as previously mentioned, will only occur in basic pH 10. Thus, the reduction must occur before the reagent breaks down. Mixing the protein solution as the Folin's reagent is simultaneously added will ensure that the reaction occurs in the desired manner. [ 4 ]
Experiments have shown that cysteine is also reactive to the reagent. Therefore, cysteine residues in protein probably also contribute to the absorbance seen in the Lowry assay. [ 5 ] The result of this reaction is an intense blue molecule known as heteropolymolybdenum Blue. [ 6 ] The concentration of the reduced Folin reagent (heteropolymolybdenum Blue) is measured by absorbance at 660 nm. [ 7 ] As a result, the total concentration of protein in the sample can be deduced from the concentration of tryptophan and tyrosine residues that reduce the Folin–Ciocalteu reagent.
The method was first proposed by Lowry in 1951. The bicinchoninic acid assay and the Hartree–Lowry assay are subsequent modifications of the original Lowry procedure. | https://en.wikipedia.org/wiki/Protein_measurement_with_the_Folin_phenol_reagent |
A protein microarray (or protein chip ) is a high-throughput method used to track the interactions and activities of proteins, and to determine their function, and determining function on a large scale. [ 1 ] Its main advantage lies in the fact that large numbers of proteins can be tracked in parallel. The chip consists of a support surface such as a glass slide, nitrocellulose membrane, bead, or microtitre plate , to which an array of capture proteins is bound. [ 2 ] Probe molecules, typically labeled with a fluorescent dye, are added to the array. Any reaction between the probe and the immobilised protein emits a fluorescent signal that is read by a laser scanner . [ 3 ] Protein microarrays are rapid, automated, economical, and highly sensitive, consuming small quantities of samples and reagents. [ 4 ] The concept and methodology of protein microarrays was first introduced and illustrated in antibody microarrays (also referred to as antibody matrix ) in 1983 in a scientific publication [ 5 ] and a series of patents. [ 6 ] The high-throughput technology behind the protein microarray was relatively easy to develop since it is based on the technology developed for DNA microarrays , [ 7 ] which have become the most widely used microarrays .
Protein microarrays were developed due to the limitations of using DNA microarrays for determining gene expression levels in proteomics . The quantity of mRNA in the cell often doesn't reflect the expression levels of the proteins they correspond to. Since it is usually the protein, rather than the mRNA, that has the functional role in cell response, a novel approach was needed. Additionally post-translational modifications , which are often critical for determining protein function, are not visible on DNA microarrays. [ 8 ] Protein microarrays replace traditional proteomics techniques such as 2D gel electrophoresis or chromatography , which were time-consuming, labor-intensive and ill-suited for the analysis of low abundant proteins.
The proteins are arrayed onto a solid surface such as microscope slides, membranes, beads or microtitre plates. The function of this surface is to provide a support onto which proteins can be immobilized. It should demonstrate maximal binding properties, whilst maintaining the protein in its native conformation so that its binding ability is retained. Microscope slides made of glass or silicon are a popular choice since they are compatible with the easily obtained robotic arrayers and laser scanners that have been developed for DNA microarray technology. Nitrocellulose film slides are broadly accepted as the highest protein binding substrate for protein microarray applications.
The chosen solid surface is then covered with a coating that must serve the simultaneous functions of immobilising the protein, preventing its denaturation , orienting it in the appropriate direction so that its binding sites are accessible, and providing a hydrophilic environment in which the binding reaction can occur. It also needs to display minimal non-specific binding in order to minimize background noise in the detection systems. Furthermore, it needs to be compatible with different detection systems. Immobilising agents include layers of aluminium or gold, hydrophilic polymers, and polyacrylamide gels , or treatment with amines , aldehyde or epoxy . Thin-film technologies like physical vapour deposition (PVD) and chemical vapour deposition (CVD) are employed to apply the coating to the support surface.
An aqueous environment is essential at all stages of array manufacture and operation to prevent protein denaturation. Therefore, sample buffers contain a high percent of glycerol (to lower the freezing point), and the humidity of the manufacturing environment is carefully regulated. Microwells have the dual advantage of providing an aqueous environment while preventing cross-contamination between samples.
In the most common type of protein array, robots place large numbers of proteins or their ligands onto a coated solid support in a pre-defined pattern. This is known as robotic contact printing or robotic spotting. Another fabrication method is ink-jetting , a drop-on-demand, non-contact method of dispersing the protein polymers onto the solid surface in the desired pattern. [ 9 ] Piezoelectric spotting is a similar method to ink-jet printing. The printhead moves across the array, and at each spot uses electric stimulation to deliver the protein molecules onto the surface via tiny jets. This is also a non-contact process. [ 10 ] Photolithography is a fourth method of arraying the proteins onto the surface. Light is used in association with photomasks , opaque plates with holes or transparencies that allow light to shine through in a defined pattern. A series of chemical treatments then enables deposition of the protein in the desired pattern upon the material underneath the photomask. [ 11 ]
The capture molecules arrayed on the solid surface may be antibodies , antigens , aptamers (nucleic acid-based ligands), affibodies (small molecules engineered to mimic monoclonal antibodies), or full length proteins. Sources of such proteins include cell-based expression systems for recombinant proteins , purification from natural sources, production in vitro by cell-free translation systems , and synthetic methods for peptides . Many of these methods can be automated for high throughput production but care must be taken to avoid conditions of synthesis or extraction that result in a denatured protein which, since it no longer recognizes its binding partner, renders the array useless.
Proteins are highly sensitive to changes in their microenvironment. This presents a challenge in maintaining protein arrays in a stable condition over extended periods of time. In situ methods — invented and published by Mingyue He and Michael Taussig in 2001 [ 12 ] [ 13 ] — involve on-chip synthesis of proteins as and when required, directly from the DNA using cell-free protein expression systems. Since DNA is a highly stable molecule it does not deteriorate over time and is therefore suited to long-term storage. This approach is also advantageous in that it circumvents the laborious and often costly processes of separate protein purification and DNA cloning , since proteins are made and immobilised simultaneously in a single step on the chip surface. Examples of in situ techniques are PISA (protein in situ array), NAPPA (nucleic acid programmable protein array) and DAPA (DNA array to protein array).
There are three types of protein microarrays that are currently used to study the biochemical activities of proteins.
Analytical microarrays are also known as capture arrays. In this technique, a library of antibodies, aptamers or affibodies is arrayed on the support surface. These are used as capture molecules since each binds specifically to a particular protein. The array is probed with a complex protein solution such as a cell lysate . Analysis of the resulting binding reactions using various detection systems can provide information about expression levels of particular proteins in the sample as well as measurements of binding affinities and specificities. This type of microarray is especially useful in comparing protein expression in different solutions. For instance the response of the cells to a particular factor can be identified by comparing the lysates of cells treated with specific substances or grown under certain conditions with the lysates of control cells. Another application is in the identification and profiling of diseased tissues.
Reverse phase protein microarray (RPPA) involve complex samples, such as tissue lysates. Cells are isolated from various tissues of interest and are lysed. The lysate is arrayed onto the microarray and probed with antibodies against the target protein of interest. These antibodies are typically detected with chemiluminescent , fluorescent or colorimetric assays. Reference peptides are printed on the slides to allow for protein quantification of the sample lysates. RPAs allow for the determination of the presence of altered proteins or other agents that may be the result of disease. Specifically, post-translational modifications, which are typically altered as a result of disease can be detected using RPAs. [ 14 ]
Functional protein microarrays (also known as target protein arrays) are constructed by immobilising large numbers of purified proteins and are used to identify protein–protein, protein–DNA, protein– RNA , protein– phospholipid , and protein–small-molecule interactions, to assay enzymatic activity and to detect antibodies and demonstrate their specificity. They differ from analytical arrays in that functional protein arrays are composed of arrays containing full-length functional proteins or protein domains. These protein chips are used to study the biochemical activities of the entire proteome in a single experiment.
The key element in any functional protein microarray-based assay is the arrayed proteins must retain their native structure, such that meaningful functional interactions can take place on the array surface. The advantages of controlling the precise mode of surface attachment through use of an appropriate affinity tag are that the immobilised proteins will have a homogeneous orientation resulting in a higher specific activity and higher signal-to-noise ratio in assays, with less interference from non-specific interactions. [ 15 ] [ 16 ]
Protein array detection methods must give a high signal and a low background. The most common and widely used method for detection is fluorescence labeling which is highly sensitive, safe and compatible with readily available microarray laser scanners. Other labels can be used, such as affinity, photochemical or radioisotope tags. These labels are attached to the probe itself and can interfere with the probe-target protein reaction. Therefore, a number of label free detection methods are available, such as surface plasmon resonance (SPR), carbon nanotubes, carbon nanowire sensors (where detection occurs via changes in conductance) and microelectromechanical system (MEMS) cantilevers. [ 17 ] All these label free detection methods are relatively new and are not yet suitable for high-throughput protein interaction detection; however, they do offer much promise for the future. Immunoassays on thiol-ene "synthetic paper" micropillar scaffolds have shown to generate a superior fluorescence signal. [ 18 ]
Protein quantitation on nitrocellulose coated glass slides can use near-IR fluorescent detection. This limits interferences due to auto-fluorescence of the nitrocellulose at the UV wavelengths used for standard fluorescent detection probes. [ 19 ]
There are five major areas where protein arrays are being applied: diagnostics, proteomics, protein functional analysis, antibody characterization, and treatment development.
Diagnostics involves the detection of antigens and antibodies in blood samples; the profiling of sera to discover new disease biomarkers ; the monitoring of disease states and responses to therapy in personalized medicine; the monitoring of environment and food. Digital bioassay is an example of using protein microarray for diagnostic purposes. In this technology, an array of microwells on a glass/polymer chip are seeded with magnetic beads (coated with fluorescent tagged antibodies), subjected to targeted antigens and then characterised by a microscope through counting fluorescing wells. A cost-effective fabrication platform (using OSTE polymers ) for such microwell arrays has been recently demonstrated and the bio-assay model system has been successfully characterised. [ 20 ]
Proteomics pertains to protein expression profiling i.e. which proteins are expressed in the lysate of a particular cell.
Protein functional analysis is the identification of protein–protein interactions (e.g. identification of members of a protein complex), protein–phospholipid interactions, small molecule targets, enzymatic substrates (particularly the substrates of kinases ) and receptor ligands.
Antibody characterization is characterizing cross-reactivity , specificity and mapping epitopes .
Treatment development involves the development of antigen-specific therapies for autoimmunity , cancer and allergies; the identification of small molecule targets that could potentially be used as new drugs.
Despite the considerable investments made by several companies, proteins chips have yet to flood the market. Manufacturers have found that proteins are actually quite difficult to handle. Production of reliable, consistent, high-throughput proteins that are correctly folded and functional is fraught with difficulties as they often result in low-yield of proteins due to decreased solubility and formation of inclusion bodies. [ citation needed ] A protein chip requires a lot more steps in its creation than does a DNA chip .
There are a number of approaches to this problem which differ fundamentally according to whether the proteins are immobilised through non-specific, poorly defined interactions, or through a specific set of known interactions. The former approach is attractive in its simplicity and is compatible with purified proteins derived from native or recombinant sources [ 21 ] [ 22 ] but suffers from a number of risks. Most notable amongst these relate to the uncontrolled nature of the interactions between each protein and the surface; at best, this might give rise to a heterogeneous population of proteins in which active sites are sometimes occluded by the surface; at worst, it might destroy activity altogether due to partial or complete surface-mediated unfolding of the immobilised protein.
Challenges include: 1) finding a surface and a method of attachment that allows the proteins to maintain their secondary or tertiary structure and thus their biological activity and their interactions with other molecules, 2) producing an array with a long shelf life so that the proteins on the chip do not denature over a short time, 3) identifying and isolating antibodies or other capture molecules against every protein in the human genome, 4) quantifying the levels of bound protein while assuring sensitivity and avoiding background noise, 5) extracting the detected protein from the chip in order to further analyze it, 6) reducing non-specific binding by the capture agents, 7) the capacity of the chip must be sufficient to allow as complete a representation of the proteome to be visualized as possible; abundant proteins overwhelm the detection of less abundant proteins such as signaling molecules and receptors, which are generally of more therapeutic interest. [ 23 ] | https://en.wikipedia.org/wiki/Protein_microarray |
A protein mimetic is a molecule such as a peptide , a modified peptide or any other molecule that biologically mimics the action or activity of some other protein . Protein mimetics are commonly used in drug design and discovery .
There are a number of different distinct classes of protein mimetics. | https://en.wikipedia.org/wiki/Protein_mimetic |
Protein misfolding cyclic amplification ( PMCA ) is an amplification technique (conceptually like polymerase chain reaction (PCR) but not involving nucleotides ) to multiply misfolded prions originally developed by Soto and colleagues. [ 1 ] It is a test for spongiform encephalopathies like chronic wasting disease (CWD) [ 2 ] or bovine spongiform encephalopathy (BSE).
The technique initially incubates a small amount of abnormal prion with an excess of normal protein, so that some conversion takes place. The growing chain of misfolded protein is then blasted with ultrasound , breaking it down into smaller chains and so rapidly increasing the amount of abnormal protein available to cause conversions. [ 1 ] [ 3 ] By repeating the cycle, the mass of normal protein is rapidly changed into the prion being tested for. [ citation needed ]
PMCA was originally developed to, in vitro , mimic prion replication with a similar efficiency to the in vivo process, but with accelerated kinetics. [ 1 ] PMCA is conceptually analogous to the polymerase chain reaction - in both systems a template grows at the expense of a substrate in a cyclic reaction, combining growing and multiplication of the template units. [ citation needed ]
PMCA has been applied to replicate the misfolded protein from diverse species. [ 4 ] [ 5 ] [ 6 ] The newly generated protein exhibits the same biochemical, biological, and structural properties as brain-derived PrP Sc and strikingly it is infectious to wild type animals, producing a disease with similar characteristics as the illness produced by brain-isolated prions. [ 7 ]
The technology has been automated, leading to a dramatic increase in the efficiency of amplification. Now, a single cycle results in a 2500-fold increase in sensitivity of detection over western blotting, [ 8 ] whereas 2 and 7 consecutive cycles result in 6 million and 3 billion-fold increases in sensitivity of detection over western blotting , a technique widely used in BSE surveillance in several countries. [ 8 ]
It has been shown that PMCA is capable of detecting as little as a single molecule of oligomeric infectious PrP Sc . [ 8 ] PMCA possesses the ability to generate millions infectious units, starting with the equivalent to one PrP Sc oligomer; well below the infectivity threshold. [ 8 ] This data demonstrates that PMCA has a similar power of amplification as PCR techniques used to amplify DNA. It opens a great promise for development of a highly sensitive detection of PrP Sc , and for understanding the molecular basis of prion replication. Indeed, PMCA has been used by various groups to PrP Sc in blood of animals experimentally infected with prions during both the symptomatic [ 9 ] and pre-symptomatic phases [ 10 ] as well as in urine. [ 11 ]
The PMCA technology has been used by several groups to understand the molecular mechanism of prion replication, the nature of the infectious agent, the phenomenon of prion strains and species barrier, the effect of cellular components, to detect PrP Sc in tissues and biological fluids and to screen for inhibitors against prion replication. [ 12 ] [ 13 ] [ 14 ] Recent studies by the groups of Supattapone and Ma were able to produce prion replication in vitro by PMCA using purified PrP C and recombinant PrP C with the sole addition of synthetic polyanions and lipids . [ 15 ] [ 16 ] These studies have shown that infectious prions can be produced in the absence of any other cellular component and constitute some of the strongest evidence in favor of the prion hypothesis.
Research in 2020 concluded that protein misfolding cyclic amplification could be used to distinguish between two progressive neurodegenerative diseases, Parkinson's disease and multiple system atrophy , being the first process to give an objective diagnosis of Multiple System Atrophy instead of just a differential diagnosis. [ 17 ] [ 18 ] | https://en.wikipedia.org/wiki/Protein_misfolding_cyclic_amplification |
Protein moonlighting is a phenomenon by which a protein can perform more than one function. [ 2 ] It is an excellent [ according to whom? ] example of gene sharing . [ 3 ]
Ancestral moonlighting proteins originally possessed a single function but, through evolution , acquired additional functions. Many proteins that moonlight are enzymes ; others are receptors , ion channels or chaperones . The most common primary function of moonlighting proteins is enzymatic catalysis , but these enzymes have acquired secondary non-enzymatic roles. Some examples of functions of moonlighting proteins secondary to catalysis include signal transduction , transcriptional regulation , apoptosis , motility , and structural. [ 4 ]
Protein moonlighting occurs widely in nature. [ 5 ] [ 6 ] [ 7 ] Protein moonlighting through gene sharing differs from the use of a single gene to generate different proteins by alternative RNA splicing , DNA rearrangement, or post-translational processing . It is also different from the multifunctionality of the protein, in which the protein has multiple domains, each serving a different function. Protein moonlighting by gene sharing means that a gene may acquire and maintain a second function without gene duplication and without loss of the primary function. Such genes are under two or more entirely different selective constraints. [ 8 ]
Various techniques have been used to reveal moonlighting functions in proteins. The detection of a protein in unexpected locations within cells, cell types, or tissues may suggest that a protein has a moonlighting function. Furthermore, the sequence or structure homology of a protein may be used to infer both primary functions as well as secondary moonlighting functions of a protein.
The most well-studied examples of gene sharing are crystallins . These proteins, when expressed at low levels in many tissues function as enzymes, but when expressed at high levels in eye tissue, become densely packed and thus form lenses. While the recognition of gene sharing is relatively recent—the term was coined in 1988, after crystallins in chickens and ducks were found to be identical to separately identified enzymes—recent studies have found many examples throughout the living world. Joram Piatigorsky has suggested that many or all proteins exhibit gene sharing to some extent, and that gene sharing is a key aspect of molecular evolution . [ 9 ] : 1–7 The genes encoding crystallins must maintain sequences for catalytic function and transparency maintenance function. [ 8 ]
Inappropriate moonlighting is a contributing factor in some genetic diseases, and moonlighting provides a possible mechanism by which bacteria may become resistant to antibiotics. [ 10 ]
The first observation of a moonlighting protein was made in the late 1980s by Joram Piatigorsky and Graeme Wistow during their research on crystallin enzymes. Piatigorsky determined that lens crystallin conservation and variance are due to other moonlighting functions outside of the lens. [ 11 ] Originally Piatigorsky called these proteins "gene sharing" proteins, but the colloquial description moonlighting was subsequently applied to proteins by Constance Jeffery in 1999 [ 12 ] to draw a similarity between multitasking proteins and people who work two jobs. [ 13 ] The phrase "gene sharing" is ambiguous since it is also used to describe horizontal gene transfer , hence the phrase "protein moonlighting" has become the preferred description for proteins with more than one function. [ 13 ]
It is believed that moonlighting proteins came about by means of evolution through which uni-functional proteins gained the ability to perform multiple functions. With alterations, much of the protein's unused space can provide new functions. [ 10 ] Many moonlighting proteins are the result of the gene fusion of two single function genes. [ 14 ] Alternatively a single gene can acquire a second function since the active site of the encoded protein typically is small compared to the overall size of the protein leaving considerable room to accommodate a second functional site. In yet a third alternative, the same active site can acquire a second function through mutations of the active site.
The development of moonlighting proteins may be evolutionarily favorable to the organism since a single protein can do the job of multiple proteins conserving amino acids and energy required to synthesize these proteins. [ 12 ] However, there is no universally agreed upon theory that explains why proteins with multiple roles evolved. [ 12 ] [ 13 ] While using one protein to perform multiple roles seems advantageous because it keeps the genome small, we can conclude that this is probably not the reason for moonlighting because of the large amount of noncoding DNA . [ 13 ]
Many proteins catalyze a chemical reaction . Other proteins fulfill structural, transport, or signaling roles. Furthermore, numerous proteins have the ability to aggregate into supramolecular assemblies . For example, a ribosome is made up of 90 proteins and RNA .
A number of the currently known moonlighting proteins are evolutionarily derived from highly conserved enzymes, also called ancient enzymes. These enzymes are frequently speculated to have evolved moonlighting functions. Since highly conserved proteins are present in many different organisms, this increases the chance that they would develop secondary moonlighting functions. [ 13 ] A high fraction of enzymes involved in glycolysis , an ancient universal metabolic pathway, exhibit moonlighting behavior. Furthermore, it has been suggested that as many as 7 out of 10 proteins in glycolysis and 7 out of 8 enzymes of the tricarboxylic acid cycle exhibit moonlighting behavior. [ 4 ]
An example of a moonlighting enzyme is pyruvate carboxylase . This enzyme catalyzes the carboxylation of pyruvate into oxaloacetate , thereby replenishing the tricarboxylic acid cycle . Surprisingly, in yeast species such as H. polymorpha and P. pastoris , pyruvate carboylase is also essential for the proper targeting and assembly of the peroxisomal protein alcohol oxidase (AO). AO, the first enzyme of methanol metabolism, is a homo-octameric flavoenzyme . In wild type cells, this enzyme is present as enzymatically active AO octamers in the peroxisomal matrix. However, in cells lacking pyruvate carboxylase, AO monomers accumulate in the cytosol, indicating that pyruvate carboxylase has a second fully unrelated function in assembly and import. The function in AO import/assembly is fully independent of the enzyme activity of pyruvate carboxylase, because amino acid substitutions can be introduced that fully inactivate the enzyme activity of pyruvate carboxylase, without affecting its function in AO assembly and import. Conversely, mutations are known that block the function of this enzyme in the import and assembly of AO, but have no effect on the enzymatic activity of the protein. [ 13 ]
The E. coli anti-oxidant thioredoxin protein is another example of a moonlighting protein. Upon infection with the bacteriophage T7 , E. coli thioredoxin forms a complex with T7 DNA polymerase , which results in enhanced T7 DNA replication, a crucial step for successful T7 infection. Thioredoxin binds to a loop in T7 DNA polymerase to bind more strongly to the DNA. The anti-oxidant function of thioredoxin is fully autonomous and fully independent of T7 DNA replication, in which the protein most likely fulfills the functional role. [ 13 ]
ADT2 and ADT5 are other examples of moonlighting proteins found in plants. Both of these proteins have roles in phenylalanine biosynthesis like all other ADTs. However ADT2, together with FtsZ is necessary in chloroplast division and ADT5 is transported by stromules into the nucleus. [ 15 ]
In many cases, the functionality of a protein not only depends on its structure, but also its location. For example, a single protein may have one function when found in the cytoplasm of a cell, a different function when interacting with a membrane, and yet a third function if excreted from the cell. This property of moonlighting proteins is known as "differential localization". [ 19 ] For example, in higher temperatures DegP ( HtrA ) will function as a protease by the directed degradation of proteins and in lower temperatures as a chaperone by assisting the non-covalent folding or unfolding and the assembly or disassembly of other macromolecular structures. [ 10 ] Furthermore, moonlighting proteins may exhibit different behaviors not only as a result of its location within a cell, but also the type of cell that the protein is expressed in. [ 19 ] Multifunctionality could also be as a consequence of differential post translational modifications (PTMs). [ 20 ] In the case of the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase ( GAPDH ) alterations in the PTMs have been shown to be associated with higher order multi functionality. [ 21 ] [ 22 ]
Other methods through which proteins may moonlight are by changing their oligomeric state, altering concentrations of the protein's ligand or substrate, use of alternative binding sites, or finally through phosphorylation . An example of a protein that displays different function in different oligomeric states is pyruvate kinase which exhibits metabolic activity as a tetramer and thyroid hormone –binding activity as a monomer. Changes in the concentrations of ligands or substrates may cause a switch in a protein's function. For example, in the presence of high iron concentrations, aconitase functions as an enzyme while at low iron concentration, aconitase functions as an iron-responsive element-binding protein (IREBP) to increase iron uptake. Proteins may also perform separate functions through the use of alternative binding sites that perform different tasks. An example of this is ceruloplasmin , a protein that functions as an oxidase in copper metabolism and moonlights as a copper-independent glutathione peroxidase . Lastly, phosphorylation may sometimes cause a switch in the function of a moonlighting protein. For example, phosphorylation of phosphoglucose isomerase (PGI) at Ser-185 by protein kinase CK2 causes it to stop functioning as an enzyme, while retaining its function as an autocrine motility factor. [ 4 ] Hence when a mutation takes place that inactivates a function of a moonlighting proteins, the other function(s) are not necessarily affected. [ 13 ]
The crystal structures of several moonlighting proteins, such as I-AniI homing endonuclease / maturase [ 23 ] and the PutA proline dehydrogenase / transcription factor , [ 24 ] have been determined. [ 25 ] An analysis of these crystal structures has demonstrated that moonlighting proteins can either perform both functions at the same time, or through conformational changes , alternate between two states, each of which is able to perform a separate function. For example, the protein DegP plays a role in proteolysis with higher temperatures and is involved in refolding functions at lower temperatures. [ 25 ] Lastly, these crystal structures have shown that the second function may negatively affect the first function in some moonlighting proteins. As seen in ƞ-crystallin, the second function of a protein can alter the structure, decreasing the flexibility, which in turn can impair enzymatic activity somewhat. [ 25 ]
Moonlighting proteins have usually been identified by chance because there is no clear procedure to identify secondary moonlighting functions. Despite such difficulties, the number of moonlighting proteins that have been discovered is rapidly increasing. Furthermore, moonlighting proteins appear to be abundant in all kingdoms of life. [ 13 ]
Various methods have been employed to determine a protein's function including secondary moonlighting functions. For example, the tissue, cellular, or subcellular distribution of a protein may provide hints as to the function. Real-time PCR is used to quantify mRNA and hence infer the presence or absence of a particular protein which is encoded by the mRNA within different cell types. Alternatively immunohistochemistry or mass spectrometry can be used to directly detect the presence of proteins and determine in which subcellular locations, cell types, and tissues a particular protein is expressed.
Mass spectrometry may be used to detect proteins based on their mass-to-charge ratio . Because of alternative splicing and posttranslational modification , identification of proteins based on the mass of the parent ion alone is very difficult. However tandem mass spectrometry in which each of the parent peaks is in turn fragmented can be used to unambiguously identify proteins. Hence tandem mass spectrometry is one of the tools used in proteomics to identify the presence of proteins in different cell types or subcellular locations. While the presence of a moonlighting protein in an unexpected location may complicate routine analyses, at the same time, the detection of a protein in unexpected multiprotein complexes or locations suggests that protein may have a moonlighting function. [ 19 ] Furthermore, mass spectrometry may be used to determine if a protein has high expression levels that do not correlate to the enzyme's measured metabolic activity. These expression levels may signify that the protein is performing a different function than previously known. [ 4 ]
The structure of a protein can also help determine its functions. Protein structure in turn may be elucidated with various techniques including X-ray crystallography or NMR . Dual-polarization interferometry may be used to measure changes in protein structure which may also give hints to the protein's function. Finally, application of systems biology approaches [ 26 ] such as interactomics give clues to a proteins function based on what it interacts with.
In the case of the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH), in addition to the large number of alternate functions it has also been observed that it can be involved in the same function by multiple means (multifunctionality within multifunctionality). For example, in its role in maintenance of cellular iron homeostasis GAPDH can function to import or extrude iron from cells. Moreover, in case of its iron import activities it can traffic into cells holo-transferrin as well as the related molecule lactoferrin by multiple pathways. [ 27 ]
In the case of crystallins , the genes must maintain sequences for catalytic function and transparency maintenance function. [ 8 ] The abundant lens crystallins have been generally viewed as static proteins serving a strictly structural role in transparency and cataract . [ 28 ] However, recent studies have shown that the lens crystallins are much more diverse than previously recognized and that many are related or identical to metabolic enzymes and stress proteins found in numerous tissues. [ 29 ] Unlike other proteins performing highly specialized tasks, such as globin or rhodopsin , the crystallins are very diverse and show numerous species differences. Essentially all vertebrate lenses contain representatives of the α and β/γ crystallins, the "ubiquitous crystallins", which are themselves heterogeneous, and only few species or selected taxonomic groups use entirely different proteins as lens crystallins. This paradox of crystallins being highly conserved in sequence while extremely diverse in number and distribution shows that many crystallins have vital functions outside the lens and cornea, and this multi-functionality of the crystallins is achieved by moonlightining. [ 30 ]
Crystallin recruitment may occur by changes in gene regulation that leads to high lens expression. One such example is gluthathione S-transferase/S11-crystallin that was specialized for lens expression by change in gene regulation and gene duplication . The fact that similar transcriptional factors such as Pax-6, and retinoic acid receptors, regulate different crystalline genes, suggests that lens-specific expression have played a crucial role for recruiting multifunctional protein as crystallins. Crystallin recruitment has occurred both with and without gene duplication, and tandem gene duplication has taken place among some of the crystallins with one of the duplicates specializing for lens expression. Ubiquitous α –crystallins and bird δ –crystallins are two examples. [ 31 ]
The α-crystallins, which contributed to the discovery of crystallins as borrowed proteins, [ 32 ] have continually supported the theory of gene sharing, and helped delineating the mechanisms used for gene sharing as well. There are two α-crystallin genes (αA and αB), which are about 55% identical in amino acid sequence. [ 29 ] Expression studies in non-lens cells showed that the αB-crystallin, other than being a functional lens protein, is a functional small heat shock protein. [ 33 ] αB-crystallin is induced by heat and other physiological stresses, and it can protect the cells from elevated temperatures [ 34 ] and hypertonic stress. [ 35 ] αB-crystallin is also overexpressed in many pathologies, including neurodegenerative diseases , fibroblasts of patients with Werner syndrome showing premature senescence, and growth abnormalities. In addition to being overexpressed under abnormal conditions, αB-crystallin is constitutively expressed in heart, skeletal muscle, kidney, lung and many other tissues. [ 36 ] In contrast to αB-crystallin, except for low-level expression in the thymus, spleen and retina, [ 37 ] αA-crystallin is highly specialized for expression in the lens [ 38 ] and is not stress-inducible. However, like αB-crystallin, it can also function as molecular chaperone and protect against thermal stress.
β/γ-crystallins are different from α-crystallins in that they are a large multigene family. Other proteins like bacterial spore coat, a slime mold cyst protein, and epidermis differentiation-specific protein, contain the same Greek key motifs and are placed under β/γ crystallin superfamily. This relationship supports the idea that β/γ- crystallins have been recruited by a gene-sharing mechanism. However, except for few reports, non-refractive function of the β/γ-crystallin is yet to be found. [ 30 ]
Similar to lens , cornea is a transparent, avascular tissue derived from the ectoderm that is responsible for focusing light onto the retina . However, unlike lens, cornea depends on the air-cell interface and its curvature for refraction. Early immunology studies have shown that BCP 54 comprises 20–40% of the total soluble protein in bovine cornea. [ 39 ] Subsequent studies have indicated that BCP 54 is ALDH3, a tumor and xenobiotic-inducible cytosolic enzyme, found in human, rat, and other mammals. [ 40 ]
While it is evident that gene sharing resulted in many of lens crystallins being multifunctional proteins, it is still uncertain to what extent the crystallins use their non-refractive properties in the lens, or on what basis they were selected. The α-crystallins provide a convincing case for a lens crystallin using its non-refractive ability within the lens to prevent protein aggregation under a variety of environmental stresses [ 41 ] and to protect against enzyme inactivation by post-translational modifications such as glycation . [ 42 ] The α-crystallins may also play a functional role in the stability and remodeling of the cytoskeleton during fiber cell differentiation in the lens. [ 43 ] In cornea, ALDH3 is also suggested to be responsible for absorbing UV-B light. [ 44 ]
Based on the similarities between lens and cornea, such as abundant water-soluble enzymes, and being derived from ectoderm, the lens and cornea are thought to be co-evolved as a "refraction unit." Gene sharing would maximize light transmission and refraction to the retina by this refraction unit. Studies have shown that many water-soluble enzymes/proteins expressed by cornea are identical to taxon-specific lens crystallins, such as ALDH1A1/ η-crystallin, α-enolase/τ-crystallin, and lactic dehydrogenase/ -crystallin. Also, the anuran corneal epithelium, which can transdifferentiate to regenerate the lens, abundantly expresses ubiquitous lens crystallins, α, β and γ, in addition to the taxon-specific crystallin α-enolase/τ-crystallin. Overall, the similarity in expression of these proteins in the cornea and lens, both in abundance and taxon-specificity, supports the idea of co-evolution of lens and cornea through gene sharing. [ 45 ]
Gene sharing is related to, but distinct from, several concepts in genetics, evolution, and molecular biology. Gene sharing entails multiple effects from the same gene, but unlike pleiotropy , it necessarily involves separate functions at the molecular level. A gene could exhibit pleiotropy when single enzyme function affects multiple phenotypic traits ; mutations of a shared gene could potentially affect only a single trait. Gene duplication followed by differential mutation is another phenomenon thought to be a key element in the evolution of protein function, but in gene sharing, there is no divergence of gene sequence when proteins take on new functions; the single polypeptide takes on new roles while retaining old ones. Alternative splicing can result in the production of multiple polypeptides (with multiple functions) from a single gene, but by definition, gene sharing involves multiple functions of a single polypeptide. [ 9 ] : 8–14
The multiple roles of moonlighting proteins complicates the determination of phenotype from genotype , [ 4 ] hampering the study of inherited metabolic disorders .
The complex phenotypes of several disorders are suspected to be caused by the involvement of moonlighting proteins. The protein GAPDH has at least 11 documented functions, one of which includes apoptosis. Excessive apoptosis is involved in many neurodegenerative diseases, such as Huntington's , Alzheimer's , and Parkinson's as well as in brain ischemia . In one case, GAPDH was found in the degenerated neurons of individuals who had Alzheimer's disease. [ 4 ]
Although there is insufficient evidence for definite conclusions, there are well documented examples of moonlighting proteins that play a role in disease. One such disease is tuberculosis . One moonlighting protein in M. tuberculosis has a function which counteracts the effects of antibiotics. [ 10 ] [ 13 ] Specifically, the bacterium gains antibiotic resistance against ciprofloxacin from overexpression of glutamate racemase in vivo . [ 10 ] GAPDH localized to the surface of pathogenic mycobacteriea has been shown to capture and traffic the mammalian iron carrier protein transferrin into cells resulting in iron acquisition by the pathogen. [ 46 ] | https://en.wikipedia.org/wiki/Protein_moonlighting |
Protein nanotechnology is a field of research that integrates the diverse physicochemical properties of proteins with nanoscale technology. This field assimilated into pharmaceutical research to give rise to a new classification of nanoparticles termed protein (or protein-based) nanoparticles (PNPs). PNPs garnered significant interest due to their favorable pharmacokinetic properties such as high biocompatibility, biodegradability, and low toxicity [ 1 ] [ 2 ] [ 3 ] [ 4 ] [ 5 ] Together, these characteristics have the potential to overcome the challenges encountered with synthetic NPs drug delivery strategies. These existing challenges including low bioavailability, a slow excretion rate, high toxicity, and a costly manufacturing process, will open the door to considerable therapeutic advancements within oncology, theranostics, and clinical translational research. [ 2 ] [ 4 ]
Continued advancement within this field is required for the clinical translation of PNPs. As of 2022, only one PNP formulation (Abraxane) and five VLPs (Gardasil, Ceravix, Mosquirix, Sci-B-Vac, Gardasil9) are approved by the FDA for clinical use. FDA approval of PNPs formulations is restrained by complications arising from in-vivo interactions between PNPs and the biological environment that jeopardize their safety or function. [ 6 ] [ 7 ] For example, PNPs may undergo protein conformation changes, form a protein corona, or induce inflammation and may risk patient well-being. [ 4 ]
To capitalize on the favorable characteristics of PNPs, improvements within PNP synthesis methods are being widely explored. Advancements or the development of new synthesis methods are desirable as existing methods (sonochemistry, thermal decomposition, and colloidal/ hydrothermal/microemulsion methods) contribute to systemic toxicity and are limited to hydrophilic drugs. As a result, recent advancements seek to overcome these challenges and achieve commercial-size production. [ 2 ] [ 8 ] [ 9 ] [ 10 ] [ 11 ]
In addition, newly developed PNP synthesis methods such as electrospray or desolvation provide a more sustainable approach as compared to traditional nanoparticle methods. [ 2 ] [ 9 ] Unlike synthetic nanoparticles, PNPs can be synthesized under mild conditions and without toxic chemicals or organic solvents. PNPs are also naturally sourced and readily degradable. Yet, despite these advantages and the addition of new synthesis methods, the methods remain relatively expensive and do not deliver full control of PNP size, greatly limiting their application in biomedicine [ 2 ] [ 12 ]
- High stability
- Shape control
- Cost-effective
- Fast
- Requires surfactants, stabilizers for thermodynamic stability
- Relative volume ratio of water and oil
- Simple
- Small NPs
- High encapsulation efficiency
- Control over shape and size
- Limited to proteins that can be diluted by transporter proteins
- Speed, pH, temperature, and addition rate of desolvating agent
- Type and concentration of desolvating agent
- Stirring rate
- Buffer type
- Ionic strength
- Temperature, pH during cross-linking
Albumin
Zein
Casein
- Simple
- Low cost
- Continuous manufacture
- High yield
- High stability
- Small size
- Mass production
- Low flow
- Magnitude of applied voltage
- Flow rate
ELPs
- Small size
- High stability
- Risk of degradation from strain
- Protein and nucleic acid interactions
Casein
Albumin
Zein
- Fast
- Simple
- Readily encapsulates hydrophilic or heat-sensitive drugs
- Control over particle size
- Difficult to incorporate hydrophobic drugs
- Flow rate
Numerous proteins are utilized in PNP synthesis. They are often sourced naturally from animal and plant sources. Accordingly, generally shared advantages of animal proteins include high biocompatibility, biodegradability, non-immunogenicity, drug loading efficiency, cell uptake, and easy and cost-effective production. [ 15 ] Tables 2–4 below compile the common proteins used in PNP synthesis. The types of PNPs share similar physical properties such as high biocompatibility, non-immunogenicity, high drug efficiency, high biodegradability, and high cell uptake. [ 3 ] [ 16 ] [ 17 ] Due to the abundance of proteins necessary for proper bodily function, the body has developed processes to update proteins into tissues and cells. PNPs take advantage of these natural processes to enhance their cellular uptake. This abundance and the natural sourcing subsequent purification of the proteins also reduce the immunogenic responses and produce low toxicity levels in the body. As the PNPs are degraded, the tissues assimilate the amino acids into energy or protein production. [ 4 ]
- Biodegradable
- FDA approved safety
- Easy to crosslink
- Easy to sterilize
- Inexpensive
- Quick degradation
- Non-immunogenic
- Biocompatible
- Biodegradable
- High binding capacity
- Versatile
- Water-soluble
- Simple preparation
Nanocapsules
- High stability
- Easy procurement
- High stability
- High binding capacity
- High-temperature resistance
- Durable to mechanical forces
- Low decomposition rate
- Mechanically flexible
- High mechanical strength
- Good stability
- Low immunogenicity
- Biodegradable
- Biocompatible
- Cost-effective
Micelles
- Can carry hydrophobic drugs
- Non-toxic
- Low water absorption
- High-temperature resistance
- Sensitive to enzymatic degradation
Nanocapsules
- Biodegradable
- Nontoxic
- High stability
- Low solubility
- Non-immunogenicity
- Rapid degradation
- Low toxicity
- Low immunogenicity
- Resistance to degradation
- High stability
- Low antigenicity
- Control over molecular weight
- Production of single-sized polymers
- Can bind to several drugs at once
- Tunable pharmacokinetic properties
- Environmentally responsive
- Safer than traditional vaccines
- Small size
- Intrinsic immunogenicity
PNPs can be chemically modified to increase particle stability, reduce degradation, and enhance favorable characteristics. Crosslinking is a common modification that can utilize synthetic or natural cross-linkers. Natural cross-linkers are significantly less toxic than synthetic cross-linkers. [ 27 ]
Driving factors in the modification of PNPs stem from their surface properties (surface charge, hydrophobicity , functional groups , etc.). Functional groups can bind to tissue-specific ligands for targeted drug delivery. Functional ligands may include protein receptors, antibodies, and smaller peptides. The purpose of ligand binding is to direct the PNP to the target cells, thereby reducing systemic toxicity, and improving the retention and excretion of the PNP within tissues. The optimal ligand for PNP modification is dependent on the target cell. Modification of a PNP surface with ligands can be achieved through chemical conjugation, though chemical dyes for imaging and peptides for immune activation can also be attached [11,33,34]. One example is the ligand anti-human epidermal growth factor receptor 2 which targets breast cancer cells. The following provides additional applications of ligand modifications and their therapeutic applications [12].
In addition to chemical conjugation, genetic modification can facilitate direct attachment of the modifying protein monomers with the PNP surface. This results in a co-assembly and a solution to existing challenges with direct attachments or large proteins. Attaching large proteins to PNPs interferes with the self-assembly process and induces steric interactions. Though, smaller protein attachments are generally tolerated by protein NPs. A significant limitation to direct attachment via genetic modification of protein monomers is that it cannot accommodate the attachment of multiple components. Enzymatic ligation helps overcome this limitation by providing a site-specific covalent link to the PNP surface following PNP assembly. This strategy can also provide greater control over the density and ratios of attached proteins. [ 26 ]
The modification of VLPs is unique due to their nanocage architecture. PNPs with cage structures can fully encapsulate functional components in their interior, termed co-encapsulation. Drug encapsulation within VLP cages can occur through two processes. This first process occurs in-vitro and requires the disassembly of the cage and reassembling it with the presence of the drug components to be encapsulated [8]. Since loading efficiency is influenced through electrostatic interactions, the drug compounds cannot be fully encapsulated without interfering with the VLP cage self-assembly. Another process is the encapsulation of drug components in-vivo. This involves direct genetic attachment of the drug components to the interior of the VLP cage. This process guides drugs for encapsulation directly to the interior of the cage. [ 28 ] [ 2 ]
Due to PNPs’ breadth of favorable pharmacokinetic properties such as high biocompatibility, high biodegradability, high modifiability, low toxicity, high cell uptake, and a fast excretion rate, PNPs are prime candidates for anti-cancer therapy. Previous anticancer therapies relied on the enhanced permeability effect to passively accumulate within tumors. This resulted in greater toxicity due to higher concentrations required to achieve critical drug efficacy levels. Newer strategies allow PNPs to actively target the tumor microenvironment via the attachment of ligands and site-specific protein receptors. Active targeting decreases the total concentration of drugs required to deliver an effective dose, thereby reducing systemic side effects. [ 29 ] [ 30 ]
In addition to active tumor targeting, PNPs can also be engineered to respond to changing external environments such as pH, temperature, or enzyme concentration. The tumor microenvironment is slightly acidic, so PNPs can be engineered to only release their drug cargo under specific tumor physiological conditions. [ 28 ]
Another application is photothermal or photodynamic therapy . PNPs selectively accumulate into the tumor microenvironment where they are subsequently irradiated using a 1064 nm wavelength laser. The light energy is transferred into heat energy, increasing the temperature of the tumor microenvironment to inhibit tumor growth. Ferritin is a favorable protein for this application due to its high thermal stability. [ 28 ] [ 14 ]
In-vivo imaging is another application of PNPs. PNPs can carry fluorescent dyes that selectively accumulate in the tumor microenvironment. This is important because a significant limitation of Green Fluorescent Protein, the standard protein for tumor imaging, is its insufficient deep tissue penetration. Due to their small size, PNPs can deliver fluorescent dyes deep into the tissue overcoming this challenge and providing more accurate tumor imaging. This strategy may also be applied to MRI imaging using PNPs carrying magnetic components to tumor microenvironments for subsequent scanning. [ 31 ] [ 32 ] [ 33 ]
Other applications include vaccine development through VLPs carrying immunogenic components. Since VLPs are not carrying any attenuated genetic material, these vaccines pose a safer alternative, especially for the immunocompromised or elderly. PNPs may also treat neurological diseases as they can cross the blood-brain barrier [28]. Lastly. PNPs may find applications within ophthalmic drug delivery as PNPs have a significantly longer circulation time in the eye than eye drops. [ 34 ]
Despite numerous pharmacokinetic advantages of PNPs, there remain several critical challenges to their clinical translation. Only two PNPs have been FDA-approved, despite over 50 PNP formulations to date (2022). The two FDA-approved drugs include Abraxane, an albumin nanoparticle carrying paclitaxel used for breast cancer, non-small cell lung cancer, and pancreatic cancer treatment. The second FDA-approved PNP is Ontak, a protein conjugate carrying L-2 and Diphtheria toxin used for cutaneous T-cell lymphoma. [ 6 ] [ 7 ] The two approved formulations are summarized in Table 5 below. The low approval rate of PNPs is due to limited existing control over drug encapsulation and the pharmacokinetic variability between PNP batches. Balancing both the repeatability of these two properties and their relative interactions is important because it ensures the predictability of their clinical outcomes, greater patient safety, and that protein loading does not interfere with the PNP's properties. [ 2 ] [ 10 ] [ 11 ] [ 7 ]
Another limitation surrounds the cost and ability of large-scale production. Many synthesis methods that can deliver greater homogeneity between produced nanoparticles are also more costly options or cannot achieve mass production. This limitation is compounded by the lower yields of PNP manufacturing. This limits the availability of PNPs to broad clinical adoption [20,29]. [ 7 ] | https://en.wikipedia.org/wiki/Protein_nanoparticles |
The protein nitrogen unit (PNU) measures the potency of the compounds used in allergy skin tests , and is equivalent to 0.01 microgram (μg) of phosphotungstic acid -precipitable protein nitrogen. [ 1 ] Potency measurements depend on the measurement technique, so that results from different manufacturers cannot be reliably compared: as a result, PNUs are being replaced by bioequivalent allergy units (BAU), which are measured by skin testing using reference preparations of standard potency. [ 2 ]
This article related to medical technology is a stub . You can help Wikipedia by expanding it . | https://en.wikipedia.org/wiki/Protein_nitrogen_unit |
In computational biology , protein p K a calculations are used to estimate the p K a values of amino acids as they exist within proteins . These calculations complement the p K a values reported for amino acids in their free state, and are used frequently within the fields of molecular modeling , structural bioinformatics , and computational biology .
p K a values of amino acid side chains play an important role in defining the pH-dependent characteristics of a protein. The pH-dependence of the activity displayed by enzymes and the pH-dependence of protein stability , for example, are properties that are determined by the p K a values of amino acid side chains.
The p K a values of an amino acid side chain in solution is typically inferred from the p K a values of model compounds (compounds that are similar to the side chains of amino acids). See Amino acid for the p K a values of all amino acid side chains inferred in such a way. There are also numerous experimental studies that have yielded such values, for example by use of NMR spectroscopy .
The table below lists the model p K a values that are often used in a protein p K a calculation, and contains a third column based on protein studies. [ 1 ]
When a protein folds, the titratable amino acids in the protein are transferred from a solution-like environment to an environment determined by the 3-dimensional structure of the protein. For example, in an unfolded protein, an aspartic acid typically is in an environment which exposes the titratable side chain to water. When the protein folds, the aspartic acid could find itself buried deep in the protein interior with no exposure to solvent.
Furthermore, in the folded protein, the aspartic acid will be closer to other titratable groups in the protein and will also interact with permanent charges (e.g. ions) and dipoles in the protein. All of these effects alter the p K a value of the amino acid side chain, and p K a calculation methods generally calculate the effect of the protein environment on the model p K a value of an amino acid side chain. [ 2 ] [ 3 ] [ 4 ] [ 5 ]
Typically, the effects of the protein environment on the amino acid p K a value are divided into pH-independent effects and pH-dependent effects. The pH-independent effects (desolvation, interactions with permanent charges and dipoles) are added to the model p K a value to give the intrinsic p K a value. The pH-dependent effects cannot be added in the same straightforward way and have to be accounted for using Boltzmann summation, Tanford–Roxby iterations or other methods.
The interplay of the intrinsic p K a values of a system with the electrostatic interaction energies between titratable groups can produce quite spectacular effects such as non-Henderson–Hasselbalch titration curves and even back-titration effects. [ 6 ]
The image on the right shows a theoretical system consisting of three acidic residues. One group is displaying a back-titration event (blue group).
Several software packages and webserver are available for the calculation of protein p K a values.
Some methods are based on solutions to the Poisson–Boltzmann equation (PBE), often referred to as FDPB-based methods ( FDPB stands for " finite difference Poisson–Boltzmann"). The PBE is a modification of Poisson's equation that incorporates a description of the effect of solvent ions on the electrostatic field around a molecule.
The H++ web server , [ 7 ] the pKD webserver , [ 8 ] MCCE2 , Karlsberg+ , [ dead link ] PETIT and GMCT use the FDPB method to compute p K a values of amino acid side chains.
FDPB-based methods calculate the change in the p K a value of an amino acid side chain when that side chain is moved from a hypothetical fully solvated state to its position in the protein. To perform such a calculation, one needs theoretical methods that can calculate the effect of the protein interior on a p K a value, and knowledge of the pKa values of amino acid side chains in their fully solvated states. [ 2 ] [ 3 ] [ 4 ] [ 5 ]
A set of empirical rules relating the protein structure to the p K a values of ionizable residues have been developed by Li, Robertson, and Jensen. [ 9 ] These rules form the basis for the web-accessible program called PROPKA for rapid predictions of p K a values. A recent empirical p K a prediction program was released by Tan KP et.al. with the online server DEPTH web server . [ 10 ]
Molecular dynamics methods of calculating p K a values make it possible to include full flexibility of the titrated molecule. [ 11 ] [ 12 ] [ 13 ]
Molecular dynamics based methods are typically much more computationally expensive, and not necessarily more accurate, ways to predict p K a values than approaches based on the Poisson–Boltzmann equation . Limited conformational flexibility can also be realized within a continuum electrostatics approach, e.g., for considering multiple amino acid sidechain rotamers. In addition, current commonly used molecular force fields do not take electronic polarizability into account, which could be an important property in determining protonation energies.
From the titration of protonatable group, one can read the so-called p K a 1 ⁄ 2 which is equal to the pH value where the group is half-protonated (i.e. when 50% such groups would be protonated). The p K a 1 ⁄ 2 is equal to the Henderson–Hasselbalch p K a (p K HH a ) if the titration curve follows the Henderson–Hasselbalch equation . [ 14 ] Most p K a calculation methods silently assume that all titration curves are Henderson–Hasselbalch shaped, and p K a values in p K a calculation programs are therefore often determined in this way. In the general case of multiple interacting protonatable sites, the p K a 1 ⁄ 2 value is not thermodynamically meaningful. In contrast, the Henderson–Hasselbalch p K a value can be computed from the protonation free energy via
p K a H H ( p H ) = p H − Δ G p r o t ( p H ) R T ln 10 {\displaystyle \mathrm {p} K_{\mathrm {a} }^{\mathrm {HH} }(\mathrm {pH} )=\mathrm {pH} -{\frac {\Delta G^{\mathrm {prot} }(\mathrm {pH} )}{\mathrm {RT} \ln 10}}}
and is thus in turn related to the protonation free energy of the site via
Δ G p r o t ( p H ) = R T ln 10 ( p H − p K a H H ) {\displaystyle \Delta G^{\mathrm {prot} }(\mathrm {pH} )=\mathrm {RT} \ln 10\;(\mathrm {pH} -\mathrm {p} K_{\mathrm {a} }^{\mathrm {HH} })}
The protonation free energy can in principle be computed from the protonation probability of the group ⟨ x ⟩ (pH) which can be read from its titration curve
Δ G p r o t ( p H ) = − R T ln [ ⟨ x ⟩ 1 − ⟨ x ⟩ ] {\displaystyle \Delta G^{\mathrm {prot} }(\mathrm {pH} )=-\mathrm {RT} \ln \left[{\frac {\langle x\rangle }{1-\langle x\rangle }}\right]}
Titration curves can be computed within a continuum electrostatics approach with formally exact but more elaborate analytical or Monte Carlo (MC) methods , or inexact but fast approximate methods. MC methods that have been used to compute titration curves [ 15 ] are Metropolis MC [ 16 ] [ 17 ] or Wang–Landau MC . [ 18 ] Approximate methods that use a mean-field approach for computing titration curves are the Tanford–Roxby method and hybrids of this method that combine an exact statistical mechanics treatment within clusters of strongly interacting sites with a mean-field treatment of intercluster interactions. [ 19 ] [ 20 ] [ 21 ] [ 22 ] [ 23 ]
In practice, it can be difficult to obtain statistically converged and accurate protonation free energies from titration curves if ⟨ x ⟩ is close to a value of 1 or 0. In this case, one can use various free energy calculation methods to obtain the protonation free energy [ 15 ] such as biased Metropolis MC, [ 24 ] free-energy perturbation , [ 25 ] [ 26 ] thermodynamic integration , [ 27 ] [ 28 ] [ 29 ] the non-equilibrium work method [ 30 ] or the Bennett acceptance ratio method. [ 31 ]
Note that the p K HH a value does in general depend on the pH value. [ 32 ]
This dependence is small for weakly interacting groups like well solvated amino acid side chains on the protein surface, but can be large for strongly interacting groups like those buried in enzyme active sites or integral membrane proteins. [ 33 ] [ 34 ] [ 35 ]
While many protein pKa prediction methods are available, their accuracies often differ significantly due to subtle and often drastic differences in strategy. [ 36 ] | https://en.wikipedia.org/wiki/Protein_pKa_calculations |
A protein phosphatase is a phosphatase enzyme that removes a phosphate group from the phosphorylated amino acid residue of its substrate protein. Protein phosphorylation is one of the most common forms of reversible protein posttranslational modification ( PTM ), with up to 30% of all proteins being phosphorylated at any given time. Protein kinases (PKs) are the effectors of phosphorylation and catalyse the transfer of a γ-phosphate from ATP to specific amino acids on proteins. Several hundred PKs exist in mammals and are classified into distinct super-families. Proteins are phosphorylated predominantly on Ser, Thr and Tyr residues, which account for 79.3, 16.9 and 3.8% respectively of the phosphoproteome, at least in mammals. In contrast, protein phosphatases (PPs) are the primary effectors of dephosphorylation and can be grouped into three main classes based on sequence, structure and catalytic function. The largest class of PPs is the phosphoprotein phosphatase (PPP) family comprising PP1, PP2A, PP2B, PP4, PP5, PP6 and PP7, and the protein phosphatase Mg 2+ - or Mn 2+ -dependent (PPM) family, composed primarily of PP2C. The protein Tyr phosphatase (PTP) super-family forms the second group, [ 1 ] and the aspartate-based protein phosphatases the third. The protein pseudophosphatases form part of the larger phosphatase family, and in most cases are thought to be catalytically inert, instead functioning as phosphate-binding proteins, integrators of signalling or subcellular traps. Examples of membrane-spanning protein phosphatases containing both active (phosphatase) and inactive (pseudophosphatase) domains linked in tandem are known, [ 1 ] conceptually similar to the kinase and pseudokinase domain polypeptide structure of the JAK pseudokinases. [ 2 ] [ 3 ] A complete comparative analysis of human phosphatases and pseudophosphatases has been completed by Manning and colleagues, [ 4 ] forming a companion piece to the ground-breaking analysis of the human kinome, which encodes the complete set of ~536 human protein kinases . [ 5 ]
Phosphorylation involves the transfer of phosphate groups from ATP to the enzyme, the energy for which comes from hydrolysing ATP into ADP or AMP . However, dephosphorylation releases phosphates into solution as free ions, because attaching them back to ATP would require energy input.
Cysteine-dependent phosphatases (CDPs) catalyse the hydrolysis of a phosphoester bond via a phospho-cysteine intermediate. [ 6 ]
The free cysteine nucleophile forms a bond with the phosphorus atom of the phosphate moiety, and the P-O bond linking the phosphate group to the tyrosine is protonated, either by a suitably positioned acidic amino acid residue (Asp in the diagram below) or a water molecule. The phospho-cysteine intermediate is then hydrolysed by another water molecule, thus regenerating the active site for another dephosphorylation reaction.
Metallo-phosphatases (e.g. PP2C) co-ordinate 2 catalytically essential metal ions within their active site. There is currently some confusion of the identity of these metal ions, as successive attempts to identify them yield different answers. There is currently evidence that these metals could be magnesium , manganese , iron , zinc , or any combination thereof. It is thought that a hydroxyl ion bridging the two metal ions takes part in nucleophilic attack on the phosphorus ion.
Phosphatases can be subdivided based upon their substrate specificity.
Protein Ser/Thr phosphatases were originally classified using biochemical assays as either, type 1 (PP1) or type 2 (PP2), and were further subdivided based on metal-ion requirement (PP2A, no metal ion; PP2B, Ca 2+ stimulated; PP2C, Mg 2+ dependent) (Moorhead et al., 2007). The protein Ser/Thr phosphatases PP1, PP2A and PP2B of the PPP family, together with PP2C of the PPM family, account for the majority of Ser/Thr PP activity in vivo (Barford et al., 1998). In the brain, they are present in different subcellular compartments in neuronal and glial cells, and contribute to different neuronal functions.
The PPM family, which includes PP2C and pyruvate dehydrogenase phosphatase, are enzymes with Mn 2+ /Mg 2+ metal ions that are resistant to classic inhibitors and toxins of the PPP family. Unlike most PPPs, PP2C exists in only one subunit but, like PTPs, it displays a wide variety of structural domains that confer unique functions. In addition, PP2C does not seem to be evolutionarily related to the major family of Ser/Thr PPs and has no sequence homology to ancient PPP enzymes. The current assumption is that PPMs evolved separately from PPPs but converged during evolutionary development.
Class I PTPs constitute the largest family. They contain the well-known classical receptor (a) and non-receptor PTPs (b), which are strictly tyrosine-specific, and the DSPs (c) which target Ser/Thr as well as Tyr and are the most diverse in terms of substrate specificity.
The third class of PTPs contains three cell cycle regulators, CDC25A, CDC25B and CDC25C, which dephosphorylate CDKs at their N-terminal, a reaction required to drive progression of the cell cycle. They are themselves regulated by phosphorylation and are degraded in response to DNA damage to prevent chromosomal abnormalities.
The haloacid dehalogenase (HAD) superfamily is a further PP group that uses Asp as a nucleophile and was recently shown to have dual-specificity. These PPs can target both Ser and Tyr, but are thought to have greater specificity towards Tyr. A subfamily of HADs, the Eyes Absent Family (Eya), are also transcription factors and can therefore regulate their own phosphorylation and that of transcriptional cofactor/s, and contribute to the control of gene transcription. The combination of these two functions in Eya reveals a greater complexity of transcriptional gene control than previously thought . A further member of this class is the RNA polymerase II C-terminal domain phosphatase. While this family remains poorly understood, it is known to play important roles in development and nuclear morphology.
Many phosphatases are promiscuous with respect to substrate type, or can evolve quickly to change substrate. An alternative structural classification [ 4 ] notes that 20 distinct protein folds have phosphatase activity, and 10 of these contain protein phosphatases.
Phosphatases act in opposition to kinases / phosphorylases , which add phosphate groups to proteins. The addition of a phosphate group may activate or de-activate an enzyme (e.g., kinase signalling pathways [ 11 ] ) or enable a protein-protein interaction to occur (e.g., SH2 domains [ 12 ] ); therefore phosphatases are integral to many signal transduction pathways. Phosphate addition and removal do not necessarily correspond to enzyme activation or inhibition, and that several enzymes have separate phosphorylation sites for activating or inhibiting functional regulation. CDK , for example, can be either activated or deactivated depending on the specific amino acid residue being phosphorylated. Phosphates are important in signal transduction because they regulate the proteins to which they are attached. To reverse the regulatory effect, the phosphate is removed. This occurs on its own by hydrolysis , or is mediated by protein phosphatases. [ 13 ] [ 14 ]
Protein phosphorylation plays a crucial role in biological functions and controls nearly every cellular process, including metabolism, gene transcription and translation, cell-cycle progression, cytoskeletal rearrangement, protein-protein interactions, protein stability, cell movement, and apoptosis . These processes depend on the highly regulated and opposing actions of PKs and PPs, through changes in the phosphorylation of key proteins. Histone phosphorylation, along with methylation, ubiquitination, sumoylation and acetylation, also regulates access to DNA through chromatin reorganisation. [ 15 ]
One of the major switches for neuronal activity is the activation of PKs and PPs by elevated intracellular calcium. The degree of activation of the various isoforms of PKs and PPs is controlled by their individual sensitivities to calcium. Furthermore, a wide range of specific inhibitors and targeting partners such as scaffolding, anchoring, and adaptor proteins also contribute to the control of PKs and PPs and recruit them into signalling complexes in neuronal cells. Such signalling complexes typically act to bring PKs and PPs in close proximity with target substrates and signalling molecules as well as enhance their selectivity by restricting accessibility to these substrate proteins. Phosphorylation events, therefore, are controlled not only by the balanced activity of PKs and PPs but also by their restricted localisation. Regulatory subunits and domains serve to restrict specific proteins to particular subcellular compartments and to modulate protein specificity. These regulators are essential for maintaining the coordinated action of signalling cascades, which in neuronal cells include short-term (synaptic) and long-term (nuclear) signalling. These functions are, in part, controlled by allosteric modification by secondary messengers and reversible protein phosphorylation. [ 16 ] [ 17 ]
It is thought that around 30% of known PPs are present in all tissues, with the rest showing some level of tissue restriction. While protein phosphorylation is a cell-wide regulatory mechanism, recent quantitative proteomics studies have shown that phosphorylation preferentially targets nuclear proteins. Many PPs that regulate nuclear events, are often enriched or exclusively present in the nucleus. In neuronal cells, PPs are present in multiple cellular compartments and play a critical role at both pre- and post-synapses, in the cytoplasm and in the nucleus where they regulate gene expression. [ 18 ]
Phosphoprotein phosphatase is activated by the hormone insulin , which indicates that there is a high concentration of glucose in the blood . The enzyme then acts to dephosphorylate other enzymes, such as phosphorylase kinase , glycogen phosphorylase , and glycogen synthase . This leads to phosphorylase kinase and glycogen phosphorylase's becoming inactive, while glycogen synthase is activated. As a result, glycogen synthesis is increased and glycogenolysis is decreased, and the net effect is for energy to enter and be stored inside the cell. [ 19 ]
In the adult brain, PPs are essential for synaptic functions and are involved in the negative regulation of higher-order brain functions such as learning and memory. Dysregulation of their activity has been linked to several disorders including cognitive ageing and neurodegeneration, as well as cancer, diabetes and obesity. [ 20 ]
Human genes that encode proteins with phosphoprotein phosphatase activity include: | https://en.wikipedia.org/wiki/Protein_phosphatase |
Protein phosphorylation is a reversible post-translational modification of proteins in which an amino acid residue is phosphorylated by a protein kinase by the addition of a covalently bound phosphate group. Phosphorylation alters the structural conformation of a protein, causing it to become activated, deactivated, or otherwise modifying its function. [ 1 ] Approximately 13,000 human proteins have sites that are phosphorylated. [ 2 ]
The reverse reaction of phosphorylation is called dephosphorylation , and is catalyzed by protein phosphatases . Protein kinases and phosphatases work independently and in a balance to regulate the function of proteins. [ 3 ]
The amino acids most commonly phosphorylated are serine , threonine , tyrosine , and histidine . [ 4 ] [ 5 ] These phosphorylations play important and well-characterized roles in signaling pathways and metabolism. However, other amino acids can also be phosphorylated post-translationally, including arginine , lysine , aspartic acid , glutamic acid and cysteine , and these phosphorylated amino acids have been identified to be present in human cell extracts and fixed human cells using a combination of antibody-based analysis (for pHis) and mass spectrometry (for all other amino acids). [ 5 ] [ 6 ] [ 7 ] [ 8 ]
Protein phosphorylation was first reported in 1906 by Phoebus Levene at the Rockefeller Institute for Medical Research with the discovery of phosphorylated vitellin . [ 9 ] However, it was nearly 50 years until the enzymatic phosphorylation of proteins by protein kinases was discovered. [ 10 ]
In 1906, Phoebus Levene at the Rockefeller Institute for Medical Research identified phosphate in the protein vitellin (phosvitin) [ 9 ] and by 1933 had detected phosphoserine in casein , with Fritz Lipmann. [ 11 ] However, it took another 20 years before Eugene P. Kennedy described the first "enzymatic phosphorylation of proteins". [ 10 ] The first phosphorylase enzyme was discovered by Carl and Gerty Cori in the late 1930s. Carl and Gerty Cori found two forms of glycogen phosphorylase which they named A and B but did not correctly understand the mechanism of the B form to A form conversion. The interconversion of phosphorylase b to phosphorylase a was later described by Edmond Fischer and Edwin Krebs , as well as, Wosilait and Sutherland , involving a phosphorylation/dephosphorylation mechanism. [ 12 ] It was found that an enzyme, named phosphorylase kinase and Mg-ATP were required to phosphorylate glycogen phosphorylase by assisting in the transfer of the γ-phosphoryl group of ATP to a serine residue on phosphorylase b. Protein phosphatase 1 is able to catalyze the dephosphorylation of phosphorylated enzymes by removing the phosphate group. Earl Sutherland explained in 1950, that the activity of phosphorylase was increased and thus glycogenolysis stimulated when liver slices were incubated with adrenalin and glucagon. Phosphorylation was considered a specific control mechanism for one metabolic pathway until the 1970s, when Lester Reed discovered that mitochondrial pyruvate dehydrogenase complex was inactivated by phosphorylation. Also in the 1970s, the term multisite phosphorylation was coined in response to the discovery of proteins that are phosphorylated on two or more residues by two or more kinases. In 1975, it was shown that cAMP-dependent proteins kinases phosphorylate serine residues on specific amino acid sequence motifs. Ray Erikson discovered that v-Src was a kinase and Tony Hunter found that v-Src phosphorylated tyrosine residues on proteins in the 1970s. [ 13 ] In the early 1980, the amino-acid sequence of the first protein kinase was determined which helped geneticists understand the functions of regulatory genes. In the late 1980s and early 1990s, the first protein tyrosine phosphatase (PTP1B) was purified and the discovery, as well as, cloning of JAK kinases was accomplished which led to many in the scientific community to name the 1990s as the decade of protein kinase cascades. [ 14 ] [ 15 ] Edmond Fischer and Edwin Krebs were awarded the Nobel prize in 1992 "for their discoveries concerning reversible protein phosphorylation as a biological regulatory mechanism". [ 16 ]
Reversible phosphorylation of proteins is abundant in both prokaryotic and even more so in eukaryotic organisms. [ 17 ] [ 18 ] [ 19 ] [ 20 ] For instance, in bacteria 5–10% of all proteins are thought to be phosphorylated. [ 21 ] [ 22 ] By contrast, it is estimated that one third of all human proteins is phosphorylated at any point in time, with 230,000, 156,000, and 40,000 unique phosphorylation sites existing in human, mouse, and yeast, respectively. [ 2 ] In yeast, about 120 kinases (out of ~6,000 proteins total) cause 8,814 known regulated phosphorylation events, generating about 3,600 phosphoproteins (about 60% of all yeast proteins). [ 23 ] [ 24 ] Hence, phosphorylation is a universal regulatory mechanism that affects a large portion of proteins. Even if a protein is not phosphorylated itself, its interactions with other proteins may be regulated by phosphorylation of these interacting proteins.
Phosphorylation introduces a charged and hydrophilic group in the side chain of amino acids, possibly changing a protein's structure by altering interactions with nearby amino acids. Some proteins such as p53 contain multiple phosphorylation sites, facilitating complex, multi-level regulation. Because of the ease with which proteins can be phosphorylated and dephosphorylated, this type of modification is a flexible mechanism for cells to respond to external signals and environmental conditions. [ 25 ]
Kinases phosphorylate proteins and phosphatases dephosphorylate proteins. Many enzymes and receptors are switched "on" or "off" by phosphorylation and dephosphorylation. Reversible phosphorylation results in a conformational change in the structure in many enzymes and receptors , causing them to become activated or deactivated. Phosphorylation usually occurs on serine , threonine , tyrosine and histidine residues in eukaryotic proteins. Histidine phosphorylation of eukaryotic proteins appears to be much more frequent than tyrosine phosphorylation. [ 26 ] In prokaryotic proteins phosphorylation occurs on the serine, threonine, tyrosine, histidine, arginine or lysine residues. [ 17 ] [ 18 ] [ 26 ] [ 27 ] The addition of a phosphate (PO 4 3- ) molecule to a non-polar R group of an amino acid residue can turn a hydrophobic portion of a protein into a polar and extremely hydrophilic portion of a molecule. In this way protein dynamics can induce a conformational change in the structure of the protein via long-range allostery with other hydrophobic and hydrophilic residues in the protein.
One such example of the regulatory role that phosphorylation plays is the p53 tumor suppressor protein. The p53 protein is heavily regulated [ 28 ] and contains more than 18 different phosphorylation sites. Activation of p53 can lead to cell cycle arrest, which can be reversed under some circumstances, or apoptotic cell death. [ 29 ] This activity occurs only in situations wherein the cell is damaged or physiology is disturbed in normal healthy individuals.
Upon the deactivating signal, the protein becomes dephosphorylated again and stops working. [ 30 ] [ citation needed ] This is the mechanism in many forms of signal transduction , for example the way in which incoming light is processed in the light-sensitive cells of the retina .
Regulatory roles of phosphorylation include:
Elucidating complex signaling pathway phosphorylation events can be difficult. In cellular signaling pathways, protein A phosphorylates protein B, and B phosphorylates C. However, in another signaling pathway, protein D phosphorylates A, or phosphorylates protein C. Global approaches such as phosphoproteomics , the study of phosphorylated proteins, which is a sub-branch of proteomics , combined with mass spectrometry -based proteomics, have been utilised to identify and quantify dynamic changes in phosphorylated proteins over time. These techniques are becoming increasingly important for the systematic analysis of complex phosphorylation networks. [ 39 ] They have been successfully used to identify dynamic changes in the phosphorylation status of more than 6,000 sites after stimulation with epidermal growth factor . [ 40 ] Another approach for understanding Phosphorylation Network, is by measuring the genetic interactions between multiple phosphorylating proteins and their targets. This reveals interesting recurring patterns of interactions – network motifs. [ 41 ] Computational methods have been developed to model phosphorylation networks [ 42 ] [ 43 ] and predict their responses under different perturbations. [ 44 ]
Eukaryotic DNA is organized with histone proteins in specific complexes called chromatin. The chromatin structure functions and facilitates the packaging, organization and distribution of eukaryotic DNA. However, it has a negative impact on several fundamental biological processes such as transcription, replication and DNA repair by restricting the accessibility of certain enzymes and proteins. Post-translational modification of histones such as histone phosphorylation has been shown to modify the chromatin structure by changing protein:DNA or protein:protein interactions. [ 45 ] Histone post-translational modifications modify the chromatin structure. The most commonly associated histone phosphorylation occurs during cellular responses to DNA damage, when phosphorylated histone H2A separates large chromatin domains around the site of DNA breakage. [ 46 ] Researchers investigated whether modifications of histones directly impact RNA polymerase II directed transcription. Researchers choose proteins that are known to modify histones to test their effects on transcription, and found that the stress-induced kinase, MSK1, inhibits RNA synthesis. Inhibition of transcription by MSK1 was most sensitive when the template was in chromatin, since DNA templates not in chromatin were resistant to the effects of MSK1. It was shown that MSK1 phosphorylated histone H2A on serine 1, and mutation of serine 1 to alanine blocked the inhibition of transcription by MSK1. Thus results suggested that the acetylation of histones can stimulate transcription by suppressing an inhibitory phosphorylation by a kinase as MSK1. [ 47 ]
Within a protein, phosphorylation can occur on several amino acids . Phosphorylation on serine is thought to be the most common, followed by threonine. Tyrosine phosphorylation is relatively rare but lies at the head of many protein phosphorylation signalling pathways (e.g. in tyrosine kinase-linked receptors) in most of the eukaryotes. Phosphorylation on amino acids, such as serine, threonine, and tyrosine results in the formation of a phosphoprotein, when the phosphate group of the phosphoprotein reacts with the -OH group of a Ser, Thr, or Tyr sidechain in an esterification reaction. [ 48 ] However, since tyrosine phosphorylated proteins are relatively easy to purify using antibodies , tyrosine phosphorylation sites are relatively well understood. Histidine and aspartate phosphorylation occurs in prokaryotes as part of two-component signaling and in some cases in eukaryotes in some signal transduction pathways. The analysis of phosphorylated histidine using standard biochemical and mass spectrometric approaches is much more challenging than that of Ser, Thr or Tyr. [ 49 ] [ 7 ] [ 5 ] and [ 50 ] In prokaryotes, archaea, and some lower eukaryotes, histidine's nitrogen act as a nucleophile and binds to a phosphate group. [ 51 ] Once histidine is phosphorylated the regulatory domain of the response regulator catalyzes the transfer of the phosphate to aspartate.
While tyrosine phosphorylation is found in relatively low abundance, it is well studied due to the ease of purification of phosphotyrosine using antibodies. Receptor tyrosine kinases are an important family of cell surface receptors involved in the transduction of extracellular signals such as hormones, growth factors, and cytokines. Binding of a ligand to a monomeric receptor tyrosine kinase stabilizes interactions between two monomers to form a dimer , after which the two bound receptors phosphorylate tyrosine residues in trans . Phosphorylation and activation of the receptor activates a signaling pathway through enzymatic activity and interactions with adaptor proteins. [ 52 ] Signaling through the epidermal growth factor receptor (EGFR) , a receptor tyrosine kinase, is critical for the development of multiple organ systems including the skin, lung, heart, and brain. Excessive signaling through the EGFR pathway is found in many human cancers. [ 53 ]
Cyclin-dependent kinases (CDKs) are serine-threonine kinases which regulate progression through the eukaryotic cell cycle . CDKs are catalytically active only when bound to a regulatory cyclin . Animal cells contain at least nine distinct CDKs which bind to various cyclins with considerable specificity. CDK inhibitors (CKIs) block kinase activity in the cyclin-CDK complex to halt the cell cycle in G1 or in response to environmental signals or DNA damage. The activity of different CDKs activate cell signaling pathways and transcription factors that regulate key events in mitosis such as the G1/S phase transition. Earlier cyclin-CDK complexes provide the signal to activate subsequent cyclin-CDK complexes. [ 54 ]
There are thousands of distinct phosphorylation sites in a given cell since:
Since phosphorylation of any site on a given protein can change the function or localization of that protein, understanding the "state" of a cell requires knowing the phosphorylation state of its proteins. For example, generally, if amino acid Serine-473 in the protein AKT is phosphorylated, AKT is functionally active as a kinase, and if it is not phosphorylated, AKT is an inactive kinase.
Phosphorylation sites are crucial for proteins and their transportation and functions. They are the covalent modification of proteins through reversible phosphorylation. This enables proteins to stay inbound within a cell since the negative phosphorylated site disallows their permeability through the cellular membrane. Protein dephosphorylation allows the cell to replenish phosphates through release of pyrophosphates which saves ATP use in the cell. [ 55 ] An example of phosphorylating enzyme is found in E. coli bacteria. It possesses alkaline phosphatase in its periplasmic region of its membrane. The outermost membrane is permeable to phosphorylated molecules however the inner cytoplasmic membrane is impermeable due to large negative charges. [ 56 ] In this way, the E. coli bacteria stores proteins and pyrophosphates in its periplasmic membrane until either are needed within the cell.
Recent advancement in phosphoproteomic identification has resulted in the discoveries of countless phosphorylation sites in proteins. This required an integrative medium for accessible data in which known phosphorylation sites of proteins are organized. A curated database of dbPAF was created, containing known phosphorylation sites in H. sapiens , M. musculus , R. norvegicus , D. melanogaster , C. elegans , S. pombe and S. cerevisiae . The database currently holds 294,370 non-redundant phosphorylation sites of 40,432 proteins. [ 57 ] Other tools of phosphorylation prediction in proteins include NetPhos [ 58 ] for eukaryotes, NetPhosBac [ 58 ] for bacteria, and ViralPhos [ 59 ] for viruses.
There are a large variety of serine residues, and the phosphorylation of each residue can lead to different metabolic consequences.
Phosphorylation of serine and threonine residues is known to crosstalk with O -GlcNAc modification of serine and threonine residues.
Tyrosine phosphorylation is a fast, reversible reaction, and one of the major regulatory mechanisms in signal transduction . Cell growth , differentiation , migration , and metabolic homeostasis are cellular processes maintained by tyrosine phosphorylation. The function of protein tyrosine kinases and protein-tyrosine phosphatase counterbalances the level of phosphotyrosine on any protein. The malfunctioning of specific chains of protein tyrosine kinases and protein tyrosine phosphatase has been linked to multiple human diseases such as obesity , insulin resistance , and type 2 diabetes mellitus . [ 64 ] Phosphorylation on tyrosine occurs in eukaryotes, select bacterial species, and is present among prokaryotes. Phosphorylation on tyrosine maintains the cellular regulation in bacteria similar to its function in eukaryotes. [ 65 ]
Arginine phosphorylation in many Gram-positive bacteria marks proteins for degradation by a Clp protease . [ 34 ]
Widespread human protein phosphorylation occurs on multiple non-canonical amino acids, including motifs containing phosphorylated histidine (1 and 3 positions), aspartate, cysteine, glutamate, arginine, and lysine in HeLa cell extracts. Due to the chemical and thermal lability of these phosphorylated residues, special procedures and separation techniques are required for preservation alongside the heat stable 'classical' Ser, Thr and Tyr phosphorylation. [ 66 ]
Antibodies can be used as powerful tool to detect whether a protein is phosphorylated at a particular site. Antibodies bind to and detect phosphorylation-induced conformational changes in the protein. Such antibodies are called phospho-specific antibodies; hundreds of such antibodies are now available. They are becoming critical reagents both for basic research and for clinical diagnosis.
Post-translational modification (PTM) isoforms are easily detected on 2D gels . Indeed, phosphorylation replaces neutral hydroxyl groups on serines, threonines, or tyrosines with negatively charged phosphates with pKs near 1.2 and 6.5. Thus, below pH 5.5, phosphates add a single negative charge; near pH 6.5, they add 1.5 negative charges; above pH 7.5, they add 2 negative charges. The relative amount of each isoform can also easily and rapidly be determined from staining intensity on 2D gels.
In some very specific cases, the detection of the phosphorylation as a shift in the protein's electrophoretic mobility is possible on simple 1-dimensional SDS-PAGE gels, as it is described for instance for a transcriptional coactivator by Kovacs et al. [ 67 ] Strong phosphorylation-related conformational changes (that persist in detergent-containing solutions) are thought to underlie this phenomenon. Most of the phosphorylation sites for which such a mobility shift has been described fall in the category of SP and TP sites (i.e. a proline residue follows the phosphorylated serine or threonine residue).
Large-scale mass spectrometry analyses have been used to determine sites of protein phosphorylation. Dozens of studies have been published, each identifying thousands of sites, many of which were previously undescribed. [ 68 ] [ 69 ] Mass spectrometry is ideally suited for such analyses using HCD or ETD fragmentation, as the addition of phosphorylation results in an increase in the mass of the protein and the phosphorylated residue. Advanced, highly accurate mass spectrometers are needed for these studies, limiting the technology to labs with high-end mass spectrometers. However, the analysis of phosphorylated peptides by mass spectrometry is still not as straightforward as for "regular", unmodified peptides. EThcD has been developed combining electron-transfer and higher-energy collision dissociation. Compared to the usual fragmentation methods, EThcD scheme provides more informative MS/MS spectra for unambiguous phosphosite localization. [ 70 ]
A detailed characterization of the sites of phosphorylation is very difficult, and the quantitation of protein phosphorylation by mass spectrometry requires isotopic internal standard approaches. [ 71 ] A relative quantitation can be obtained with a variety of differential isotope labeling technologies. [ 72 ] There are also several quantitative protein phosphorylation methods, including fluorescence immunoassays, microscale thermophoresis , FRET , TRF, fluorescence polarization, fluorescence-quenching, mobility shift, bead-based detection, and cell-based formats. [ 73 ] [ 74 ]
Protein phosphorylation is common among all clades of life, including all animals, plants, fungi, bacteria, and archaea. The origins of protein phosphorylation mechanisms are ancestral and have diverged greatly between different species. In eukaryotes, it is estimated that between 30 – 65% of all proteins may be phosphorylated, with tens or even hundreds of thousands of distinct phosphorylation sites. [ 75 ] [ 2 ] Some phosphorylation sites appear to have evolved as conditional "off" switches, blocking the active site of an enzyme, such as in the prokaryotic metabolic enzyme isocitrate dehydrogenase. However, in the case of proteins that must be phosphorylated to be active, it is less clear how they could have emerged from non-phosphorylated ancestors. It has been shown that a subset of serine phosphosites are often replaced by acidic residues such as aspartate and glutamate between different species. These anionic residues can interact with cationic residues such as lysine and arginine to form salt bridges , stable non-covalent interactions that alter a protein's structure. These phosphosites often participate in salt bridges, suggesting that some phosphorylation sites evolved as conditional "on" switches for salt bridges, allowing these proteins to adopt an active conformation only in response to a specific signal. [ 76 ]
There are around 600 known eukaryotic protein kinases, making them one of the largest eukaryotic gene families. Most phosphorylation is carried out by a single superfamily of protein kinases that share a conserved kinase domain. Protein phosphorylation is highly conserved in pathways central to cell survival, such as cell cycle progression relying on cyclin-dependent kinases (CDKs), but individual phosphorylation sites are often flexible. Targets of CDK phosphorylation often have phosphosites in disordered segments , which are found in non-identical locations even in close species. Conversely, targets of CDK phosphorylation in structurally defined regions are more highly conserved. While CDK activity is critical for cell growth and survival in all eukaryotes, only very few phosphosites show strong conservation of their precise positions. Positioning is likely to be highly important for phosphates that allosterically regulate protein structure, but much more flexible for phosphates that interact with phosphopeptide-binding domains to recruit regulatory proteins. [ 77 ]
Protein phosphorylation is a reversible post-translational modification of proteins. In eukaryotes, protein phosphorylation functions in cell signaling, gene expression, and differentiation. It is also involved in DNA replication during the cell cycle, and the mechanisms that cope with stress-induced replication blocks. Compared to eukaryotes, prokaryotes use Hanks-type kinases and phosphatases for signal transduction. Whether or not the phosphorylation of proteins in bacteria can also regulate processes like DNA repair or replication still remains unclear. [ 78 ]
Compared to the protein phosphorylation of prokaryotes, studies of protein phosphorylation in eukaryotes from yeast to human cells have been rather extensive. It is known that eukaryotes rely on the phosphorylation of the hydroxyl group on the side chains of serine, threonine, and tyrosine for cell signaling. These are the main regulatory post-translational modifications in eukaryotic cells but the protein phosphorylation of prokaryotes are less intensely studied. While serine, threonine, and tyrosine are phosphorylated in eukaryotes, histidine and aspartate is phosphorylated in prokaryotes and eukaryotes. In bacteria, histidine phosphorylation occurs in the phosphoenolpyruvate-dependent phosphotransferase systems (PTSs), which are involved in the process of internalization as well as the phosphorylation of sugars. [ 79 ]
Protein phosphorylation by protein kinase was first shown in E. coli and Salmonella typhimurium and has since been demonstrated in many other bacterial cells. [ 80 ] It was found that bacteria use histidine and aspartate phosphorylation as a model for bacterial signaling transduction. Serine, threonine and tyrosine phosphorylation are also present in bacteria. Bacteria carry kinases and phosphatases similar to that of their eukaryotic equivalent and have also developed unique kinases and phosphatases not found in eukaryotes. [ 79 ]
Abnormal protein phosphorylation has been implicated in a number of diseases, including cancer , Alzheimer's disease , Parkinson's disease , and other degenerative disorders .
Tau protein belongs to a group of microtubule associated proteins (MAPs) which help stabilize microtubules in cells, including neurons. [ 81 ] Association and stabilizing activity of tau protein depends on its phosphorylated state. In Alzheimer's disease, due to misfoldings and abnormal conformational changes in tau protein structure, it is rendered ineffective at binding to microtubules and unable to keep the neural cytoskeletal structure organized during neural processes. Abnormal tau inhibits and disrupts microtubule organization and disengages normal tau from microtubules into cytosolic phase. [ 82 ] The misfoldings lead to the abnormal aggregation into fibrillary tangles inside the neurons. The tau protein needs to be phosphorylated to function, but hyperphosphorylation of tau protein is one of the major influences on its incapacity to associate. [ 82 ] Phosphatases PP1, PP2A, PP2B, and PP2C dephosphorylate tau protein in vitro , and their activities are reduced in areas of the brain in Alzheimer patients. [ 82 ] [ 83 ] Tau phosphoprotein is three to fourfold hyperphosphorylated in an Alzheimer patient compared to an aged non-afflicted individual. Alzheimer disease tau seems to remove MAP1 and MAP2 (two other major associated proteins) from microtubules and this deleterious effect is reversed when dephosphorylation is performed, evidencing hyperphosphorylation as the sole cause of the crippling activity. [ 82 ]
α-Synuclein is a protein that is associated with Parkinson's disease. [ 84 ] In humans, this protein is encoded by the SNCA gene . [ 85 ] α-Synuclein is involved in recycling synaptic vesicles that carry neurotransmitters and naturally occurs in an unfolded form. Elevated levels of α-Synuclein are found in patients with Parkinson's disease. There is a correlation between the concentration of unphosphorylated α-Synuclein present in the patient and the severity of Parkinson's disease. [ 86 ] Specifically, phosphorylation of Ser129 in α-Synuclein has an impact on severity. Healthy patients have higher levels of unphosphorylated α-Synuclein than patients with Parkinson's disease. The measurement of change in the ratio of concentrations of phosphorylated α-Synuclein to unphosphorylated α-Synuclein within a patient could be a marker of the disease progression. Antibodies that target α-Synuclein at phosphorylated Ser129 are used to study the molecular aspects of synucleinopathies. [ 87 ] [ 88 ]
Phosphorylation of Ser129 is associated with the aggregation of the protein and further damage to the nervous system. The aggregation of phosphorylated α-Synuclein can be enhanced if a presynaptic scaffold protein, Sept4, is present in insufficient quantities. Direct interaction of α-Synuclein with Sept4 inhibits the phosphorylation of Ser129. [ 89 ] [ 90 ] [ 91 ] However, phosphorylation of Ser129 can be observed without synuclein aggregation in conditions of overexpression. [ 92 ] | https://en.wikipedia.org/wiki/Protein_phosphorylation |
Protein precipitation is widely used in downstream processing of biological products in order to concentrate proteins and purify them from various contaminants. For example, in the biotechnology industry protein precipitation is used to eliminate contaminants commonly contained in blood. [ 1 ] The underlying mechanism of precipitation is to alter the solvation potential of the solvent, more specifically, by lowering the solubility of the solute by addition of a reagent.
The solubility of proteins in aqueous buffers depends on the distribution of hydrophilic and hydrophobic amino acid residues on the protein's surface. Hydrophobic residues predominantly occur in the globular protein core, but some exist in patches on the surface. Proteins that have high hydrophobic amino acid content on the surface have low solubility in an aqueous solvent. Charged and polar surface residues interact with ionic groups in the solvent and increase the solubility of a protein. Knowledge of a protein's amino acid composition will aid in determining an ideal precipitation solvent and methods.
Repulsive electrostatic forces form when proteins are dissolved in an electrolyte solution. These repulsive forces between proteins prevent aggregation and facilitate dissolution. Upon dissolution in an electrolyte solution, solvent counterions migrate towards charged surface residues on the protein, forming a rigid matrix of counterions on the protein's surface. Next to this layer is another solvation layer that is less rigid and, as one moves away from the protein surface, contains a decreasing concentration of counterions and an increasing concentration of co-ions. The presence of these solvation layers cause the protein to have fewer ionic interactions with other proteins and decreases the likelihood of aggregation.
Repulsive electrostatic forces also form when proteins are dissolved in water. Water forms a solvation layer around the hydrophilic surface residues of a protein. Water establishes a concentration gradient around the protein, with the highest concentration at the protein surface. This water network has a damping effect on the attractive forces between proteins.
Dispersive or attractive forces exist between proteins through permanent and induced dipoles . For example, basic residues on a protein can have electrostatic interactions with acidic residues on another protein. However, solvation by ions in an electrolytic solution or water will decrease protein–protein attractive forces. Therefore, to precipitate or induce accumulation of proteins, the hydration layer around the protein should be reduced. The purpose of the added reagents in protein precipitation is to reduce the hydration layer.
Protein precipitate formation occurs in a stepwise process. First, a precipitating agent is added and the solution is steadily mixed. Mixing causes the precipitant and protein to collide. Enough mixing time is required for molecules to diffuse across the fluid eddies. Next, proteins undergo a nucleation phase, where submicroscopic sized protein aggregates, or particles, are generated. Growth of these particles is under Brownian diffusion control. Once the particles reach a critical size (0.1 μm to 10 μm for high and low shear fields, respectively), by diffusive addition of individual protein molecules to it, they continue to grow by colliding into each other and sticking or flocculating . This phase occurs at a slower rate. During the final step, called aging in a shear field, the precipitate particles repeatedly collide and stick, then break apart, until a stable mean particle size is reached, which is dependent upon individual proteins. The mechanical strength of the protein particles correlates with the product of the mean shear rate and the aging time, which is known as the Camp number. Aging helps particles withstand the fluid shear forces encountered in pumps and centrifuge feed zones without reducing in size.
Salting out is the most common method used to precipitate a protein. Addition of a neutral salt, such as ammonium sulfate , compresses the solvation layer and increases protein–protein interactions. As the salt concentration of a solution is increased, the charges on the surface of the protein interact with the salt, not the water, thereby exposing hydrophobic patches on the protein surface and causing the protein to fall out of solution (aggregate and precipitate).
Salting out is a spontaneous process when the right concentration of the salt is reached in solution. The hydrophobic patches on the protein surface generate highly ordered water shells. This results in a small decrease in enthalpy , Δ H , and a larger decrease in entropy , Δ S, of the ordered water molecules relative to the molecules in the bulk solution. The overall free energy change, Δ G , of the process is given by the Gibbs free energy equation:
Δ G = Free energy change, Δ H = Enthalpy change upon precipitation, Δ S = Entropy change upon precipitation, T = Absolute temperature.
When water molecules in the rigid solvation layer are brought back into the bulk phase through interactions with the added salt, their greater freedom of movement causes a significant increase in their entropy. Thus, Δ G becomes negative and precipitation occurs spontaneously.
Kosmotropes or "water structure stabilizers" are salts which promote the dissipation / dispersion of water from the solvation layer around a protein. Hydrophobic patches are then exposed on the protein's surface, and they interact with hydrophobic patches on other proteins. These salts enhance protein aggregation and precipitation. Chaotropes or "water structure breakers," have the opposite effect of Kosmotropes. These salts promote an increase in the solvation layer around a protein. The effectiveness of the kosmotropic salts in precipitating proteins follows the order of the Hofmeister series:
Most precipitation P O 4 3 − > S O 4 2 − > C O O − > C l − {\displaystyle \mathrm {PO_{4}^{3-}>SO_{4}^{2-}>COO^{-}>Cl^{-}} } least precipitation
Most precipitation N H 4 + > K + > N a + {\displaystyle \mathrm {NH_{4}^{+}>K^{+}>Na^{+}} } least precipitation
The decrease in protein solubility follows a normalized solubility curve of the type shown. The relationship between the solubility of a protein and increasing ionic strength of the solution can be represented by the Cohn equation:
S = solubility of the protein, B is idealized solubility, K is a salt-specific constant and I is the ionic strength of the solution, which is attributed to the added salt.
I = 1 2 ∑ i = 1 n c i z i 2 {\displaystyle I={\begin{matrix}{\frac {1}{2}}\end{matrix}}\sum _{i=1}^{n}c_{i}z_{i}^{2}}
z i is the ion charge of the salt and c i is the salt concentration. The ideal salt for protein precipitation is most effective for a particular amino acid composition, inexpensive, non-buffering, and non-polluting. The most commonly used salt is ammonium sulfate . There is a low variation in salting out over temperatures 0 °C to 30 °C. Protein precipitates left in the salt solution can remain stable for years-protected from proteolysis and bacterial contamination by the high salt concentrations.
The isoelectric point (pI) is the pH of a solution at which the net primary charge of a protein becomes zero. At a solution pH that is above the pI the surface of the protein is predominantly negatively charged and therefore like-charged molecules will exhibit repulsive forces. Likewise, at a solution pH that is below the pI, the surface of the protein is predominantly positively charged and repulsion between proteins occurs. However, at the pI the negative and positive charges cancel, repulsive electrostatic forces are reduced and the attraction forces predominate. The attraction forces will cause aggregation and precipitation. The pI of most proteins is in the pH range of 4–6. Mineral acids, such as hydrochloric and sulfuric acid are used as precipitants. The greatest disadvantage to isoelectric point precipitation is the irreversible denaturation caused by the mineral acids. For this reason isoelectric point precipitation is most often used to precipitate contaminant proteins, rather than the target protein. The precipitation of casein during cheesemaking, or during production of sodium caseinate, is an isoelectric precipitation.
Addition of miscible solvents such as ethanol or methanol to a solution may cause proteins in the solution to precipitate. The solvation layer around the protein will decrease as the organic solvent progressively displaces water from the protein surface and binds it in hydration layers around the organic solvent molecules. With smaller hydration layers, the proteins can aggregate by attractive electrostatic and dipole forces. Important parameters to consider are temperature, which should be less than 0 °C to avoid denaturation , pH and protein concentration in solution. Miscible organic solvents decrease the dielectric constant of water, which in effect allows two proteins to come close together. At the isoelectric point the relationship between the dielectric constant and protein solubility is given by:
S 0 is an extrapolated value of S , e is the dielectric constant of the mixture and k is a constant that relates to the dielectric constant of water. The Cohn process for plasma protein fractionation relies on solvent precipitation with ethanol to isolate individual plasma proteins.
a clinical application for the use of methanol as a protein precipitating agent is in the estimation of bilirubin.
Polymers , such as dextrans and polyethylene glycols , are frequently used to precipitate proteins because they have low flammability and are less likely to denature biomaterials than isoelectric precipitation. These polymers in solution attract water molecules away from the solvation layer around the protein. This increases the protein–protein interactions and enhances precipitation. For the specific case of polyethylene glycol, precipitation can be modeled by the equation:
C is the polymer concentration, P is a protein–protein interaction coefficient, a is a protein–polymer interaction coefficient and
μ is the chemical potential of component I, R is the universal gas constant and T is the absolute temperature.
Alginate , carboxymethylcellulose, polyacrylic acid, tannic acid and polyphosphates can form extended networks between protein molecules in solution. The effectiveness of these polyelectrolytes depend on the pH of the solution. Anionic polyelectrolytes are used at pH values less than the isoelectric point. Cationic polyelectrolytes are at pH values above the pI. It is important to note that an excess of polyelectrolytes will cause the precipitate to dissolve back into the solution. An example of polyelectrolyte flocculation is the removal of protein cloud from beer wort using Irish moss .
Metal salts can be used at low concentrations to precipitate enzymes and nucleic acids from solutions. Polyvalent metal ions frequently used are Ca 2+ , Mg 2+ , Mn 2+ or Fe 2+ .
There are numerous industrial scaled reactors than can be used to precipitate large amounts of proteins, such as recombinant DNA polymerases from a solution. [1]
Batch reactors are the simplest type of precipitation reactor. The precipitating agent is slowly added to the protein solution under mixing. The aggregating protein particles tend to be compact and regular in shape. Since the particles are exposed to a wide range of shear stresses for a long period of time, they tend to be compact, dense and mechanically stable.
In tubular reactors, feed protein solution and the precipitating reagent are contacted in a zone of efficient mixing then fed into long tubes where precipitation takes place. The fluid in volume elements approach plug flow as they move though the tubes of the reactor. Turbulent flow is promoted through wire mesh inserts in the tube. The tubular reactor does not require moving mechanical parts and is inexpensive to build. However, the reactor can become impractically long if the particles aggregate slowly.
CSTR reactors run at steady state with a continuous flow of reactants and products in a well-mixed tank. Fresh protein feed contacts slurry that already contains precipitate particles and the precipitation reagents. | https://en.wikipedia.org/wiki/Protein_precipitation |
Protein primary structure is the linear sequence of amino acids in a peptide or protein . [ 1 ] By convention, the primary structure of a protein is reported starting from the amino -terminal (N) end to the carboxyl -terminal (C) end. Protein biosynthesis is most commonly performed by ribosomes in cells. Peptides can also be synthesized in the laboratory. Protein primary structures can be directly sequenced , or inferred from DNA sequences .
Amino acids are polymerised via peptide bonds to form a long backbone , with the different amino acid side chains protruding along it. In biological systems, proteins are produced during translation by a cell's ribosomes . Some organisms can also make short peptides by non-ribosomal peptide synthesis , which often use amino acids other than the encoded 22, and may be cyclised, modified and cross-linked.
Peptides can be synthesised chemically via a range of laboratory methods. Chemical methods typically synthesise peptides in the opposite order (starting at the C-terminus) to biological protein synthesis (starting at the N-terminus).
Protein sequence is typically notated as a string of letters, listing the amino acids starting at the amino -terminal end through to the carboxyl -terminal end. Either a three letter code or single letter code can be used to represent the 22 naturally encoded amino acids, as well as mixtures or ambiguous amino acids (similar to nucleic acid notation ). [ 1 ] [ 2 ] [ 3 ]
Peptides can be directly sequenced , or inferred from DNA sequences . Large sequence databases now exist that collate known protein sequences.
In general, polypeptides are unbranched polymers, so their primary structure can often be specified by the sequence of amino acids along their backbone. However, proteins can become cross-linked, most commonly by disulfide bonds , and the primary structure also requires specifying the cross-linking atoms, e.g., specifying the cysteines involved in the protein's disulfide bonds. Other crosslinks include desmosine .
The chiral centers of a polypeptide chain can undergo racemization . Although it does not change the sequence, it does affect the chemical properties of the sequence. In particular, the L -amino acids normally found in proteins can spontaneously isomerize at the C α {\displaystyle \mathrm {C^{\alpha }} } atom to form D -amino acids, which cannot be cleaved by most proteases . Additionally, proline can form stable trans-isomers at the peptide bond.
Additionally, the protein can undergo a variety of post-translational modifications , which are briefly summarized here.
The N-terminal amino group of a polypeptide can be modified covalently, e.g.,
The C-terminal carboxylate group of a polypeptide can also be modified, e.g.,
Finally, the peptide side chains can also be modified covalently, e.g.,
Most of the polypeptide modifications listed above occur post-translationally , i.e., after the protein has been synthesized on the ribosome , typically occurring in the endoplasmic reticulum , a subcellular organelle of the eukaryotic cell.
Many other chemical reactions (e.g., cyanylation) have been applied to proteins by chemists, although they are not found in biological systems.
In addition to those listed above, the most important modification of primary structure is peptide cleavage (by chemical hydrolysis or by proteases ). Proteins are often synthesized in an inactive precursor form; typically, an N-terminal or C-terminal segment blocks the active site of the protein, inhibiting its function. The protein is activated by cleaving off the inhibitory peptide.
Some proteins even have the power to cleave themselves. Typically, the hydroxyl group of a serine (rarely, threonine) or the thiol group of a cysteine residue will attack the carbonyl carbon of the preceding peptide bond, forming a tetrahedrally bonded intermediate [classified as a hydroxyoxazolidine (Ser/Thr) or hydroxythiazolidine (Cys) intermediate]. This intermediate tends to revert to the amide form, expelling the attacking group, since the amide form is usually favored by free energy, (presumably due to the strong resonance stabilization of the peptide group). However, additional molecular interactions may render the amide form less stable; the amino group is expelled instead, resulting in an ester (Ser/Thr) or thioester (Cys) bond in place of the peptide bond. This chemical reaction is called an N-O acyl shift .
The ester/thioester bond can be resolved in several ways:
The compression of amino acid sequences is a comparatively challenging task. The existing specialized amino acid sequence compressors are low compared with that of DNA sequence compressors, mainly because of the characteristics of the data. For example, modeling inversions is harder because of the reverse information loss (from amino acids to DNA sequence). The current lossless data compressor that provides higher compression is AC2. [ 5 ] AC2 mixes various context models using Neural Networks and encodes the data using arithmetic encoding.
The proposal that proteins were linear chains of α-amino acids was made nearly simultaneously by two scientists at the same conference in 1902, the 74th meeting of the Society of German Scientists and Physicians, held in Karlsbad. Franz Hofmeister made the proposal in the morning, based on his observations of the biuret reaction in proteins. Hofmeister was followed a few hours later by Emil Fischer , who had amassed a wealth of chemical details supporting the peptide-bond model. For completeness, the proposal that proteins contained amide linkages was made as early as 1882 by the French chemist E. Grimaux. [ 6 ]
Despite these data and later evidence that proteolytically digested proteins yielded only oligopeptides, the idea that proteins were linear, unbranched polymers of amino acids was not accepted immediately. Some scientists such as William Astbury doubted that covalent bonds were strong enough to hold such long molecules together; they feared that thermal agitations would shake such long molecules asunder. Hermann Staudinger faced similar prejudices in the 1920s when he argued that rubber was composed of macromolecules . [ 6 ]
Thus, several alternative hypotheses arose. The colloidal protein hypothesis stated that proteins were colloidal assemblies of smaller molecules. This hypothesis was disproved in the 1920s by ultracentrifugation measurements by Theodor Svedberg that showed that proteins had a well-defined, reproducible molecular weight and by electrophoretic measurements by Arne Tiselius that indicated that proteins were single molecules. A second hypothesis, the cyclol hypothesis advanced by Dorothy Wrinch , proposed that the linear polypeptide underwent a chemical cyclol rearrangement C=O + HN → {\displaystyle \rightarrow } C(OH)-N that crosslinked its backbone amide groups, forming a two-dimensional fabric . Other primary structures of proteins were proposed by various researchers, such as the diketopiperazine model of Emil Abderhalden and the pyrrol/piperidine model of Troensegaard in 1942. Although never given much credence, these alternative models were finally disproved when Frederick Sanger successfully sequenced insulin [ when? ] and by the crystallographic determination of myoglobin and hemoglobin by Max Perutz and John Kendrew [ when? ] .
Any linear-chain heteropolymer can be said to have a "primary structure" by analogy to the usage of the term for proteins, but this usage is rare compared to the extremely common usage in reference to proteins. In RNA , which also has extensive secondary structure , the linear chain of bases is generally just referred to as the "sequence" as it is in DNA (which usually forms a linear double helix with little secondary structure). Other biological polymers such as polysaccharides can also be considered to have a primary structure, although the usage is not standard.
The primary structure of a biological polymer to a large extent determines the three-dimensional shape ( tertiary structure ). Protein sequence can be used to predict local features , such as segments of secondary structure, or trans-membrane regions. However, the complexity of protein folding currently prohibits predicting the tertiary structure of a protein from its sequence alone. Knowing the structure of a similar homologous sequence (for example a member of the same protein family ) allows highly accurate prediction of the tertiary structure by homology modeling . If the full-length protein sequence is available, it is possible to estimate its general biophysical properties , such as its isoelectric point .
Sequence families are often determined by sequence clustering , and structural genomics projects aim to produce a set of representative structures to cover the sequence space of possible non-redundant sequences. | https://en.wikipedia.org/wiki/Protein_primary_structure |
Protein production is the biotechnological process of generating a specific protein . It is typically achieved by the manipulation of gene expression in an organism such that it expresses large amounts of a recombinant gene . This includes the transcription of the recombinant DNA to messenger RNA ( mRNA ), the translation of mRNA into polypeptide chains, which are ultimately folded into functional proteins and may be targeted to specific subcellular or extracellular locations. [ 1 ]
Protein production systems (also known as expression systems ) are used in the life sciences , biotechnology , and medicine . Molecular biology research uses numerous proteins and enzymes, many of which are from expression systems; particularly DNA polymerase for PCR , reverse transcriptase for RNA analysis, restriction endonucleases for cloning, and to make proteins that are screened in drug discovery as biological targets or as potential drugs themselves. There are also significant applications for expression systems in industrial fermentation , notably the production of biopharmaceuticals such as human insulin to treat diabetes , and to manufacture enzymes .
Commonly used protein production systems include those derived from bacteria , [ 2 ] [ 3 ] yeast , [ 4 ] [ 5 ] baculovirus / insect , [ 6 ] mammalian cells, [ 7 ] [ 8 ] and more recently filamentous fungi such as Myceliophthora thermophila . [ 9 ] When biopharmaceuticals are produced with one of these systems, process-related impurities termed host cell proteins also arrive in the final product in trace amounts. [ 10 ]
The oldest and most widely used expression systems are cell-based and may be defined as the " combination of an expression vector , its cloned DNA, and the host for the vector that provide a context to allow foreign gene function in a host cell, that is, produce proteins at a high level ". [ 11 ] [ 12 ] Overexpression is an abnormally and excessively high level of gene expression which produces a pronounced gene-related phenotype . [ 13 ] [ 14 ] [ clarification needed ]
There are many ways to introduce foreign DNA to a cell for expression, and many different host cells may be used for expression — each expression system has distinct advantages and liabilities. Expression systems are normally referred to by the host and the DNA source or the delivery mechanism for the genetic material. For example, common hosts are bacteria (such as E. coli , B. subtilis ), yeast (such as S. cerevisiae [ 5 ] ) or eukaryotic cell lines . Common DNA sources and delivery mechanisms are viruses (such as baculovirus , retrovirus , adenovirus ), plasmids , artificial chromosomes and bacteriophage (such as lambda ). The best expression system depends on the gene involved, for example the Saccharomyces cerevisiae is often preferred for proteins that require significant posttranslational modification . Insect or mammal cell lines are used when human-like splicing of mRNA is required. Nonetheless, bacterial expression has the advantage of easily producing large amounts of protein, which is required for X-ray crystallography or nuclear magnetic resonance experiments for structure determination.
Because bacteria are prokaryotes , they are not equipped with the full enzymatic machinery to accomplish the required post-translational modifications or molecular folding. Hence, multi-domain eukaryotic proteins expressed in bacteria often are non-functional. Also, many proteins become insoluble as inclusion bodies that are difficult to recover without harsh denaturants and subsequent cumbersome protein-refolding.
To address these concerns, expressions systems using multiple eukaryotic cells were developed for applications requiring the proteins be conformed as in, or closer to eukaryotic organisms: cells of plants (i.e. tobacco), of insects or mammalians (i.e. bovines) are transfected with genes and cultured in suspension and even as tissues or whole organisms, to produce fully folded proteins. Mammalian in vivo expression systems have however low yield and other limitations (time-consuming, toxicity to host cells,..). To combine the high yield/productivity and scalable protein features of bacteria and yeast, and advanced epigenetic features of plants, insects and mammalians systems, other protein production systems are developed using unicellular eukaryotes (i.e. non-pathogenic ' Leishmania ' cells).
E. coli is one of the most widely used expression hosts, and DNA is normally introduced in a plasmid expression vector. The techniques for overexpression in E. coli are well developed and work by increasing the number of copies of the gene or increasing the binding strength of the promoter region so assisting transcription. [ 3 ]
For example, a DNA sequence for a protein of interest could be cloned or subcloned into a high copy-number plasmid containing the lac (often LacUV5 ) promoter, which is then transformed into the bacterium E. coli . Addition of IPTG (a lactose analog) activates the lac promoter and causes the bacteria to express the protein of interest. [ 2 ]
E. coli strain BL21 and BL21(DE3) are two strains commonly used for protein production. As members of the B lineage, they lack lon and OmpT proteases, protecting the produced proteins from degradation. The DE3 prophage found in BL21(DE3) provides T7 RNA polymerase (driven by the LacUV5 promoter), allowing for vectors with the T7 promoter to be used instead. [ 15 ]
Non-pathogenic species of the gram-positive Corynebacterium are used for the commercial production of various amino acids. The C. glutamicum species is widely used for producing glutamate and lysine , [ 16 ] components of human food, animal feed and pharmaceutical products.
Expression of functionally active human epidermal growth factor has been done in C. glutamicum , [ 17 ] thus demonstrating a potential for industrial-scale production of human proteins. Expressed proteins can be targeted for secretion through either the general, secretory pathway (Sec) or the twin-arginine translocation pathway (Tat). [ 18 ]
Unlike gram-negative bacteria , the gram-positive Corynebacterium lack lipopolysaccharides that function as antigenic endotoxins in humans. [ citation needed ]
The non-pathogenic and gram-negative bacteria, Pseudomonas fluorescens , is used for high level production of recombinant proteins; commonly for the development bio-therapeutics and vaccines. P. fluorescens is a metabolically versatile organism, allowing for high throughput screening and rapid development of complex proteins. P. fluorescens is most well known for its ability to rapid and successfully produce high titers of active, soluble protein. [ 19 ]
Expression systems using either S. cerevisiae or Pichia pastoris allow stable and lasting production of proteins that are processed similarly to mammalian cells, at high yield, in chemically defined media of proteins. [ 4 ] [ 5 ]
Filamentous fungi, especially Aspergillus and Trichoderma , have long been used to produce diverse industrial enzymes from their own genomes ("native", "homologous") and from recombinant DNA ("heterologous"). [ 9 ]
More recently, Myceliophthora thermophila C1 has been developed into an expression platform for screening and production of native and heterologous proteins.The expression system C1 shows a low viscosity morphology in submerged culture, enabling the use of complex growth and production media. C1 also does not "hyperglycosylate" heterologous proteins, as Aspergillus and Trichoderma tend to do. [ 9 ]
Baculovirus -infected insect cells [ 20 ] ( Sf9 , Sf21 , High Five strains) or mammalian cells [ 21 ] ( HeLa , HEK 293 ) allow production of glycosylated or membrane proteins that cannot be produced using fungal or bacterial systems. [ 20 ] [ 6 ] It is useful for production of proteins in high quantity. Genes are not expressed continuously because infected host cells eventually lyse and die during each infection cycle. [ 22 ]
Non-lytic insect cell expression is an alternative to the lytic baculovirus expression system. In non-lytic expression, vectors are transiently or stably transfected into the chromosomal DNA of insect cells for subsequent gene expression. [ 23 ] [ 24 ] This is followed by selection and screening of recombinant clones. [ 25 ] The non-lytic system has been used to give higher protein yield and quicker expression of recombinant genes compared to baculovirus-infected cell expression. [ 24 ] Cell lines used for this system include: Sf9 , Sf21 from Spodoptera frugiperda cells, Hi-5 from Trichoplusia ni cells, and Schneider 2 cells and Schneider 3 cells from Drosophila melanogaster cells. [ 23 ] [ 25 ] With this system, cells do not lyse and several cultivation modes can be used. [ 23 ] Additionally, protein production runs are reproducible. [ 23 ] [ 24 ] This system gives a homogeneous product. [ 24 ] A drawback of this system is the requirement of an additional screening step for selecting viable clones . [ 25 ]
Leishmania tarentolae (cannot infect mammals) expression systems allow stable and lasting production of proteins at high yield, in chemically defined media. Produced proteins exhibit fully eukaryotic post-translational modifications, including glycosylation and disulfide bond formation. [ citation needed ]
The most common mammalian expression systems are Chinese Hamster ovary (CHO) and Human embryonic kidney (HEK) cells. [ 26 ] [ 27 ] [ 28 ]
Cell-free production of proteins is performed in vitro using purified RNA polymerase, ribosomes, tRNA and ribonucleotides. These reagents may be produced by extraction from cells or from a cell-based expression system. Due to the low expression levels and high cost of cell-free systems, cell-based systems are more widely used. [ 29 ] | https://en.wikipedia.org/wiki/Protein_production |
Protein purification is a series of processes intended to isolate one or a few proteins from a complex mixture, usually cells , tissues , or whole organisms. Protein purification is vital for the specification of the function, structure, and interactions of the protein of interest. The purification process may separate the protein and non-protein parts of the mixture, and finally separate the desired protein from all other proteins. Ideally, to study a protein of interest, it must be separated from other components of the cell so that contaminants will not interfere in the examination of the protein of interest's structure and function. [ 1 ] Separation of one protein from all others is typically the most laborious aspect of protein purification. Separation steps usually exploit differences in protein size, physico-chemical properties, binding affinity, and biological activity . The pure result may be termed protein isolate .
The protein manufacturing cost remains high and there is a growing demand to develop cost efficient and rapid protein purification methods. Understanding the different protein purification methods and optimizing the downstream processing is critical to minimize production costs while maintaining the quality of acceptable standards of homogeneity. [ 2 ] Protein purification is either preparative or analytical .
Preparative purifications aim to produce a relatively large quantity of purified proteins for subsequent use. Examples include the preparation of commercial products such as enzymes (e.g. lactase ), nutritional proteins (e.g. soy protein isolate), and certain biopharmaceuticals (e.g. insulin ). Several preparative purification steps are often deployed to remove bi-products, such as host cell proteins , which pose a potential threat to the patient's health. [ 3 ]
Analytical purification produces a relatively small amount of a protein for a variety of research or analytical purposes, including identification, quantification, and studies of the protein's structure , post-translational modifications , and function. Each step of a protein purification scheme is monitored and takes into consideration purification levels and yield. A high purification level and a poor yield leaves hardly any protein with which to experiment. On the other hand, a high yield with low purification levels leaves many contaminants (proteins other than the one interest) which interfere with research purposes. [ 1 ]
If the protein of interest is not secreted by the organism into the surrounding solution, the first step of each purification process is the disruption of the cells containing the protein. Depending on how fragile the protein is and how stable the cells are, one could, for instance, use one of the following methods: i) repeated freezing and thawing, ii) sonication , iii) homogenization by high pressure ( French press ), iv) homogenization by grinding (bead mill), and v) permeabilization by detergents (e.g. Triton X-100 ) and/or enzymes (e.g. lysozyme ). [ 4 ] Finally, the cell debris can be removed by differential centrifugation, which is a procedure where the homogenate is centrifuged at low speed, then again at a greater force to yield a pellet consisting of nuclei and supernatant. This yields several fractions of decreasing density where more discriminating purification techniques are applied to one fraction. [ 1 ]
Also, proteases are released during cell lysis , which will start digesting the proteins in the solution. If the protein of interest is sensitive to proteolysis , it is recommended to proceed quickly, and to keep the extract cooled, to slow down the digestion. Alternatively, one or more protease inhibitors can be added to the lysis buffer immediately before cell disruption. Sometimes it is also necessary to add DNAse in order to reduce the viscosity of the cell lysate caused by a high DNA content.
Centrifugation is a process that uses centrifugal force to separate mixtures of particles of varying masses or densities suspended in a liquid. When a vessel (typically a tube or bottle) containing a mixture of proteins or other particulate matter, such as bacterial cells, is rotated at high speeds, the inertia of each particle yields a force in the direction of the particles velocity that is proportional to its mass. The tendency of a given particle to move through the liquid because of this force is offset by the resistance the liquid exerts on the particle. The net effect of "spinning" the sample in a centrifuge is that massive, small, and dense particles move outward faster than less massive particles or particles with more "drag" in the liquid. When suspensions of particles are "spun" in a centrifuge, a "pellet" may form at the bottom of the vessel that is enriched for the most massive particles with low drag in the liquid.
Non-compacted particles remain mostly in the liquid called "supernatant" and can be removed from the vessel thereby separating the supernatant from the pellet. The rate of centrifugation is determined by the angular acceleration applied to the sample, typically measured in comparison to the g-force . If samples are centrifuged long enough, the particles in the vessel will reach equilibrium wherein the particles accumulate specifically at a point in the vessel where their buoyant density is balanced with centrifugal force. Such an "equilibrium" centrifugation can allow extensive purification of a given particle.
Sucrose gradient centrifugation —a linear concentration gradient of sugar (typically sucrose, glycerol, or a silica-based density gradient media, like Percoll )—is generated in a tube such that the highest concentration is on the bottom and lowest on top. A protein sample is then layered on top of the gradient and spun at high speeds in an ultracentrifuge . This causes heavy macromolecules to migrate towards the bottom of the tube faster than lighter material. During centrifugation in the absence of sucrose, as particles move farther and farther from the center of rotation, they experience more and more centrifugal force (the further they move, the faster they move). The problem with this is that the useful separation range within the vessel is restricted to a small observable window. Spinning a sample twice as long does not mean the particle of interest will go twice as far; in fact, it will go significantly further. However, when the proteins are moving through a sucrose gradient, they encounter liquid of increasing density and viscosity. A properly designed sucrose gradient will counteract the increasing centrifugal force so the particles move in close proportion to the time they have been in the centrifugal field. Samples separated by these gradients are referred to as "rate zonal" centrifugations. After separating the protein/particles, the gradient is then fractionated and collected. In biochemistry, ultracentrifugation is valuable for separating biomolecules and analyzing their physical properties. [ 1 ]
The choice of starting material is key to the design of a purification process. In a plant or animal, a particular protein usually is not distributed homogeneously throughout the body; different organs or tissues have higher or lower concentrations of the protein. The use of only the tissues or organs with the highest concentration decreases the volumes needed to produce a given amount of purified protein. If the protein is present in low abundance, or if it has a high value, scientists may use recombinant DNA technology to develop cells that will produce large quantities of the desired protein (this is known as an expression system ). Recombinant expression allows the protein to be tagged, e.g. by a His-tag [ 5 ] or Strep-tag [ 6 ] to facilitate purification, reducing the number of purification steps required.
Analytical purification generally utilizes three properties to separate proteins. First, proteins may be purified according to their isoelectric points by running them through a pH-graded gel or an ion exchange column. Second, proteins can be separated according to their size or molecular weight via size exclusion chromatography or by SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) analysis. Proteins are often purified by using 2D-PAGE and are then analysed by peptide mass fingerprinting to establish the protein identity. This is very useful for scientific purposes and the detection limits for protein are nowadays very low and nanogram amounts of protein are sufficient for their analysis. Thirdly, proteins may be separated by polarity/hydrophobicity via high-performance liquid chromatography or reversed-phase chromatography .
Usually, a protein purification protocol contains one or more chromatographic steps. The basic procedure in chromatography is to flow the solution containing the protein through a column packed with various materials. Different proteins interact differently with the column material, and can thus be separated by the time required to pass the column, or the conditions required to elute the protein from the column. Proteins are typically detected as they are coming off the column by their absorbance at 280 nm. Many different chromatographic methods exist:
Most proteins require some salt to dissolve in water, a process called salting in . As the salt concentration is increased, proteins can precipitate, a process called salting out which involves changing protein solubility. [ 1 ] For example, in bulk protein purification, a common first step to isolate proteins is precipitation with ammonium sulfate (NH 4 ) 2 SO 4 . [ 7 ] This is performed by adding increasing amounts of ammonium sulfate and collecting the different fractions of precipitated protein. Subsequently, ammonium sulfate can be removed using dialysis (separating proteins from small molecules through a semipermeable membrane). [ 1 ] During the ammonium sulfate precipitation step, hydrophobic groups present on the proteins are exposed to the atmosphere, attracting other hydrophobic groups; the result is the formation of an aggregate of hydrophobic components. In this case, the protein precipitate will typically be visible to the naked eye . One advantage of this method is that it can be performed inexpensively, even with very large volumes.
The first proteins to be purified are water-soluble proteins. Purification of integral membrane proteins requires disruption of the cell membrane in order to isolate any one particular protein from others that are in the same membrane compartment. Sometimes a particular membrane fraction can be isolated first, such as isolating mitochondria from cells before purifying a protein located in a mitochondrial membrane. A detergent such as sodium dodecyl sulfate (SDS) can be used to dissolve cell membranes and keep membrane proteins in solution during purification; however, because SDS causes denaturation , milder detergents such as Triton X-100 or CHAPS can be used to retain the protein's native conformation during complete purification.
Chromatography can be used to separate protein in solution or denaturing conditions by using porous gels. This technique is a more discriminating separation and is known as size exclusion chromatography . The principle is that smaller molecules have to traverse a larger volume in a porous matrix. Consequentially, proteins of a certain range in size will require a variable volume of eluent (solvent) before being collected at the other end of the column of gel. Larger molecules (or proteins) will travel through less volume and elute prior to smaller molecules.
In the context of protein purification, the eluent is usually pooled in different test tubes. All test tubes containing no measurable trace of the protein to purify are discarded. The remaining solution is thus made of the protein to purify and any other similarly-sized proteins.
One chromatography technique based on molecular properties is usually not sufficient in obtaining a protein of high purity. [ 1 ] In addition to size, ion exchange chromatography separates compounds according to the nature and degree of their ionic charge. The column to be used is selected according to its type and strength of charge. Anion exchange resins have a positive charge and are used to retain and separate negatively charged compounds (anions), while cation exchange resins have a negative charge and are used to separate positively charged molecules (cations).
Before the separation begins a buffer is pumped through the column to equilibrate the opposing charged ions. Upon injection of the sample, solute molecules will exchange with the buffer ions as each competes for the binding sites on the resin. The length of retention for each solute depends upon the strength of its charge. The most weakly charged compounds will elute first, followed by those with successively stronger charges. Because of the nature of the separating mechanism, pH, buffer type, buffer concentration, and temperature all play important roles in controlling the separation.
Ion exchange chromatography is a very powerful tool for use in protein purification and is frequently used in both analytical and preparative separations. It is especially useful when purifying nucleic-acid binding proteins, where separation of the protein from the bound nucleic acid is required to obtain a pure sample devoid of nucleic acids co-purified from the expression system or the native source.
Free-flow electrophoresis (FFE) is a carrier-free electrophoresis technique that allows preparative protein separation in a laminar buffer stream by using an orthogonal electric field. By making use of a pH-gradient, that can for example be induced by ampholytes , this technique allows to separate protein isoforms up to a resolution of < 0.02 delta-pI.
HIC media is amphiphilic, with both hydrophobic and hydrophilic regions, allowing for the separation of proteins based on their surface hydrophobicity. Target proteins and their product aggregate species tend to have different hydrophobic properties and removing them via HIC further purifies the protein of interest. [ 8 ] Additionally, the environment used typically employs less harsh denaturing conditions than other chromatography techniques, thus helping to preserve the protein of interest in its native and functional state. In pure water, the interactions between the resin and the hydrophobic regions of protein would be very weak, but this interaction is enhanced by applying a protein sample to HIC resin in a high ionic strength buffer. The ionic strength of the buffer is then reduced to elute proteins in order of decreasing hydrophobicity. [ 9 ]
Affinity Chromatography is another powerful separation technique that is highly selective for the protein of interest based upon molecular conformation, which frequently utilizes application specific resins. These resins have ligands attached to their surfaces which are specific for the compounds to be separated. Most frequently, these ligands function in a fashion similar to that of antibody-antigen interactions. This "lock and key" fit between the ligand and its target compound makes it highly specific, frequently generating a single peak, while all else in the sample is unretained.
Many membrane proteins are glycoproteins and can be purified by lectin affinity chromatography. Detergent-solubilized proteins can be allowed to bind to a chromatography resin that has been modified to have a covalently attached lectin. Proteins that do not bind to the lectin are washed away and then specifically bound glycoproteins can be eluted by adding a high concentration of a sugar that competes with the bound glycoproteins at the lectin binding site. Some lectins have high affinity binding to oligosaccharides of glycoproteins that is hard to compete with sugars, and bound glycoproteins need to be released by denaturing the lectin.
Immunoaffinity chromatography uses the specific binding of an antibody -antigen to selectively purify the target protein. The procedure involves immobilizing a protein to a solid substrate (e.g. a porous bead or a membrane), which then selectively binds the target, while everything else flows through. The target protein can be eluted by changing the pH or the salinity . The immobilized ligand can be an antibody (such as immunoglobulin G ) or it can be a protein (such as protein A ). Because this method does not involve engineering in a tag, it can be used for proteins from natural sources. [ 10 ]
High-performance liquid chromatography or high-pressure liquid chromatography is a form of chromatography applying high pressure to drive the solutes through the column faster. This means that the diffusion is limited and the resolution is improved. The most common form is "reversed phase" HPLC, where the column material is hydrophobic . The proteins are eluted by a gradient of increasing amounts of an organic solvent , such as acetonitrile. The proteins elute according to their hydrophobicity. After purification by HPLC the protein is in a solution that only contains volatile compounds, and can easily be lyophilized. [ 11 ] HPLC purification frequently results in denaturation of the purified proteins and is thus not applicable to proteins that do not spontaneously refold.
Another way to tag proteins is to engineer an antigen peptide tag onto the protein, and then purify the protein on a column or by incubating with a loose resin that is coated with an immobilized antibody . This particular procedure is known as immunoprecipitation . Immunoprecipitation is capable of generating an extremely specific interaction which usually results in binding only the desired protein. The purified tagged proteins can then easily be separated from the other proteins in solution and later eluted back into clean solution.
When the tags are not needed anymore, they can be cleaved off by a protease. This often involves engineering a protease cleavage site between the tag and the protein.
Self-cleaving tags eliminate the need for proteases to separate tag from target protein of interest during purification process ( e.g. i CapTag™). [ 12 ] [ 13 ] The main component of the tag is an intein, which cleaves off simply after a pH change. Tagless and pure target protein is then released into the elution buffer.
At the end of a protein purification, the protein often has to be concentrated. Different methods exist.
If the solution doesn't contain any other soluble component than the protein in question the protein can be lyophilized (dried). This is commonly done after an HPLC run. This simply removes all volatile components, leaving the proteins behind.
Ultrafiltration concentrates a protein solution using selective permeable membranes. The function of the membrane is to let the water and small molecules pass through while retaining the protein. The solution is forced against the membrane by mechanical pump, gas pressure, or centrifugation.
The most general method to monitor the purification process is by running a SDS-PAGE of the different steps. This method only gives a rough measure of the amounts of different proteins in the mixture, and it is not able to distinguish between proteins with similar apparent molecular weight .
If the protein has a distinguishing spectroscopic feature or an enzymatic activity , this property can be used to detect and quantify the specific protein, and thus to select the fractions of the separation, that contains the protein. If antibodies against the protein are available then western blotting and ELISA can specifically detect and quantify the amount of desired protein. Some proteins function as receptors and can be detected during purification steps by a ligand binding assay, often using a radioactive ligand .
In order to evaluate the process of multistep purification, the amount of the specific protein has to be compared to the amount of total protein. The latter can be determined by the Bradford total protein assay or by absorbance of light at 280 nm , however some reagents used during the purification process may interfere with the quantification. For example, imidazole (commonly used for purification of polyhistidine-tagged recombinant proteins) is an amino acid analogue and at low concentrations will interfere with the bicinchoninic acid (BCA) assay for total protein quantification. Impurities in low-grade imidazole will also absorb at 280 nm, resulting in an inaccurate reading of protein concentration from UV absorbance.
Another method to be considered is surface plasmon resonance (SPR). SPR can detect binding of label free molecules on the surface of a chip. If the desired protein is an antibody, binding can be translated directly to the activity of the protein. One can express the active concentration of the protein as the percent of the total protein. SPR can be a powerful method for quickly determining protein activity and overall yield. It is a powerful technology that requires an instrument to perform.
Gel electrophoresis is a common laboratory technique that can be used both as a preparative and analytical method. The principle of electrophoresis relies on the movement of a charged ion in an electric field. In practice, the proteins are denatured in a solution containing a detergent ( SDS ). In these conditions, the proteins are unfolded and coated with negatively charged detergent molecules. The proteins in SDS-PAGE are separated on the sole basis of their size.
In analytical methods, the protein migrate as bands based on size. Each band can be detected using stains such as Coomassie blue dye or silver stain . Preparative methods to purify large amounts of protein, require the extraction of the protein from the electrophoretic gel. This extraction may involve excision of the gel containing a band, or eluting the band directly off the gel as it runs off the end of the gel.
In the context of a purification strategy, denaturing condition electrophoresis provides an improved resolution over size exclusion chromatography, but does not scale to large quantity of proteins in a sample as well as the late chromatography columns .
A non-denaturing electrophoretic procedure for isolating bioactive metalloproteins in complex protein mixtures is preparative native PAGE . The intactness or the structural integrity of the isolated protein has to be confirmed by an independent method. [ 14 ] | https://en.wikipedia.org/wiki/Protein_purification |
Protein quaternary structure [ a ] is the fourth (and highest) classification level of protein structure . Protein quaternary structure refers to the structure of proteins which are themselves composed of two or more smaller protein chains (also referred to as subunits). Protein quaternary structure describes the number and arrangement of multiple folded protein subunits in a multi-subunit complex . It includes organizations from simple dimers to large homooligomers and complexes with defined or variable numbers of subunits. [ 1 ] In contrast to the first three levels of protein structure, not all proteins will have a quaternary structure since some proteins function as single units. Protein quaternary structure can also refer to biomolecular complexes of proteins with nucleic acids and other cofactors .
Many proteins are actually assemblies of multiple polypeptide chains. The quaternary structure refers to the number and arrangement of the protein subunits with respect to one another. [ 2 ] Examples of proteins with quaternary structure include hemoglobin , DNA polymerase , ribosomes , antibodies , and ion channels .
Enzymes composed of subunits with diverse functions are sometimes called holoenzymes , in which some parts may be known as regulatory subunits and the functional core is known as the catalytic subunit. Other assemblies referred to instead as multiprotein complexes also possess quaternary structure. Examples include nucleosomes and microtubules . Changes in quaternary structure can occur through conformational changes within individual subunits or through reorientation of the subunits relative to each other. It is through such changes, which underlie cooperativity and allostery in "multimeric" enzymes, that many proteins undergo regulation and perform their physiological function.
The above definition follows a classical approach to biochemistry, established at times when the distinction between a protein and a functional, proteinaceous unit was difficult to elucidate. More recently, people refer to protein–protein interaction when discussing quaternary structure of proteins and consider all assemblies of proteins as protein complexes .
The number of subunits in an oligomeric complex is described using names that end in -mer (Greek for "part, subunit"). Formal and Greco-Latinate names are generally used for the first ten types and can be used for up to twenty subunits, whereas higher order complexes are usually described by the number of subunits, followed by -meric.
The smallest unit forming a homo-oligomer, i.e. one protein chain or subunit , is designated as a monomer, subunit or protomer . The latter term was originally devised to specify the smallest unit of hetero-oligomeric proteins, but is also applied to homo-oligomeric proteins in current literature. The subunits usually arrange in cyclic symmetry to form closed point group symmetries .
Although complexes higher than octamers are rarely observed for most proteins, there are some important exceptions. Viral capsids are often composed of multiples of 60 proteins. Several molecular machines are also found in the cell, such as the proteasome (four heptameric rings = 28 subunits), the transcription complex and the spliceosome . The ribosome is probably the largest molecular machine, and is composed of many RNA and protein molecules.
In some cases, proteins form complexes that then assemble into even larger complexes. In such cases, one uses the nomenclature, e.g., "dimer of dimers" or "trimer of dimers". This may suggest that the complex might dissociate into smaller sub-complexes before dissociating into monomers. This usually implies that the complex consists of different oligomerisation interfaces. For example, a tetrameric protein may have one four-fold rotation axis, i.e. point group symmetry 4 or C 4 . In this case the four interfaces between the subunits are identical. It may also have point group symmetry 222 or D 2 . This tetramer has different interfaces and the tetramer can dissociate into two identical homodimers. Tetramers of 222 symmetry are "dimer of dimers". Hexamers of 32 point group symmetry are "trimer of dimers" or "dimer of trimers". Thus, the nomenclature "dimer of dimers" is used to specify the point group symmetry or arrangement of the oligomer, independent of information relating to its dissociation properties.
Another distinction often made when referring to oligomers is whether they are homomeric or heteromeric, referring to whether the smaller protein subunits that come together to make the protein complex are the same (homomeric) or different (heteromeric) from each other. For example, two identical protein monomers would come together to form a homo-dimer, whereas two different protein monomers would create a hetero-dimer.
Protein quaternary structure can be determined using a variety of experimental techniques that require a sample of protein in a variety of experimental conditions. The experiments often provide an estimate of the mass of the native protein and, together with knowledge of the masses and/or stoichiometry of the subunits, allow the quaternary structure to be predicted with a given accuracy. It is not always possible to obtain a precise determination of the subunit composition for a variety of reasons.
The number of subunits in a protein complex can often be determined by measuring the hydrodynamic molecular volume or mass of the intact complex, which requires native solution conditions. For folded proteins, the mass can be inferred from its volume using the partial specific volume of 0.73 ml/g. However, volume measurements are less certain than mass measurements, since unfolded proteins appear to have a much larger volume than folded proteins; additional experiments are required to determine whether a protein is unfolded or has formed an oligomer.
Methods that measure the mass or volume under unfolding conditions (such as MALDI-TOF mass spectrometry and SDS-PAGE ) are generally not useful, since non-native conditions usually cause the complex to dissociate into monomers. However, these may sometimes be applicable; for example, the experimenter may apply SDS-PAGE after first treating the intact complex with chemical cross-link reagents.
Some bioinformatics methods have been developed for predicting the quaternary structural attributes of proteins based on their sequence information by using various modes of pseudo amino acid composition . [ 2 ] [ 8 ] [ 9 ]
Protein folding prediction programs used to predict protein tertiary structure have also been expanding to better predict protein quaternary structure. One such development is AlphaFold-Multimer [ 10 ] built upon the AlphaFold model for predicting protein tertiary structure.
Protein quaternary structure also plays an important role in certain cell signaling pathways. The G-protein coupled receptor pathway involves a heterotrimeric protein known as a G-protein. G-proteins contain three distinct subunits known as the G-alpha, G-beta, and G-gamma subunits. When the G-protein is activated, it binds to the G-protein coupled receptor protein and the cell signaling pathway is initiated. Another example is the receptor tyrosine kinase (RTK) pathway, which is initiated by the dimerization of two receptor tyrosine kinase monomers. When the dimer is formed, the two kinases can phosphorylate each other and initiate a cell signaling pathway. [ 11 ]
Proteins are capable of forming very tight but also only transient complexes. For example, ribonuclease inhibitor binds to ribonuclease A with a roughly 20 fM dissociation constant . Other proteins have evolved to bind specifically to unusual moieties on another protein, e.g., biotin groups (avidin), phosphorylated tyrosines ( SH2 domains ) or proline-rich segments ( SH3 domains ). Protein–protein interactions can be engineered to favor certain oligomerization states. [ 12 ]
When multiple copies of a polypeptide encoded by a gene form a quaternary complex, this protein structure is referred to as a multimer. [ 13 ] When a multimer is formed from polypeptides produced by two different mutant alleles of a particular gene, the mixed multimer may exhibit greater functional activity than the unmixed multimers formed by each of the mutants alone. In such a case, the phenomenon is referred to as intragenic complementation (also called inter-allelic complementation). Intragenic complementation appears to be common and has been studied in many different genes in a variety of organisms including the fungi Neurospora crassa , Saccharomyces cerevisiae and Schizosaccharomyces pombe ; the bacterium Salmonella typhimurium ; the virus bacteriophage T4 , [ 14 ] an RNA virus, [ 15 ] and humans. [ 16 ] The intermolecular forces likely responsible for self-recognition and multimer formation were discussed by Jehle. [ 17 ]
Direct interaction of two nascent proteins emerging from nearby ribosomes appears to be a general mechanism for oligomer formation. [ 18 ] Hundreds of protein oligomers were identified that assemble in human cells by such an interaction. [ 18 ] The most prevalent form of interaction was between the N-terminal regions of the interacting proteins. Dimer formation appears to be able to occur independently of dedicated assembly machines. | https://en.wikipedia.org/wiki/Protein_quaternary_structure |
Protein quinary structure refers to the features of protein surfaces that are shaped by evolutionary adaptation to the physiological context of living cells . [ 1 ] [ 2 ] [ 3 ] [ 4 ] Quinary structure is thus the fifth level of protein complexity, additional to protein primary , secondary , tertiary and quaternary structures. As opposed to the first four levels of protein structure, which are relevant to isolated proteins in dilute conditions, quinary structure emerges from the crowdedness of the cellular context, [ 5 ] in which transient encounters among macromolecules are constantly occurring.
In order to perform their functions, proteins often need to find a specific counterpart to which they will bind in a relatively long encounter. In a very crowded cytosol, in which proteins engage in a vast and complex network of attracting and repelling interactions, such search becomes challenging, because it involves sampling a huge space of possible partners, of which very few will be productive. A solution to this challenge requires that proteins spend as little time as possible on each encounter, so that they can explore a larger number of surfaces, while simultaneously making this interaction as intimate as possible, so if they do come across the right partner, they will not miss it. [ 6 ] In this sense, quinary structure is the result of a series of adaptations present in protein surfaces, which allow proteins to navigate the complexity of the cellular environment.
With the sense with which it is used today, the term quinary structure first appeared in the work of McConkey, in 1989. [ 7 ] In his work, McConkey runs 2D electrophoresis gels on the total protein content of hamster ( CHO ) and human ( HeLa ) cells. In a 2D electrophoresis gel experiment, the coordinates of a protein depend on its molecular weight and its isoelectric point . Given the evolutionary distance between humans and hamsters, and considering evolutionary rates typical of mammals, one would expect a large number of substitutions to have occurred between hamsters and humans, a fraction of which would involve acidic ( aspartate and glutamate ) and basic ( arginine and lysine ) residues, resulting in changes in the isoelectric point of many proteins. Strikingly, hamster and human cells yielded almost identical fingerprints in the experiment, implying that many fewer of those substitutions actually took place. McConkey suggested in that paper [ 7 ] that the reason why the proteins of humans and hamsters had not diverged as much he anticipated was that an additional selective pressure must have been related to the many non-specific “interactions that are inherently transient” experienced by proteins in the cytoplasm and which “constitute the fifth level of protein organization”.
Despite the crudeness of McConkey's experiment, his interpretation of the results have proved to be accurate. Rather than simply being hydrophilic , protein surfaces must have carefully been modulated by evolution and adapted to this network of weak interactions, often called quinary interactions . It is important to note that protein-protein interactions responsible for the emergence of quinary structure are fundamentally different from specific protein encounters. The latter are the result of relatively high-stability binding, often linked to functionally meaningful events –many of which have already been described [ 8 ] – while the former are often interpreted as some background noise of physiologically unproductive misinteractions that complicate the interpretation of protein networks and need to be avoided, so that normal cellular functions can proceed. [ 9 ] [ 10 ] [ 11 ]
The transient nature of these protein encounters complicates the study of quinary structure. Indeed, the interactions responsible for this upper level of protein organisation are weak and short-lived, and hence would not produce protein-protein complexes that could be isolated by conventional biochemical methods. Therefore, quinary structure can only be understood in vivo . [ 12 ]
In-cell NMR is an experimental technique prominent in the research field of protein quinary structure. The physical principle of in-cell NMR measurements is identical to that of conventional protein NMR , but the experiments rely on expressing high concentrations of the probe protein, which should remain soluble and contained in the cellular space; which introduces additional difficulties and limitations. However, these experiments provide critical insights about the cross-talk between a probe protein and the intracellular environment.
Early attempts at using in-cell NMR to study protein quinary structure were hindered by a limitation caused by the very phenomenon they were trying to understand. Many probe proteins tested in these experiments turned out to produce broad signals, near the detection limit of the method, when measured inside cells of Escherichia coli . In particular, these proteins seemed to tumble as if they had molecular weights much larger than those corresponding to their size. These observations seemed to indicate that the proteins were sticking to other macromolecules, which would have led to poor relaxation properties [ 13 ]
Other in-cell NMR experiments showed that single amino acid changes of surface residues could be used to consistently modulate the tumbling of three different proteins inside bacterial cells. [ 14 ] Charged and hydrophobic residues were shown to have the largest impact in protein intracellular mobility. In particular, more negatively charged proteins would tumble faster in comparison with near-null or positively charged proteins. In contrast, the presence of many hydrophobic residues in the protein surface would slow down protein intracellular tumbling. Protein dipole moment , a measure of charge separation across the protein, was shown to have a significant contribution to protein mobility, where high dipole moments would correlate with slower tumbling. | https://en.wikipedia.org/wiki/Protein_quinary_structure |
Protein secondary structure is the local spatial conformation of the polypeptide backbone excluding the side chains. [ 1 ] The two most common secondary structural elements are alpha helices and beta sheets , though beta turns and omega loops occur as well. Secondary structure elements typically spontaneously form as an intermediate before the protein folds into its three dimensional tertiary structure .
Secondary structure is formally defined by the pattern of hydrogen bonds between the amino hydrogen and carboxyl oxygen atoms in the peptide backbone . Secondary structure may alternatively be defined based on the regular pattern of backbone dihedral angles in a particular region of the Ramachandran plot regardless of whether it has the correct hydrogen bonds.
The concept of secondary structure was first introduced by Kaj Ulrik Linderstrøm-Lang at Stanford in 1952. [ 2 ] [ 3 ] Other types of biopolymers such as nucleic acids also possess characteristic secondary structures .
The most common secondary structures are alpha helices and beta sheets . Other helices, such as the 3 10 helix and π helix , are calculated to have energetically favorable hydrogen-bonding patterns but are rarely observed in natural proteins except at the ends of α helices due to unfavorable backbone packing in the center of the helix. Other extended structures such as the polyproline helix and alpha sheet are rare in native state proteins but are often hypothesized as important protein folding intermediates. Tight turns and loose, flexible loops link the more "regular" secondary structure elements. The random coil is not a true secondary structure, but is the class of conformations that indicate an absence of regular secondary structure.
Amino acids vary in their ability to form the various secondary structure elements. Proline and glycine are sometimes known as "helix breakers" because they disrupt the regularity of the α helical backbone conformation; however, both have unusual conformational abilities and are commonly found in turns . Amino acids that prefer to adopt helical conformations in proteins include methionine , alanine , leucine , glutamate and lysine ("MALEK" in amino-acid 1-letter codes); by contrast, the large aromatic residues ( tryptophan , tyrosine and phenylalanine ) and C β -branched amino acids ( isoleucine , valine , and threonine ) prefer to adopt β-strand conformations. However, these preferences are not strong enough to produce a reliable method of predicting secondary structure from sequence alone.
Low frequency collective vibrations are thought to be sensitive to local rigidity within proteins, revealing beta structures to be generically more rigid than alpha or disordered proteins. [ 6 ] [ 7 ] Neutron scattering measurements have directly connected the spectral feature at ~1 THz to collective motions of the secondary structure of beta-barrel protein GFP. [ 8 ]
Hydrogen bonding patterns in secondary structures may be significantly distorted, which makes automatic determination of secondary structure difficult. There are several methods for formally defining protein secondary structure (e.g., DSSP , [ 9 ] DEFINE, [ 10 ] STRIDE , [ 11 ] ScrewFit, [ 12 ] SST [ 13 ] ).
The Dictionary of Protein Secondary Structure, in short DSSP, is commonly used to describe the protein secondary structure with single letter codes. The secondary structure is assigned based on hydrogen bonding patterns as those initially proposed by Pauling et al. in 1951 (before any protein structure had ever been experimentally determined). There are eight types of secondary structure that DSSP defines:
'Coil' is often codified as ' ' (space), C (coil) or '–' (dash). The helices (G, H and I) and sheet conformations are all required to have a reasonable length. This means that 2 adjacent residues in the primary structure must form the same hydrogen bonding pattern. If the helix or sheet hydrogen bonding pattern is too short they are designated as T or B, respectively. Other protein secondary structure assignment categories exist (sharp turns, Omega loops , etc.), but they are less frequently used.
Secondary structure is defined by hydrogen bonding , so the exact definition of a hydrogen bond is critical. The standard hydrogen-bond definition for secondary structure is that of DSSP , which is a purely electrostatic model. It assigns charges of ± q 1 ≈ 0.42 e to the carbonyl carbon and oxygen, respectively, and charges of ± q 2 ≈ 0.20 e to the amide hydrogen and nitrogen, respectively. The electrostatic energy is
According to DSSP, a hydrogen-bond exists if and only if E is less than −0.5 kcal/mol (−2.1 kJ/mol). Although the DSSP formula is a relatively crude approximation of the physical hydrogen-bond energy, it is generally accepted as a tool for defining secondary structure.
SST [ 14 ] [ 13 ] is a Bayesian method to assign secondary structure to protein coordinate data using the Shannon information criterion of Minimum Message Length ( MML ) inference. SST treats any assignment of secondary structure as a potential hypothesis that attempts to explain ( compress ) given protein coordinate data. The core idea is that the best secondary structural assignment is the one that can explain ( compress ) the coordinates of a given protein coordinates in the most economical way, thus linking the inference of secondary structure to lossless data compression . SST accurately delineates any protein chain into regions associated with the following assignment types: [ 15 ]
SST [ 16 ] detects π and 3 10 helical caps to standard α -helices, and automatically assembles the various extended strands into consistent β-pleated sheets. It provides a readable output of dissected secondary structural elements, and a corresponding PyMol -loadable script to visualize the assigned secondary structural elements individually.
The rough secondary-structure content of a biopolymer (e.g., "this protein is 40% α-helix and 20% β-sheet .") can be estimated spectroscopically . [ 17 ] For proteins, a common method is far-ultraviolet (far-UV, 170–250 nm) circular dichroism . A pronounced double minimum at 208 and 222 nm indicate α-helical structure, whereas a single minimum at 204 nm or 217 nm reflects random-coil or β-sheet structure, respectively. A less common method is infrared spectroscopy , which detects differences in the bond oscillations of amide groups due to hydrogen-bonding. Finally, secondary-structure contents may be estimated accurately using the chemical shifts of an initially unassigned NMR spectrum. [ 18 ]
Predicting protein tertiary structure from only its amino sequence is a very challenging problem (see protein structure prediction ), but using the simpler secondary structure definitions is more tractable.
Early methods of secondary-structure prediction were restricted to predicting the three predominate states: helix, sheet, or random coil. These methods were based on the helix- or sheet-forming propensities of individual amino acids, sometimes coupled with rules for estimating the free energy of forming secondary structure elements. The first widely used techniques to predict protein secondary structure from the amino acid sequence were the Chou–Fasman method [ 19 ] [ 20 ] [ 21 ] and the GOR method . [ 22 ] Although such methods claimed to achieve ~60% accurate in predicting which of the three states (helix/sheet/coil) a residue adopts, blind computing assessments later showed that the actual accuracy was much lower. [ 23 ]
A significant increase in accuracy (to nearly ~80%) was made by exploiting multiple sequence alignment ; knowing the full distribution of amino acids that occur at a position (and in its vicinity, typically ~7 residues on either side) throughout evolution provides a much better picture of the structural tendencies near that position. [ 24 ] [ 25 ] For illustration, a given protein might have a glycine at a given position, which by itself might suggest a random coil there. However, multiple sequence alignment might reveal that helix-favoring amino acids occur at that position (and nearby positions) in 95% of homologous proteins spanning nearly a billion years of evolution. Moreover, by examining the average hydrophobicity at that and nearby positions, the same alignment might also suggest a pattern of residue solvent accessibility consistent with an α-helix. [ 26 ] Taken together, these factors would suggest that the glycine of the original protein adopts α-helical structure, rather than random coil. Several types of methods are used to combine all the available data to form a 3-state prediction, including neural networks , hidden Markov models and support vector machines . Modern prediction methods also provide a confidence score for their predictions at every position.
Secondary-structure prediction methods were evaluated by the Critical Assessment of protein Structure Prediction (CASP) experiments and continuously benchmarked, e.g. by EVA (benchmark) . Based on these tests, the most accurate methods were Psipred , SAM, [ 27 ] PORTER, [ 28 ] PROF, [ 29 ] and SABLE. [ 30 ] The chief area for improvement appears to be the prediction of β-strands; residues confidently predicted as β-strand are likely to be so, but the methods are apt to overlook some β-strand segments (false negatives). There is likely an upper limit of ~90% prediction accuracy overall, due to the idiosyncrasies of the standard method ( DSSP ) for assigning secondary-structure classes (helix/strand/coil) to PDB structures, against which the predictions are benchmarked. [ 31 ]
Accurate secondary-structure prediction is a key element in the prediction of tertiary structure , in all but the simplest ( homology modeling ) cases. For example, a confidently predicted pattern of six secondary structure elements βαββαβ is the signature of a ferredoxin fold. [ 32 ]
Both protein and nucleic acid secondary structures can be used to aid in multiple sequence alignment . These alignments can be made more accurate by the inclusion of secondary structure information in addition to simple sequence information. This is sometimes less useful in RNA because base pairing is much more highly conserved than sequence. Distant relationships between proteins whose primary structures are unalignable can sometimes be found by secondary structure. [ 24 ]
It has been shown that α-helices are more stable, robust to mutations, and designable than β-strands in natural proteins, [ 33 ] thus designing functional all-α proteins is likely to be easier that designing proteins with both helices and strands; this has been recently confirmed experimentally. [ 34 ] | https://en.wikipedia.org/wiki/Protein_secondary_structure |
Protein sequencing is the practical process of determining the amino acid sequence of all or part of a protein or peptide . This may serve to identify the protein or characterize its post-translational modifications . Typically, partial sequencing of a protein provides sufficient information (one or more sequence tags) to identify it with reference to databases of protein sequences derived from the conceptual translation of genes .
The two major direct methods of protein sequencing are mass spectrometry and Edman degradation using a protein sequenator (sequencer). Mass spectrometry methods are now the most widely used for protein sequencing and identification but Edman degradation remains a valuable tool for characterizing a protein's N -terminus .
It is often desirable to know the unordered amino acid composition of a protein prior to attempting to find the ordered sequence, as this knowledge can be used to facilitate the discovery of errors in the sequencing process or to distinguish between ambiguous results. Knowledge of the frequency of certain amino acids may also be used to choose which protease to use for digestion of the protein. The misincorporation of low levels of non-standard amino acids (e.g. norleucine) into proteins may also be determined. [ 1 ] A generalized method often referred to as amino acid analysis [ 2 ] for determining amino acid frequency is as follows:
Hydrolysis is done by heating a sample of the protein in 6 M hydrochloric acid to 100–110 °C for 24 hours or longer. Proteins with many bulky hydrophobic groups may require longer heating periods. However, these conditions are so vigorous that some amino acids ( serine , threonine , tyrosine , tryptophan , glutamine , and cysteine ) are degraded. To circumvent this problem, Biochemistry Online suggests heating separate samples for different times, analysing each resulting solution, and extrapolating back to zero hydrolysis time. Rastall suggests a variety of reagents to prevent or reduce degradation, such as thiol reagents or phenol to protect tryptophan and tyrosine from attack by chlorine, and pre-oxidising cysteine. He also suggests measuring the quantity of ammonia evolved to determine the extent of amide hydrolysis .
The amino acids can be separated by ion-exchange chromatography then derivatized to facilitate their detection. More commonly, the amino acids are derivatized then resolved by reversed phase HPLC .
An example of the ion-exchange chromatography is given by the NTRC using sulfonated polystyrene as a matrix, adding the amino acids in acid solution and passing a buffer of steadily increasing pH through the column. Amino acids are eluted when the pH reaches their respective isoelectric points . Once the amino acids have been separated, their respective quantities are determined by adding a reagent that will form a coloured derivative. If the amounts of amino acids are in excess of 10 nmol, ninhydrin can be used for this; it gives a yellow colour when reacted with proline, and a vivid purple with other amino acids. The concentration of amino acid is proportional to the absorbance of the resulting solution. With very small quantities, down to 10 pmol, fluorescent derivatives can be formed using reagents such as ortho-phthaldehyde (OPA) or fluorescamine .
Pre-column derivatization may use the Edman reagent to produce a derivative that is detected by UV light. Greater sensitivity is achieved using a reagent that generates a fluorescent derivative. The derivatized amino acids are subjected to reversed phase chromatography, typically using a C8 or C18 silica column and an optimised elution gradient. The eluting amino acids are detected using a UV or fluorescence detector and the peak areas compared with those for derivatised standards in order to quantify each amino acid in the sample.
Determining which amino acid forms the N -terminus of a peptide chain is useful for two reasons: to aid the ordering of individual peptide fragments' sequences into a whole chain, and because the first round of Edman degradation is often contaminated by impurities and therefore does not give an accurate determination of the N -terminal amino acid. A generalised method for N -terminal amino acid analysis follows:
There are many different reagents which can be used to label terminal amino acids. They all react with amine groups and will therefore also bind to amine groups in the side chains of amino acids such as lysine - for this reason it is necessary to be careful in interpreting chromatograms to ensure that the right spot is chosen. Two of the more common reagents are Sanger's reagent ( 1-fluoro-2,4-dinitrobenzene ) and dansyl derivatives such as dansyl chloride . Phenylisothiocyanate , the reagent for the Edman degradation, can also be used. The same questions apply here as in the determination of amino acid composition, with the exception that no stain is needed, as the reagents produce coloured derivatives and only qualitative analysis is required. So the amino acid does not have to be eluted from the chromatography column, just compared with a standard. Another consideration to take into account is that, since any amine groups will have reacted with the labelling reagent, ion exchange chromatography cannot be used, and thin-layer chromatography or high-pressure liquid chromatography should be used instead.
The number of methods available for C-terminal amino acid analysis is much smaller than the number of available methods of N-terminal analysis. The most common method is to add carboxypeptidases to a solution of the protein, take samples at regular intervals, and determine the terminal amino acid by analysing a plot of amino acid concentrations against time. This method will be very useful in the case of polypeptides and protein-blocked N termini. C-terminal sequencing would greatly help in verifying the primary structures of proteins predicted from DNA sequences and to detect any posttranslational processing of gene products from known codon sequences.
The Edman degradation is a very important reaction for protein sequencing, because it allows the ordered amino acid composition of a protein to be discovered. Automated Edman sequencers are now in widespread use, and are able to sequence peptides up to approximately 50 amino acids long. A reaction scheme for sequencing a protein by the Edman degradation follows; some of the steps are elaborated on subsequently.
Peptides longer than about 50–70 amino acids long cannot be sequenced reliably by the Edman degradation. Because of this, long protein chains need to be broken up into small fragments that can then be sequenced individually. Digestion is done either by endopeptidases such as trypsin or pepsin or by chemical reagents such as cyanogen bromide . Different enzymes give different cleavage patterns, and the overlap between fragments can be used to construct an overall sequence.
The peptide to be sequenced is adsorbed onto a solid surface. One common substrate is glass fibre coated with polybrene , a cationic polymer . The Edman reagent, phenylisothiocyanate (PITC), is added to the adsorbed peptide, together with a mildly basic buffer solution of 12% trimethylamine . This reacts with the amine group of the N-terminal amino acid.
The terminal amino acid can then be selectively detached by the addition of anhydrous acid. The derivative then isomerises to give a substituted phenylthiohydantoin , which can be washed off and identified by chromatography, and the cycle can be repeated. The efficiency of each step is about 98%, which allows about 50 amino acids to be reliably determined.
A protein sequenator [ 3 ] is a machine that performs Edman degradation in an automated manner. A sample of the protein or peptide is immobilized in the reaction vessel of the protein sequenator and the Edman degradation is performed. Each cycle releases and derivatises one amino acid from the protein or peptide's N -terminus and the released amino-acid derivative is then identified by HPLC. The sequencing process is done repetitively for the whole polypeptide until the entire measurable sequence is established or for a pre-determined number of cycles.
Protein identification is the process of assigning a name to a protein of interest (POI), based on its amino-acid sequence. Typically, only part of the protein’s sequence needs to be determined experimentally in order to identify the protein with reference to databases of protein sequences deduced from the DNA sequences of their genes. Further protein characterization may include confirmation of the actual N- and C-termini of the POI, determination of sequence variants and identification of any post-translational modifications present.
A general scheme for protein identification is described. [ 4 ] [ 5 ]
The pattern of fragmentation of a peptide allows for direct determination of its sequence by de novo sequencing . This sequence may be used to match databases of protein sequences or to investigate post-translational or chemical modifications. It may provide additional evidence for protein identifications performed as above.
The peptides matched during protein identification do not necessarily include the N- or C-termini predicted for the matched protein. This may result from the N- or C-terminal peptides being difficult to identify by MS (e.g. being either too short or too long), being post-translationally modified (e.g. N-terminal acetylation) or genuinely differing from the prediction. Post-translational modifications or truncated termini may be identified by closer examination of the data (i.e. de novo sequencing). A repeat digest using a protease of different specificity may also be useful.
Whilst detailed comparison of the MS data with predictions based on the known protein sequence may be used to define post-translational modifications, targeted approaches to data acquisition may also be used. For instance, specific enrichment of phosphopeptides may assist in identifying phosphorylation sites in a protein. Alternative methods of peptide fragmentation in the mass spectrometer, such as ETD or ECD , may give complementary sequence information.
The protein’s whole mass is the sum of the masses of its amino-acid residues plus the mass of a water molecule and adjusted for any post-translational modifications. Although proteins ionize less well than the peptides derived from them, a protein in solution may be able to be subjected to ESI-MS and its mass measured to an accuracy of 1 part in 20,000 or better. This is often sufficient to confirm the termini (thus that the protein’s measured mass matches that predicted from its sequence) and infer the presence or absence of many post-translational modifications.
Proteolysis does not always yield a set of readily analyzable peptides covering the entire sequence of POI. The fragmentation of peptides in the mass spectrometer often does not yield ions corresponding to cleavage at each peptide bond. Thus, the deduced sequence for each peptide is not necessarily complete. The standard methods of fragmentation do not distinguish between leucine and isoleucine residues since they are isomeric.
Because the Edman degradation proceeds from the N-terminus of the protein, it will not work if the N-terminus has been chemically modified (e.g. by acetylation or formation of Pyroglutamic acid). Edman degradation is generally not useful to determine the positions of disulfide bridges. It also requires peptide amounts of 1 picomole or above for discernible results, making it less sensitive than mass spectrometry .
In biology, proteins are produced by translation of messenger RNA (mRNA) with the protein sequence deriving from the sequence of codons in the mRNA. The mRNA is itself formed by the transcription of genes and may be further modified. These processes are sufficiently understood to use computer algorithms to automate predictions of protein sequences from DNA sequences, such as from whole-genome DNA-sequencing projects, and have led to the generation of large databases of protein sequences such as UniProt . Predicted protein sequences are an important resource for protein identification by mass spectrometry.
Historically, short protein sequences (10 to 15 residues) determined by Edman degradation were back-translated into DNA sequences that could be used as probes or primers to isolate molecular clones of the corresponding gene or complementary DNA. The sequence of the cloned DNA was then determined and used to deduce the full amino-acid sequence of the protein.
Bioinformatics tools exist to assist with interpretation of mass spectra (see de novo peptide sequencing ), to compare or analyze protein sequences (see sequence analysis ), or search databases using peptide or protein sequences (see BLAST ).
The difficulty of protein sequencing was recently proposed as a basis for creating k-time programs, programs that run exactly k times before self-destructing. Such a thing is impossible to build purely in software because all software is inherently clonable an unlimited number of times. | https://en.wikipedia.org/wiki/Protein_sequencing |
A protein skimmer or foam fractionator is a device used to remove organic compounds such as food and waste particles from water. It is most commonly used in commercial applications like municipal water treatment facilities, public aquariums , and aquaculture facilities. Smaller protein skimmers are also used for filtration of home saltwater aquariums and even freshwater aquariums and ponds.
Protein skimming removes certain organic compounds, including proteins and amino acids found in food particles and fish waste, by using the polarity of the protein itself. Due to their intrinsic charge, water-borne proteins are either repelled or attracted by the air–water interface and these molecules can be described as hydrophobic (such as fats or oils) or hydrophilic (such as salt, sugar, ammonia, most amino acids, and most inorganic compounds). However, some larger organic molecules can have both hydrophobic and hydrophilic portions. These molecules are called amphipathic or amphiphilic . Commercial protein skimmers work by generating a large air–water interface, specifically by injecting large numbers of bubbles into the water column. In general, the smaller the bubbles the more effective the protein skimming is because the surface area of small bubbles occupying the same volume is much greater than the same volume of larger bubbles. [ 1 ] Large numbers of small bubbles present an enormous air–water interface for hydrophobic organic molecules and amphipathic organic molecules to collect on the bubble surface (the air–water interface). Water movement hastens diffusion of organic molecules, which effectively brings more organic molecules to the air–water interface and lets the organic molecules accumulate on the surface of the air bubbles. This process continues until the interface is saturated, unless the bubble is removed from the water or it bursts, in which case the accumulated molecules release back into the water column. However, it is important to note that further exposure of a saturated air bubble to organic molecules may continue to result in changes as compounds that bind more strongly may replace those molecules with a weaker binding that have already accumulated on the interface. Although some aquarists believe that increasing the contact time (or dwell time as it is sometimes called) is always good, it is incorrect to claim that it is always better to increase the contact time between bubbles and the aquarium water. [ 2 ] As the bubbles increase near the top of the protein skimmer water column, they become denser and the water begins to drain and create the foam that will carry the organic molecules to the skimmate collection cup or to a separate skimmate waste collector and the organic molecules, and any inorganic molecules that may have become bound to the organic molecules, will be exported from the water system.
In addition to the proteins removed by skimming, there are a number of other organic and inorganic molecules that are typically removed. These include a variety of fats, fatty acids , carbohydrates, metals such as copper, and trace elements such as iodine. Particulates, phytoplankton , bacteria, and detritus are also removed; this is desired by some aquarists, and is often enhanced by placement of the skimmer before other forms of filtration, lessening the burden on the filtration system as a whole. There is at least one published study that provides a detailed list of the export products removed by the skimmer. [ 3 ] Aquarists who keep filter-feeding invertebrates, however, sometimes prefer to keep these particulates in the water to serve as natural food. [ 4 ] [ 5 ]
Protein skimmers are used to harvest algae and phytoplankton gently enough to maintain viability for culturing or commercial sale as live cultures.
Alternative forms of water filtration have recently come into use, including the algae scrubber , which leaves food particles in the water for corals and small fish to consume, but removes the noxious compounds including ammonia, nitrite, nitrate, and phosphate that protein skimmers do not remove.
All skimmers have key features in common: water flows through a chamber and is brought into contact with a column of fine bubbles. The bubbles collect proteins and other substances and carry them to the top of the device where the foam, but not the water, collects in a cup. Here the foam condenses to a liquid, which can be easily removed from the system. The material that collects in the cup can range from pale greenish-yellow, watery liquid to a thick black tar.
Consider this summary of optimal protein skimmer design by Randy Holmes-Farley: [ 6 ]
For a skimmer to function maximally, the following things must take place: 1. A large amount of air–water interface must be generated. 2. Organic molecules must be allowed to collect at the air–water interface. 3. The bubbles forming this air–water interface must come together to form a foam. 4. The water in the foam must partially drain without the bubbles popping prematurely. 5. The drained foam must be separated from the bulk water and discarded.
Also under considerable recent attention has been the general shape of a skimmer as well. In particular, much attention has been given to the introduction of cone shaped skimmer units. Originally designed by Klaus Jensen in 2004, the concept was founded on the principle that a conical body allows the foam to accumulate more steadily through a gently sloping transition. It was claimed that this reduces the overall turbulence, resulting in more efficient skimming. However, this design reduces the overall volume inside the skimmer, reducing dwell time. Cylindrical-shaped protein skimmers are the most popular design and allow for the largest volume of air and water. [ 7 ]
Overall, protein skimmers can be classed in two ways depending on whether they operate by co-current flow or counter-current flow . In a co-current flow system, air is introduced at the bottom of the chamber and is in contact with the water as it rises upwards towards the collection chamber. In a counter-current system, air is forced into the system under pressure and moves against the flow of the water for a while before it rises up towards the collection cup. Because the air bubbles may be in contact with the water for a longer period in a counter-current flow system, protein skimmers of this type are considered by some to be more effective at removing organic wastes. [ 8 ]
The original method of protein skimming, running pressurized air through a diffuser to produce large quantities of microbubbles, remains a viable, effective, and economic choice, although newer technologies may require lower maintenance. The air stone is most often an oblong, partially hollowed block of wood, most often of the genus Tilia . The most popular wooden air-stones for skimmers are made from limewood ( Tilia europaea or European limewood) although basswood ( Tilia americana or American linden), works as well, may be cheaper and is often more readily available. The wooden blocks are drilled, tapped, fitted with an air fitting, and connected by air tubing to one or more air pumps delivering at least 1 cfm. The wooden air stone is placed at the bottom of a tall column of water. The tank water is pumped into the column, allowed to pass by the rising bubbles, and back into the tank. To get enough contact time with the bubble, these units can be many feet in height.
Air stone protein skimmers may be constructed as a DIY project from pvc pipes and fittings at low cost [1] [2] and with varying degrees of complexity [3] .
Air stone protein skimmers require powerful air pumps which are often power hungry, loud, and hot, leading to an increase in the aquarium water temperatures. While this method has been around for many years, due to more efficient technologies emerging, many regard it as inefficient current uses in larger systems or systems with large bio-loads.
The premise behind these skimmers is that a high-pressure pump, combined with a venturi , can be used to introduce the bubbles into the water stream. The tank water is pumped through the venturi, in which fine bubbles are introduced via pressure differential, then enters the skimmer body. This method was popular due to its compact size and high efficiency for the time but venturi designs are now outdated and surpassed by more efficient needle-wheel designs.
This basic concept is more correctly known as an aspirating skimmer, since some skimmer designs using an aspirator do not use a pin-wheel / Adrian-wheel or needle-wheel . Pin-wheel / Adrian-wheel describes the look of an impeller that consists of a disk with pins mounted perpendicular (90°) to the disc and parallel to the rotor. Needle-wheel describes the look of an impeller that consists of a series of pins projecting out perpendicular to the rotor from a central axis. Mesh-wheel describes the look of an impeller that consists of a mesh material attached to a plate or central axis on the rotor. The purpose of these modified impellers is to chop or shred the air that is introduced via an air aspirator apparatus or external air pump into very fine bubbles. The mesh-wheel design provides excellent results in the short term because of its ability to create fine bubbles with its thin cutting surfaces, but its propensity for clogging makes it an unreliable design.
The air aspirator differs from the venturi by the positioning of the water pump. With a venturi, the water is pushed through the unit, creating a vacuum to draw in air. With an air aspirator, the water is pulled through the unit, creating a vacuum to draw in air. These terms, however, are often incorrectly interchanged.
This style of protein skimmer has become very popular with public aquariums and is believed to be the most popular type of skimmer used with residential reef aquariums today. It has been particularly successful in smaller aquariums due to its usually compact size, ease of set up and use, and quiet operation. Since the pump is pushing a mixture of air and water, the power required to turn the rotor can be decreased and may result in a lower power requirement for that pump vs. the same pump with a different impeller when it is only pumping water.
The downdraft skimmer is both a proprietary skimmer design and a style of protein skimmer that injects water under high pressure into tubes that have a foam or bubble generating mechanism and carry the air–water mixture down into the skimmer and into a separate chamber. The proprietary design is protected in the United States with patents and commercial skimmer products in the US are limited to that single company. Their design uses one or more tubes with plastic media such as bio balls inside to mix water under high pressure and air in the body of the skimmer resulting in foam that collects protein waste in a collection cup. This was one of the earlier high performance protein skimmer designs and large models were produced that saw success in large and public aquariums.
The Beckett skimmer has some similarities to the downdraft skimmer but introduced a foam nozzle to produce the flow of air bubbles. The name Beckett comes from the patented foam nozzle developed and sold by the Beckett Corporation (United States), although similar foam nozzle designs are sold by other companies outside the United States (e.g. Sicce (Italy)). Instead of using the plastic media that is found in downdraft skimmer designs, the Beckett skimmer uses design concepts from previous generations of skimmers, specifically the downdraft skimmer and the venturi skimmer (the Beckett 1408 Foam Nozzle is a modified 4 port venturi) to produce a hybrid that is capable of using powerful pressure rated water pumps and quickly processing large amounts of aquarium water in a short period of time. Commercial Beckett skimmers come in single Beckett, dual Beckett, and quad Beckett designs. Well engineered Beckett skimmers are quiet and reliable. Due to the advances in pump technologies and introduction of DC pumps, the concerns of powerful pumps taking up additional space, introducing additional noise, and using more electricity have all been alleviated. Unlike the Downdraft and Spray Induction skimmers, Beckett skimmer designs are produced by a number of companies in the United States and elsewhere and are not known to be restricted by patents.
This method is related to the downdraft, but uses a pump to power a spray nozzle, fixed a few inches above the water level. The spray action entraps and shreds the air in the base of the unit, similar to holding your thumb over a garden hose, which then rises to the collection chamber. In the United States, one company has patented the spray induction technology and the commercial product offerings are limited to that single company.
A recent trend is to change the method by which the skimmer is fed 'dirty' water from the aquarium as a means to recirculate water within the skimmer multiple times before it is returned to the sump or the aquarium. Aspirating pump skimmers are the most popular type of skimmer to use recirculating designs although other types of skimmers, such as Beckett skimmers, are also available in recirculating versions. While there is a popular belief among some aquarist that this recirculation increases the dwell or contact time of the generated air bubbles within the skimmer there is no authoritative evidence that this is true. Each time water is recirculated within the skimmer any air bubbles in that water sample are destroyed and new bubbles are generated by the recirculating pump venturi apparatus so the air-water contact time begins again for these newly created bubbles. In non-recirculating skimmer designs, a skimmer has one inlet supplied by a pump that pulls water in from the aquarium and injects it with air into the skimmer and releasing the foam or air–water mix into the reaction chamber. With a recirculating design, the one inlet is usually driven by a separate feed pump, or in some cases may be gravity fed, to receive the dirty water to process, while the pump providing the foam or air–water mix into the reaction chamber is set up separately in a closed loop on the side of the skimmer. The recirculating pump pulls water out of the skimmer and injects air to generate the foam or air–water mix before returning it to the skimmer reaction chamber—thus 'recirculating' it. The feed pump in a recirculating design typically injects a smaller amount of dirty water than co/counter-current designs. The separate feed pump allows easy control of the rate of water exchange through the skimmer and for many aquarists this is one of the important attractions of recirculating skimmer designs. Because the pump configuration of these skimmers is similar to that of aspirating pump skimmers, the power consumption advantages are also similar. | https://en.wikipedia.org/wiki/Protein_skimmer |
Protein subcellular localization prediction (or just protein localization prediction) involves the prediction of where a protein resides in a cell , its subcellular localization .
In general, prediction tools take as input information about a protein, such as a protein sequence of amino acids , and produce a predicted location within the cell as output, such as the nucleus , Endoplasmic reticulum , Golgi apparatus , extracellular space , or other organelles . The aim is to build tools that can accurately predict the outcome of protein targeting in cells.
Prediction of protein subcellular localization is an important component of bioinformatics based prediction of protein function and genome annotation , and it can aid the identification of drug targets.
Experimentally determining the subcellular localization of a protein can be a laborious and time consuming task. Immunolabeling or tagging (such as with a green fluorescent protein ) to view localization using fluorescence microscope are often used. A high throughput alternative is to use prediction.
Through the development of new approaches in computer science, coupled with an increased dataset of proteins of known localization, computational tools can now provide fast and accurate localization predictions for many organisms. This has resulted in subcellular localization prediction becoming one of the challenges being successfully aided by bioinformatics , and machine learning .
Many prediction methods now exceed the accuracy of some high-throughput laboratory methods for the identification of protein subcellular localization. [ 1 ] [ 2 ] [ 3 ] Particularly, some predictors have been developed [ 4 ] that can be used to deal with proteins that may simultaneously exist, or move between, two or more different subcellular locations. Experimental validation is typically required to confirm the predicted localizations.
In 1999 PSORT was the first published program to predict subcellular localization. [ 5 ] Subsequent tools and websites have been released using techniques such as artificial neural networks , support vector machine and protein motifs . Predictors can be specialized for proteins in different organisms. Some are specialized for eukaryotic proteins, [ 6 ] some for human proteins, [ 7 ] and some for plant proteins. [ 8 ] Methods for the prediction of bacterial localization predictors, and their accuracy, have been reviewed. [ 9 ] In 2021, SCLpred-MEM, a membrane protein prediction tool powered by artificial neural networks was published. [ 10 ] SCLpred-EMS is another tool powered by Artificial neural networks that classify proteins into endomembrane system and secretory pathway (EMS) versus all others. [ 11 ] Similarly, Light-Attention uses machine learning methods to predict ten different common subcellular locations. [ 12 ]
The first model to generalize protein subcellular localization to all cell line does so by leveraging images of subcellular landmark stains (i.e., nuclear, plasma membrane, and endoplasmic reticulum markers) across multiple cell stains. Coupling multimodal data of landmark stains along with a pre-trained protein language model, the Prediction of Unseen Proteins' Subcellular Localization (PUPS) model is capable of generative subcellular localization prediction of any protein in any cell line given the protein's amino acid sequence and reference stains of the cell line. [ 13 ]
The development of protein subcellular location prediction has been summarized in two comprehensive review articles. [ 14 ] [ 15 ] Recent tools and an experience report can be found in a recent paper by Meinken and Min (2012) .
Knowledge of the subcellular localization of a protein can significantly improve target identification during the drug discovery process. For example, secreted proteins and plasma membrane proteins are easily accessible by drug molecules due to their localization in the extracellular space or on the cell surface.
Bacterial cell surface and secreted proteins are also of interest for their potential as vaccine candidates or as diagnostic targets. Aberrant subcellular localization of proteins has been observed in the cells of several diseases, such as cancer and Alzheimer's disease . Secreted proteins from some archaea that can survive in unusual environments have industrially important applications.
By using prediction a high number of proteins can be assessed in order to find candidates that are trafficked to the desired location.
The results of subcellular localization prediction can be stored in databases. Examples include the multi-species database Compartments , FunSecKB2, a fungal database; [ 16 ] PlantSecKB, a plant database; [ 17 ] MetazSecKB, an animal and human database; [ 18 ] and ProtSecKB, a protist database. [ 19 ] | https://en.wikipedia.org/wiki/Protein_subcellular_localization_prediction |
Protein subfamily is a level of protein classification, based on their close evolutionary relationship . It is below the larger levels of protein superfamily and protein family . [ 1 ]
Proteins typically share greater sequence and function similarities with other subfamily members than they do with members of their wider family. [ 1 ] [ 2 ] For example, in the Structural Classification of Proteins database classification system, members of a subfamily share the same interaction interfaces and interaction partners . [ 3 ] These are stricter criteria than for a family, where members have similar structures, but may be more distantly related and so have different interfaces. Subfamilies are assigned by a variety of methods, including sequence similarity , [ 4 ] motifs linked to function, [ 5 ] or phylogenetic clade. [ 6 ] [ 7 ] There is no exact and consistent distinction between a subfamily and a family. The same group of proteins may sometimes be described as a family or a subfamily, depending on the context.
This protein -related article is a stub . You can help Wikipedia by expanding it . | https://en.wikipedia.org/wiki/Protein_subfamily |
A protein superfamily is the largest grouping ( clade ) of proteins for which common ancestry can be inferred (see homology ). Usually this common ancestry is inferred from structural alignment [ 1 ] and mechanistic similarity, even if no sequence similarity is evident. [ 2 ] Sequence homology can then be deduced even if not apparent (due to low sequence similarity). Superfamilies typically contain several protein families which show sequence similarity within each family. The term protein clan is commonly used for protease and glycosyl hydrolases superfamilies based on the MEROPS and CAZy classification systems. [ 2 ] [ 3 ]
Superfamilies of proteins are identified using a number of methods. Closely related members can be identified by different methods to those needed to group the most evolutionarily divergent members.
Historically, the similarity of different amino acid sequences has been the most common method of inferring homology . [ 5 ] Sequence similarity is considered a good predictor of relatedness, since similar sequences are more likely the result of gene duplication and divergent evolution , rather than the result of convergent evolution . Amino acid sequence is typically more conserved than DNA sequence (due to the degenerate genetic code ), so it is a more sensitive detection method. Since some of the amino acids have similar properties (e.g., charge, hydrophobicity, size), conservative mutations that interchange them are often neutral to function. The most conserved sequence regions of a protein often correspond to functionally important regions like catalytic sites and binding sites, since these regions are less tolerant to sequence changes.
Using sequence similarity to infer homology has several limitations. There is no minimum level of sequence similarity guaranteed to produce identical structures. Over long periods of evolution, related proteins may show no detectable sequence similarity to one another. Sequences with many insertions and deletions can also sometimes be difficult to align and so identify the homologous sequence regions. In the PA clan of proteases , for example, not a single residue is conserved through the superfamily, not even those in the catalytic triad . Conversely, the individual families that make up a superfamily are defined on the basis of their sequence alignment, for example the C04 protease family within the PA clan.
Nevertheless, sequence similarity is the most commonly used form of evidence to infer relatedness, since the number of known sequences vastly outnumbers the number of known tertiary structures . [ 6 ] In the absence of structural information, sequence similarity constrains the limits of which proteins can be assigned to a superfamily. [ 6 ]
Structure is much more evolutionarily conserved than sequence, such that proteins with highly similar structures can have entirely different sequences. [ 7 ] Over very long evolutionary timescales, very few residues show detectable amino acid sequence conservation, however secondary structural elements and tertiary structural motifs are highly conserved. Some protein dynamics [ 8 ] and conformational changes of the protein structure may also be conserved, as is seen in the serpin superfamily . [ 9 ] Consequently, protein tertiary structure can be used to detect homology between proteins even when no evidence of relatedness remains in their sequences. Structural alignment programs, such as DALI , use the 3D structure of a protein of interest to find proteins with similar folds. [ 10 ] However, on rare occasions, related proteins may evolve to be structurally dissimilar [ 11 ] and relatedness can only be inferred by other methods. [ 12 ] [ 13 ] [ 14 ]
The catalytic mechanism of enzymes within a superfamily is commonly conserved, although substrate specificity may be significantly different. [ 15 ] Catalytic residues also tend to occur in the same order in the protein sequence. [ 16 ] For the families within the PA clan of proteases, although there has been divergent evolution of the catalytic triad residues used to perform catalysis, all members use a similar mechanism to perform covalent, nucleophilic catalysis on proteins, peptides or amino acids. [ 17 ] However, mechanism alone is not sufficient to infer relatedness. Some catalytic mechanisms have been convergently evolved multiple times independently, and so form separate superfamilies, [ 18 ] [ 19 ] [ 20 ] and in some superfamilies display a range of different (though often chemically similar) mechanisms. [ 15 ] [ 21 ]
Protein superfamilies represent the current limits of our ability to identify common ancestry. [ 22 ] They are the largest evolutionary grouping based on direct evidence that is currently possible. They are therefore amongst the most ancient evolutionary events currently studied. Some superfamilies have members present in all kingdoms of life , indicating that the last common ancestor of that superfamily was in the last universal common ancestor of all life (LUCA). [ 23 ]
Superfamily members may be in different species, with the ancestral protein being the form of the protein that existed in the ancestral species ( orthology ). Conversely, the proteins may be in the same species, but evolved from a single protein whose gene was duplicated in the genome ( paralogy ).
A majority of proteins contain multiple domains. Between 66-80% of eukaryotic proteins have multiple domains while about 40-60% of prokaryotic proteins have multiple domains. [ 5 ] Over time, many of the superfamilies of domains have mixed together. In fact, it is very rare to find “consistently isolated superfamilies”. [ 5 ] [ 1 ] When domains do combine, the N- to C-terminal domain order (the "domain architecture") is typically well conserved. Additionally, the number of domain combinations seen in nature is small compared to the number of possibilities, suggesting that selection acts on all combinations. [ 5 ]
Several biological databases document protein superfamilies and protein folds, for example:
Similarly there are algorithms that search the PDB for proteins with structural homology to a target structure, for example: | https://en.wikipedia.org/wiki/Protein_superfamily |
Protein tags are peptide sequences genetically grafted onto a recombinant protein . Tags are attached to proteins for various purposes. They can be added to either end of the target protein, so they are either C-terminus or N-terminus specific or are both C-terminus and N-terminus specific. Some tags are also inserted at sites within the protein of interest; they are known as internal tags. [ 1 ]
Affinity tags are appended to proteins so that they can be purified from their crude biological source using an affinity technique. Affinity tags include chitin binding protein (CBP), maltose binding protein (MBP), Strep-tag [ 2 ] and glutathione-S-transferase (GST). The poly(His) tag is a widely used protein tag, which binds to matrices bearing immobilized metal ions.
Solubilization tags are used, especially for recombinant proteins expressed in species such as E. coli , to assist in the proper folding in proteins and keep them from aggregating in inclusion bodies . These tags include thioredoxin (TRX) and poly(NANP). Some affinity tags have a dual role as a solubilization agent, such as MBP and GST.
Chromatography tags are used to alter chromatographic properties of the protein to afford different resolution across a particular separation technique. Often, these consist of polyanionic amino acids, such as FLAG-tag or polyglutamate tag. [ 3 ]
Epitope tags are short peptide sequences which are chosen because high-affinity antibodies can be reliably produced in many different species. These are usually derived from viral genes, which explain their high immunoreactivity. Epitope tags include ALFA-tag, V5-tag , Myc-tag , HA-tag , Spot-tag , T7-tag and NE-tag . These tags are particularly useful for western blotting , immunofluorescence and immunoprecipitation experiments, although they also find use in antibody purification.
Fluorescence tags are used to give visual readout on a protein. Green fluorescent protein (GFP) and its variants are the most commonly used fluorescence tags. [ 4 ] More advanced applications of GFP include using it as a folding reporter (fluorescent if folded, colorless if not).
Protein tags may allow specific enzymatic modification (such as biotinylation by biotin ligase) or chemical modification (such as coupling to other proteins through SpyCatcher or reaction with FlAsH-EDT2 for fluorescence imaging). Often tags are combined, in order to connect proteins to multiple other components. However, with the addition of each tag comes the risk that the native function of the protein may be compromised by interactions with the tag. Therefore, after purification, tags are sometimes removed by specific proteolysis (e.g. by TEV protease , Thrombin , Factor Xa or Enteropeptidase ) or intein splicing.
(See Proteinogenic amino acid#Chemical properties for the A-Z amino-acid codes)
HiBiT-tag was developed by Scientists at Promega . It is an 11-amino-acid peptide tag, and it can be fused to the N- or C-terminus or internal locations of proteins. [ 29 ] Its small size leads to a rapid knock-in of this tag with other proteins through CRISPR/Cas9 technology. [ 29 ] | https://en.wikipedia.org/wiki/Protein_tag |
Protein topology is a property of protein molecule that does not change under deformation (without cutting or breaking a bond).
Two main topology frameworks have been developed and applied to protein molecules.
Knot theory which categorises chain entanglements. The usage of knot theory is limited to a small percentage of proteins as most of them are unknot.
Circuit topology categorises intra-chain contacts based on their arrangements. Circuit topology is a determinant of protein folding kinetics [ 1 ] and stability. [ 2 ]
In biology literature, the term topology is also used to refer to mutual orientation of regular secondary structures , such as alpha-helices and beta strands in protein structure [ 3 ] [1] . For example, two adjacent interacting alpha-helices or beta-strands can go in the same or in opposite directions. Topology diagrams of different proteins with known three-dimensional structure are provided by PDBsum ( an example ).
This protein -related article is a stub . You can help Wikipedia by expanding it . | https://en.wikipedia.org/wiki/Protein_topology |
In molecular biology , Proteinase K ( EC 3.4.21.64 , protease K , endopeptidase K , Tritirachium alkaline proteinase , Tritirachium album serine proteinase , Tritirachium album proteinase K ) is a broad-spectrum serine protease . [ 2 ] [ 3 ] [ 4 ] The enzyme was discovered in 1974 in extracts of the fungus Parengyodontium album (formerly Engyodontium album or Tritirachium album ). [ 5 ] Proteinase K is able to digest hair ( keratin ), hence, the name "Proteinase K". The predominant site of cleavage is the peptide bond adjacent to the carboxyl group of aliphatic and aromatic amino acids with blocked alpha amino groups . It is commonly used for its broad specificity. This enzyme belongs to Peptidase family S8 ( subtilisin ). The molecular weight of Proteinase K is 28,900 daltons (28.9 kDa).
Activated by calcium, the enzyme digests proteins preferentially after hydrophobic amino acids (aliphatic, aromatic and other hydrophobic amino acids). Although calcium ions do not affect the enzyme activity, they do contribute to its stability.
Proteins will be completely digested if the incubation time is long and the protease concentration high enough. Upon removal of the calcium ions, the stability of the enzyme is reduced, but the proteolytic activity remains. [ 6 ] Proteinase K has two binding sites for Ca 2+ , which are located close to the active center, but are not directly involved in the catalytic mechanism. The residual activity is sufficient to digest proteins, which usually contaminate nucleic acid preparations. Therefore, the digestion with Proteinase K for the purification of nucleic acids is usually performed in the presence of EDTA (inhibition of metal-ion dependent enzymes such as nucleases).
Proteinase K is also stable over a wide pH range (4–12), with a pH optimum of pH 8.0. [ 5 ] An elevation of the reaction temperature from 37 °C to 50–60 °C may increase the activity several times, like the addition of 0.5–1% sodium dodecyl sulfate (SDS) or Guanidinium chloride (3 M), Guanidinium thiocyanate (1 M) and urea (4 M) [ disputed (for: no source cited for temperature) – discuss ] . The above-mentioned conditions enhance proteinase K activity by making its substrate cleavage sites more accessible. Temperatures above 65 °C, trichloroacetic acid (TCA) or the serine protease-inhibitors AEBSF , PMSF or DFP inhibit the activity.
Proteinase K will not be inhibited by Guanidinium chloride , Guanidinium thiocyanate , urea , Sarkosyl , Triton X-100 , Tween 20 , SDS , citrate , iodoacetic acid , EDTA or by other serine protease inhibitors like Nα-Tosyl-Lys Chloromethyl Ketone (TLCK) and Nα-Tosyl-Phe Chloromethyl Ketone (TPCK) [ citation needed ] .
Protease K activity in commonly used buffers [ 7 ]
Proteinase K is commonly used in molecular biology to digest protein and remove contamination from preparations of nucleic acid . Addition of Proteinase K to nucleic acid preparations rapidly inactivates nucleases that might otherwise degrade the DNA or RNA during purification. It is highly suited to this application since the enzyme is active in the presence of chemicals that denature proteins, such as SDS and urea , chelating agents such as EDTA , sulfhydryl reagents, as well as trypsin or chymotrypsin inhibitors.
Proteinase K is used for the destruction of proteins in cell lysates (tissue, cell culture cells) and for the release of nucleic acids, since it very effectively inactivates DNases and RNases. Some examples for applications:
Proteinase K is very useful in the isolation of highly native, undamaged DNAs or RNAs, since most microbial or mammalian DNases and RNases are rapidly inactivated by the enzyme, particularly in the presence of 0.5–1% SDS.
The enzyme's activity towards native proteins is stimulated by denaturants such as SDS. In contrast, when measured using peptide substrates, denaturants inhibit the enzyme. The reason for this result is that the denaturing agents unfold the protein substrates and make them more accessible to the protease. [ 8 ]
Proteinase K has two disulfide bonds, [ 9 ] but it exhibits higher proteolytic activity in the presence of reducing agents (e.g. 5 mM DTT ), [ 10 ] suggesting that the presumed reduction of its own disulfide bonds does not lead to its irreversible inactivation. Proteinase K is inhibited by serine protease inhibitors such as phenylmethylsulfonyl fluoride ( PMSF ), diisopropylfluorophosphate ( DFP ), or 4-(2-aminoethyl)benzenesulfonyl fluoride ( AEBSF ). Proteinase K activity is unaffected by the sulfhydryl modifying reagents: para-chloromercuribenzoic acid ( PCMB ), N-alpha-tosyl-L-lysyl-chloromethyl-ketone ( TLCK ), or N-alpha-Tosyl-l-phenylalanine Chloromethyl Ketone ( TPCK ), [ 10 ] although presumably if these reagents were included alongside disulfide reducing reagents which exposed the typically-unavailable Proteinase K thiols, it may then become inhibited. | https://en.wikipedia.org/wiki/Proteinase_K |
In plant biology , proteinase inhibitors are a family of small proteins that serve an integral role in the plant’s defense mechanisms against herbivory from insects or microorganisms that may compromise the integrity of the plant.
The proteinase inhibitors work to disrupt the enzymatic ability of the digestive or microbial enzymes that are present in the stomach of the attacker resulting in the inability to properly digest the plant material. This causes an interference of proper growth and discourages further wounding of the plant by the attacker. [ 1 ] Studies have also recently [ when? ] revealed that some proteinase inhibitors also provide defense for the plant through the possession of antimicrobial properties providing for the inhibition of pathogen growth. [ 2 ]
While proteinase inhibitors are present in plants naturally, production of these proteins for defense is often induced by either wounding of the plant or by chemical signaling through molecules such as methyl jasmonate . [ 3 ] Both wounding of the plant as well as signaling molecules result in the formation of jasmonic acid , which then induces the gene expression of proteinase inhibitors. Many other signal cascades as well as the translocation of signal molecules through the phloem and xylem of the plant are also necessary for the production of these inhibitors.
Once the proteinase inhibitor has been ingested by the insect, it presents itself as a normal substrate for the digestive enzymes binding to the active site on the enzyme. This binding of the inhibitor to the proteinase creates a new complex that is very unlikely to dissociate. Once bound, the active site can no longer be accessed by any other substrate and the enzyme can no longer digest the amino acids of the plant. [ 4 ] Without proper digestion, the insect is unable to grow, and may starve if it chooses to remain at the wounded plant. Similar inhibition of growth can be seen in pathogens that interact with these inhibitors.
In order to discover how the production of the inhibitors was induced, scientists exposed tomato plants to different forms of methyl jasmonate and then assayed using radial immunodiffusion for proteinase inhibitors in leaf juices. A group of tomato plants was sprayed with a solution containing methyl jasmonate, while another group of tomato plants was exposed to methyl jasmonate vapor in air-tight glass chambers. Control groups were sprayed with a solution or exposed to a vapor that did not contain methyl jasmonate. Both experimental groups revealed increased proteinase inhibitor production as a result of exposure to volatile methyl jasmonate in comparison to control groups. [ 3 ]
The production of proteinase inhibitors reveals that plants have the ability to alter their defense behavior in response to a threat or direct attack on plant integrity. This complex defense mechanism serves to not only protect the plant from being eaten, but also from pathogen infection requiring both coordination and communication. | https://en.wikipedia.org/wiki/Proteinase_inhibitors_in_plants |
In medicine , proteinopathy ([ pref . protein]; -pathy [ suff . disease]; proteinopathies pl .; proteinopathic adj ), or proteopathy , protein conformational disorder , or protein misfolding disease , is a class of diseases in which certain proteins become structurally abnormal, and thereby disrupt the function of cells , tissues and organs of the body. [ 1 ] [ 2 ]
Often the proteins fail to fold into their normal configuration; in this misfolded state , the proteins can become toxic in some way (a toxic gain-of-function ) or they can lose their normal function. [ 3 ] The proteinopathies include such diseases as Creutzfeldt–Jakob disease (and a variant associated with mad cow disease ) and other prion diseases , Alzheimer's disease , Parkinson's disease , amyloidosis , multiple system atrophy , and a wide range of other disorders. [ 2 ] [ 4 ] [ 5 ] [ 6 ] [ 7 ] [ 8 ] The term proteopathy was first proposed in 2000 by Lary Walker and Harry LeVine. [ 1 ]
The concept of proteopathy can trace its origins to the mid-19th century, when, in 1854, Rudolf Virchow coined the term amyloid ("starch-like") to describe a substance in cerebral corpora amylacea that exhibited a chemical reaction resembling that of cellulose . In 1859, Friedreich and Kekulé demonstrated that, rather than consisting of cellulose, "amyloid" actually is rich in protein. [ 9 ] Subsequent research has shown that many different proteins can form amyloid, and that all amyloids show birefringence in cross- polarized light after staining with the dye Congo red , as well as a fibrillar ultrastructure when viewed with an electron microscope . [ 9 ] However, some proteinaceous lesions lack birefringence and contain few or no classical amyloid fibrils, such as the diffuse deposits of amyloid beta (Aβ) protein in the brains of people with Alzheimer's. [ 10 ] Furthermore, evidence has emerged that small, non-fibrillar protein aggregates known as oligomers are toxic to the cells of an affected organ, and that amyloidogenic proteins in their fibrillar form may be relatively benign. [ 11 ] [ 12 ]
In most, if not all proteinopathies, a change in the 3-dimensional folding conformation increases the tendency of a specific protein to bind to itself. [ 5 ] In this aggregated form, the protein is resistant to clearance and can interfere with the normal capacity of the affected organs. In some cases, misfolding of the protein results in a loss of its usual function. For example, cystic fibrosis is caused by a defective cystic fibrosis transmembrane conductance regulator (CFTR) protein, [ 3 ] and in amyotrophic lateral sclerosis / frontotemporal lobar degeneration (FTLD), certain gene-regulating proteins inappropriately aggregate in the cytoplasm, and thus are unable to perform their normal tasks within the nucleus. [ 13 ] [ 14 ]
Because proteins share a common structural feature known as the polypeptide backbone, all proteins have the potential to misfold under some circumstances. [ 15 ] However, only a relatively small number of proteins are linked to proteopathic disorders, possibly due to structural idiosyncrasies of the vulnerable proteins. For example, proteins that are normally unfolded or relatively unstable as monomers (that is, as single, unbound protein molecules) are more likely to misfold into an abnormal conformation. [ 5 ] [ 15 ] [ 16 ] In nearly all instances, the disease-causing molecular configuration involves an increase in beta-sheet secondary structure of the protein. [ 5 ] [ 15 ] [ 17 ] [ 18 ] [ 19 ]
The abnormal proteins in some proteopathies have been shown to fold into multiple 3-dimensional shapes; these variant, proteinaceous structures are defined by their different pathogenic, biochemical, and conformational properties. [ 20 ] They have been most thoroughly studied with regard to prion disease , and are referred to as protein strains . [ 21 ] [ 22 ]
The likelihood that proteinopathy will develop is increased by certain risk factors that promote the self-assembly of a protein. These include destabilizing changes in the primary amino acid sequence of the protein, post-translational modifications (such as hyperphosphorylation ), changes in temperature or pH , an increase in production of a protein, or a decrease in its clearance. [ 1 ] [ 5 ] [ 15 ] Advancing age is a strong risk factor, [ 1 ] as is traumatic brain injury. [ 23 ] [ 24 ] In the aging brain, multiple proteopathies can overlap. [ 25 ] For example, in addition to tauopathy and Aβ-amyloidosis (which coexist as key pathologic features of Alzheimer's disease), many Alzheimer patients have concomitant synucleinopathy ( Lewy bodies ) in the brain. [ 26 ]
It is hypothesized that chaperones and co-chaperones (proteins that assist protein folding ) may antagonize proteotoxicity during aging and in protein misfolding-diseases to maintain proteostasis . [ 27 ] [ 28 ] [ 29 ]
Some proteins can be induced to form abnormal assemblies by exposure to the same (or similar) protein that has folded into a disease-causing conformation, a process called 'seeding' or 'permissive templating'. [ 30 ] [ 31 ] In this way, the disease state can be brought about in a susceptible host by the introduction of diseased tissue extract from an affected donor. The best known forms of inducible proteopathy are prion diseases , [ 32 ] which can be transmitted by exposure of a host organism to purified prion protein in a disease-causing conformation. [ 33 ] [ 34 ]
There is now evidence that other proteinopathies can be induced by a similar mechanism, including Aβ amyloidosis, amyloid A (AA) amyloidosis, and apolipoprotein AII amyloidosis, [ 31 ] [ 35 ] tauopathy, [ 36 ] synucleinopathy, [ 37 ] [ 38 ] [ 39 ] [ 40 ] and the aggregation of superoxide dismutase -1 (SOD1), [ 41 ] [ 42 ] polyglutamine, [ 43 ] [ 44 ] and TAR DNA-binding protein-43 ( TDP-43 ). [ 45 ]
In all of these instances, an aberrant form of the protein itself appears to be the pathogenic agent. In some cases, the deposition of one type of protein can be experimentally induced by aggregated assemblies of other proteins that are rich in β-sheet structure, possibly because of structural complementarity of the protein molecules. For example, AA amyloidosis can be stimulated in mice by such diverse macromolecules as silk, the yeast amyloid Sup35, and curli fibrils from the bacterium Escherichia coli . [ 46 ] AII amyloid can be induced in mice by a variety of β-sheet rich amyloid fibrils, [ 47 ] and cerebral tauopathy can be induced by brain extracts that are rich in aggregated Aβ. [ 48 ] There is also experimental evidence for cross-seeding between prion protein and Aβ. [ 49 ] In general, such heterologous seeding is less efficient than is seeding by a corrupted form of the same protein.
The development of effective treatments for many proteopathies has been challenging. [ 75 ] [ 76 ] Because the proteopathies often involve different proteins arising from different sources, treatment strategies must be customized to each disorder; however, general therapeutic approaches include maintaining the function of affected organs, reducing the formation of the disease-causing proteins, preventing the proteins from misfolding and/or aggregating, or promoting their removal. [ 77 ] [ 75 ] [ 78 ] For example, in Alzheimer's disease, researchers are seeking ways to reduce the production of the disease-associated protein Aβ by inhibiting the enzymes that free it from its parent protein. [ 76 ] Another strategy is to use antibodies to neutralize specific proteins by active or passive immunization . [ 79 ] In some proteopathies, inhibiting the toxic effects of protein oligomers might be beneficial. [ 80 ]
For example, Amyloid A (AA) amyloidosis can be reduced by treating the inflammatory state that increases the amount of the protein in the blood (referred to as serum amyloid A, or SAA). [ 75 ] In immunoglobulin light chain amyloidosis (AL amyloidosis), chemotherapy can be used to lower the number of the blood cells that make the light chain protein that forms amyloid in various bodily organs. [ 81 ] Transthyretin (TTR) amyloidosis (ATTR) results from the deposition of misfolded TTR in multiple organs. [ 82 ] Because TTR is mainly produced in the liver , TTR amyloidosis can be slowed in some hereditary cases by liver transplantation . [ 83 ] TTR amyloidosis also can be treated by stabilizing the normal assemblies of the protein (called tetramers because they consist of four TTR molecules bound together). Stabilization prevents individual TTR molecules from escaping, misfolding, and aggregating into amyloid. [ 84 ] [ 85 ]
Several other treatment strategies for proteopathies are being investigated, including small molecules and biologic medicines such as small interfering RNAs , antisense oligonucleotides , peptides , and engineered immune cells . [ 84 ] [ 81 ] [ 86 ] [ 87 ] In some cases, multiple therapeutic agents may be combined to improve effectiveness. [ 81 ] [ 88 ] | https://en.wikipedia.org/wiki/Proteinopathy |
Proteinoplasts (sometimes called proteoplasts , aleuroplasts , and aleuronaplasts ) are specialized organelles found only in plant cells . Proteinoplasts belong to a broad category of organelles known as plastids . Plastids are specialized double-membrane organelles found in plant cells. [ 1 ] [ 2 ] Plastids perform a variety of functions such as metabolism of energy, and biological reactions. [ 2 ] [ 3 ] There are multiple types of plastids recognized including Leucoplasts , Chromoplasts , and Chloroplasts . [ 2 ] Plastids are broken up into different categories based on characteristics such as size, function and physical traits. [ 2 ] Chromoplasts help to synthesize and store large amounts of carotenoids. [ 4 ] Chloroplasts are photosynthesizing structures that help to make light energy for the plant. [ 4 ] Leucoplasts are a colorless type of plastid which means that no photosynthesis occurs here. [ 3 ] The colorless pigmentation of the leucoplast is due to not containing the structural components of thylakoids unlike what is found in chloroplasts and chromoplasts that gives them their pigmentation. [ 4 ] From leucoplasts stems the subtype, proteinoplasts, which contain proteins for storage. They contain crystalline bodies of protein and can be the sites of enzyme activity involving those proteins. Proteinoplasts are found in many seeds, such as brazil nuts , peanuts and pulses . Although all plastids contain high concentrations of protein, proteinoplasts were identified in the 1960s and 1970s as having large protein inclusions that are visible with both light microscopes and electron microscopes . Other subtypes of Leucoplasts include amyloplast , and elaioplasts . Amyloplasts help to store and synthesize starch molecules found in plants, while elaioplasts synthesize and store lipids in plant cells. [ 1 ]
This cell biology article is a stub . You can help Wikipedia by expanding it . | https://en.wikipedia.org/wiki/Proteinoplast |
proteins@home [ 1 ] was a volunteer computing project that used the BOINC architecture. The project was run by the Department of Biology at École Polytechnique . The project began on December 28, 2006, and ended in June 2008.
proteins@home was a large-scale non-profit protein structure prediction project utilizing volunteer computing to perform intensive computations in a small amount of time. From their website:
The amino acid sequence of a protein determines its three-dimensional structure, or 'fold'. Conversely, the three-dimensional structure is compatible with a large, but limited set of amino acid sequences. Enumerating the allowed sequences for a given fold is known as the 'inverse protein folding problem'. We are working to solve this problem for a large number of known protein folds (a representative subset: about 1500 folds). The most expensive step is to build a database of energy functions that describe all these structures. For each structure, we consider all possible sequences of amino acids. Surprisingly, this is computationally tractable, because our energy functions are sums over pairs of interactions. Once this is done, we can explore the space of amino acid sequences in a fast and efficient way, and retain the most favorable sequences. This large-scale mapping of protein sequence space will have applications for predicting protein structure and function, for understanding protein evolution, and for designing new proteins. By joining the project, you will help to build the database of energy functions and advance an important area of science with potential biomedical applications. [ 2 ]
This computer science article is a stub . You can help Wikipedia by expanding it .
This computing article is a stub . You can help Wikipedia by expanding it . | https://en.wikipedia.org/wiki/Proteins@home |
DNA-binding proteins are proteins that have DNA-binding domains and thus have a specific or general affinity for single- or double-stranded DNA . [ 3 ] [ 4 ] [ 5 ] Sequence-specific DNA-binding proteins generally interact with the major groove of B-DNA , because it exposes more functional groups that identify a base pair . [ 6 ] [ 7 ]
DNA-binding proteins include transcription factors which modulate the process of transcription, various polymerases , nucleases which cleave DNA molecules, and histones which are involved in chromosome packaging and transcription in the cell nucleus . DNA-binding proteins can incorporate such domains as the zinc finger , the helix-turn-helix , and the leucine zipper (among many others) that facilitate binding to nucleic acid. There are also more unusual examples such as transcription activator like effectors .
Structural proteins that bind DNA are well-understood examples of non-specific DNA-protein interactions. Within chromosomes, DNA is held in complexes with structural proteins. These proteins organize the DNA into a compact structure called chromatin . In eukaryotes , this structure involves DNA binding to a complex of small basic proteins called histones . In prokaryotes , multiple types of proteins are involved. [ 8 ] [ 9 ] The histones form a disk-shaped complex called a nucleosome , which contains two complete turns of double-stranded DNA wrapped around its surface. These non-specific interactions are formed through basic residues in the histones making ionic bonds to the acidic sugar-phosphate backbone of the DNA, and are therefore largely independent of the base sequence. [ 10 ] Chemical modifications of these basic amino acid residues include methylation , phosphorylation and acetylation . [ 11 ] These chemical changes alter the strength of the interaction between the DNA and the histones, making the DNA more or less accessible to transcription factors and changing the rate of transcription. [ 12 ] Other non-specific DNA-binding proteins in chromatin include the high-mobility group (HMG) proteins, which bind to bent or distorted DNA. [ 13 ] Biophysical studies show that these architectural HMG proteins bind, bend and loop DNA to perform its biological functions. [ 14 ] [ 15 ] These proteins are important in bending arrays of nucleosomes and arranging them into the larger structures that form chromosomes. [ 16 ] Recently FK506 binding protein 25 (FBP25) was also shown to non-specifically bind to DNA which helps in DNA repair. [ 17 ]
A distinct group of DNA-binding proteins are the DNA-binding proteins that specifically bind single-stranded DNA. In humans, replication protein A is the best-understood member of this family and is used in processes where the double helix is separated, including DNA replication, recombination and DNA repair. [ 18 ] These binding proteins seem to stabilize single-stranded DNA and protect it from forming stem-loops or being degraded by nucleases .
In contrast, other proteins have evolved to bind to specific DNA sequences. The most intensively studied of these are the various transcription factors , which are proteins that regulate transcription. Each transcription factor binds to one specific set of DNA sequences and activates or inhibits the transcription of genes that have these sequences near their promoters. The transcription factors do this in two ways. Firstly, they can bind the RNA polymerase responsible for transcription, either directly or through other mediator proteins; this locates the polymerase at the promoter and allows it to begin transcription. [ 19 ] Alternatively, transcription factors can bind enzymes that modify the histones at the promoter. This alters the accessibility of the DNA template to the polymerase. [ 20 ]
These DNA targets can occur throughout an organism's genome. Thus, changes in the activity of one type of transcription factor can affect thousands of genes. [ 21 ] Thus, these proteins are often the targets of the signal transduction processes that control responses to environmental changes or cellular differentiation and development. The specificity of these transcription factors' interactions with DNA come from the proteins making multiple contacts to the edges of the DNA bases, allowing them to read the DNA sequence. Most of these base-interactions are made in the major groove, where the bases are most accessible. [ 22 ] Mathematical descriptions of protein-DNA binding taking into account sequence-specificity, and competitive and cooperative binding of proteins of different types are usually performed with the help of the lattice models . [ 23 ] Computational methods to identify the DNA binding sequence specificity have been proposed to make a good use of the abundant sequence data in the post-genomic era. [ 24 ] In addition, progress has happened on structure-based prediction of binding specificity across protein families using deep learning. [ 25 ]
Protein–DNA interactions occur when a protein binds a molecule of DNA , often to regulate the biological function of DNA, usually the expression of a gene . Among the proteins that bind to DNA are transcription factors that activate or repress gene expression by binding to DNA motifs and histones that form part of the structure of DNA and bind to it less specifically. Also proteins that repair DNA such as uracil-DNA glycosylase interact closely with it.
In general, proteins bind to DNA in the major groove ; however, there are exceptions. [ 26 ] Protein–DNA interaction are of mainly two types, either specific interaction, or non-specific interaction. Recent single-molecule experiments showed that DNA binding proteins undergo of rapid rebinding in order to bind in correct orientation for recognizing the target site. [ 27 ]
Designing DNA-binding proteins that have a specified DNA-binding site has been an important goal for biotechnology. Zinc finger proteins have been designed to bind to specific DNA sequences and this is the basis of zinc finger nucleases . Recently transcription activator-like effector nucleases (TALENs) have been created which are based on natural proteins secreted by Xanthomonas bacteria via their type III secretion system when they infect various plant species. [ 28 ]
There are many in vitro and in vivo techniques which are useful in detecting DNA-Protein Interactions. The following lists some methods currently in use: [ 29 ] Electrophoretic mobility shift assay (EMSA) is a widespread qualitative technique to study protein–DNA interactions of known DNA binding proteins. [ 30 ] [ 31 ] DNA-Protein-Interaction - Enzyme-Linked ImmunoSorbant Assay (DPI-ELISA) allows the qualitative and quantitative analysis of DNA-binding preferences of known proteins in vitro . [ 32 ] [ 33 ] This technique allows the analysis of protein complexes that bind to DNA (DPI-Recruitment-ELISA) or is suited for automated screening of several nucleotide probes due to its standard ELISA plate formate. [ 34 ] [ 35 ] DNase footprinting assay can be used to identify the specific sites of binding of a protein to DNA at basepair resolution. [ 36 ] Chromatin immunoprecipitation is used to identify the in vivo DNA target regions of a known transcription factor. This technique when combined with high throughput sequencing is known as ChIP-Seq and when combined with microarrays it is known as ChIP-chip . Yeast one-hybrid System (Y1H) is used to identify which protein binds to a particular DNA fragment. Bacterial one-hybrid system (B1H) is used to identify which protein binds to a particular DNA fragment. Structure determination using X-ray crystallography has been used to give a highly detailed atomic view of protein–DNA interactions.
Besides these methods, other techniques such as SELEX, PBM (protein binding microarrays), DNA microarray screens, DamID, FAIRE or more recently DAP-seq are used in the laboratory to investigate DNA-protein interaction in vivo and in vitro .
The protein–DNA interactions can be modulated using stimuli like ionic strength of the buffer, macromolecular crowding, [ 27 ] temperature, pH and electric field. This can lead to reversible dissociation/association of the protein–DNA complex. [ 37 ] [ 38 ] | https://en.wikipedia.org/wiki/Protein–DNA_interaction |
Carbohydrate–protein interactions are the intermolecular and intramolecular interactions between protein and carbohydrate moieties. These interactions form the basis of specific recognition of carbohydrates by lectins. Carbohydrates are important biopolymers and have a variety of functions. Often carbohydrates serve a function as a recognition element. That is, they are specifically recognized by other biomolecules. Proteins which bind carbohydrate structures are known as lectins . Compared to the study of protein–protein and protein–DNA interaction, it is relatively recent that scientists get to know the protein–carbohydrate binding. [ 1 ]
Many of these interactions involved carbohydrates found at the cell surface, as part of a membrane glycoprotein or glycolipid . These interactions can play a role in cellular adhesion and other cellular recognition events. Intramolecular carbohydrate–protein interactions refer to interactions between glycan and polypeptide moieties in glycoproteins or the glycosylated proteins . [ 2 ]
Generally, there are two types of protein carbohydrate binding important in biological processes: Lectin and antibody.
Lectin is a kind of protein that can bind to carbohydrate with their carbohydrate recognition domains (CRDs). We could use different CRD to classify them. [ 3 ]
Ca 2+ is required to activate the binding. Ca 2+ binds to the protein and carbohydrate by non covalent bond. Mannose-binding protein (MBP) contains the C-type CRD.
Two types mannose-6-phosphate can recognize phosphorylated saccharide. One is cation-dependent and the other does not require cation to activate.
I-type lectin named from the immunoglobulin-like domain. Sialoadhesin is one of the I-type lectin, which binds specifically to sialic acid.
Most antibodies have the similar structure except the hypervariable region which is called the antigen binding site. This region is constituted by the combination of various amino acids. When the antigen is a kind of carbohydrate ( Polysaccharide ), the binding could be regarded as a protein-carbohydrate interaction.
Protein–carbohydrate interactions play an important role in biological function.
Just like other organic molecule study, X-ray crystallography is a very useful tool to know the detail information on the interaction between carbohydrate and protein. [ 8 ]
By using titration, NOESY( N uclear O verhauser E ffect S pectroscop Y ), CIDNP experiments, the specificity and affinity of binding, association constants and equilibrium thermodynamic parameters of carbohydrate–protein binding can be studied. [ 9 ]
In many cases, the conformation information is required, however, sometimes it is not able to get directly from the experiments. So the knowledge-based model building approach is used.
Fluorescence spectrometry is a useful tool and has its advantages: no procedure for separation and plenty of ways to get fluorophore source: there are some of amino acids and ligands that have fluorophore after they are activated. [ 10 ]
Dual polarisation interferometry is a label free analytical technique for measuring interactions and associated conformational changes . [ 11 ]
Recently, studies by using metal nanoparticle probes to detect the carbohydrate–protein interactions were reported. [ 12 ] Use of gold and silver nanoparticle probes in resonant light scattering (RLS) gives particular high sensitivity. Zhenxin Wang and coworker the same principle applied this method to detect the interaction between carbohydrate and protein.
As Lectin can strongly bind to specific carbohydrate, scientists develop several lectin-based carbohydrate biosensors. [ 13 ] Designed lectin contains specific groups can be detected by analytical method. | https://en.wikipedia.org/wiki/Protein–carbohydrate_interaction |
Protein–ligand docking is a molecular modelling technique. The goal of protein–ligand docking is to predict the position and orientation of a ligand (a small molecule) when it is bound to a protein receptor or enzyme. [ 1 ] Pharmaceutical research employs docking techniques for a variety of purposes, most notably in the virtual screening of large databases of available chemicals in order to select likely drug candidates. There has been rapid development in computational ability to determine protein structure with programs such as AlphaFold , [ 2 ] and the demand for the corresponding protein-ligand docking predictions is driving implementation of software that can find accurate models. Once the protein folding can be predicted accurately along with how the ligands of various structures will bind to the protein, the ability for drug development to progress at a much faster rate becomes possible.
Computer-aided drug design (CADD) was introduced in the 1980s in order to screen for novel drugs. [ 3 ] The underlying premise is that by parsing an extremely large data set for chemical compounds which may be viable to make a certain pharmaceutical, researchers were able to minimize the amount of novel without testing them all experimentally. The ability to accurately predict target binding sites is a new phenomena, however, which expands on the ability to simply parse a data set of chemical compounds; now due to increasing computational capability, it is possible to inspect the actual geometries of the protein-ligand binding site in vitro. Hardware advancements in computation have made these structure-oriented methods of drug discovery the next frontier in the 21st century biopharma. In order to finely train the new algorithms to capture the accurate geometry of the protein-ligand binding capability, an experimentally gathered dataset can be used by applying techniques such as X-ray crystallography or NMR spectroscopy . [ 3 ]
Several protein–ligand docking software applications that calculate the site, geometry and energy of small molecules or peptides interacting with proteins are available, such as AutoDock and AutoDock Vina , rDock , FlexAID , Molecular Operating Environment , and Glide . Peptides are a highly flexible type of ligand that has proven to be a difficult type of structure to predict in protein bonding programs. DockThor implements up to 40 rotatable bonds to help model these complex physicochemical bindings at the target site. [ 4 ] Root Mean Square Deviation is the standard method of evaluating various software performance within the binding mode of the protein-ligand structure. Specifically, it is the root-mean-squared deviation between the software-predicted docking pose of the ligand and the experimental binding mode. The RMSD measurement is computed for all of the computer-generated poses of the possible bindings between the protein and ligand. The program does not always perfectly predict the actual physical pose when evaluating the RMSD between candidates. In order to then evaluate the strength of a computer algorithm to predict protein docking, the ranking of RMSD among computer-generated candidates must be examined to determine whether the experimental pose actually was generated but not selected.
Computational capacity has increased dramatically over the last two decades [ when? ] making possible the use of more sophisticated and computationally intensive methods in computer-assisted drug design. However, dealing with receptor flexibility in docking methodologies is still a thorny issue. [ 5 ] The main reason behind this difficulty is the large number of degrees of freedom that have to be considered in this kind of calculations. However, in most of the cases, neglecting it leads to poor docking results in terms of binding pose prediction in real-world settings. [ 6 ] Using coarse grained protein models to overcome this problem seems to be a promising approach. [ 5 ] Coarse-grained models are often implemented in the case of protein-peptide docking , as they frequently involve large-scale conformation transitions of the protein receptor . [ 7 ] [ 8 ]
AutoDock is one of the computational tools frequently used to model the interactions between proteins and ligands during the drug discovery process. Although the classically used algorithms to search for effective poses often assume the receptor proteins to be rigid while the ligand is moderately flexible, newer approaches are implementing models with limited receptor flexibility as well. AutoDockFR is a newer model that is able to simulate this partial flexibility within the receptor protein by letting side-chains of the protein to take various poses among their conformational space. This allows the algorithm to explore a vastly larger space of energetically relevant poses for each ligand tested. [ 9 ]
In order to simplify the complexity of the search space for prediction algorithms, various hypotheses have been tested. One such hypothesis is that side-chain conformational changes that contain more atoms and rotations of greater magnitude are actually less likely to occur than the smaller rotations due to the energy barriers that arise. Steric hindrance and rotational energy cost that are introduced with these larger changes made it less likely that they were included in the actual protein-ligand pose. [ 10 ] Findings such as these can make it easier for scientists to develop heuristics that can lower the complexity of the search space and improve the algorithms.
The original method of testing the molecular models of various binding sites was introduced in the 1980s where the receptor was estimated in a rough manner by spheres which occupied the surface clefts. [ 11 ] The ligand was approximated by more spheres which would occupy the relevant volume. Then a search was executed for maximizing the steric overlap between the spheres of both the binding and receptor spheres.
However, the new scoring functions to evaluate molecular dynamics and protein-ligand docking potential are implementing supervised molecular dynamic [ 3 ] approach. Essentially, the simulations are sequences of small time windows by which the distance between the center of mass of the ligand and protein is computed. The distance values are updated at regular frequencies and then regressively fitted linearly. When the slope is negative, the ligand is getting nearer to the binding site, and vice versa. When the ligand is departing from the binding site, the tree of possibilities is pruned right at that moment so as to avoid unnecessary computation. The advantage of this method is speed without the introduction of any energetic bias which could foul the model from accurate mappings to the experimental truths. [ 3 ] | https://en.wikipedia.org/wiki/Protein–ligand_docking |
Protein–lipid interaction is the influence of membrane proteins on the lipid physical state or vice versa.
The questions which are relevant to understanding of the structure and function of the membrane are: 1) Do intrinsic membrane proteins bind tightly to lipids (see annular lipid shell ), and what is the nature of the layer of lipids adjacent to the protein? 2) Do membrane proteins have long-range effects on the order or dynamics of membrane lipids? 3) How do the lipids influence the structure and/or function of membrane proteins? 4) How do peripheral membrane proteins which bind to the layer surface interact with lipids and influence their behavior?
A large research effort involves approaches to know whether proteins have binding sites which are specific for particular lipids and whether the protein–lipid complexes can be considered to be long-lived, on the order of the time required for the turnover a typical enzyme , that is 10 −3 sec. This is now known through the use of 2 H-NMR , ESR , and fluorescent methods.
There are two approaches used to measure the relative affinity of lipids binding to specific membrane proteins. These involve the use of lipid analogues in reconstituted phospholipid vesicles containing the protein of interest:
1) Spin-labeled phospholipids are motionally restricted when they are adjacent to membrane proteins. The result is a component in the ESR spectrum which is broadened. The experimental spectrum can be analyzed as the sum of the two components, a rapidly tumbling species in the "bulk" lipid phase with a sharp spectrum, and a motionally restricted component adjacent to the protein. Membrane protein denaturation causes further broadening of ESR spin label spectrum and throws more light on membrane lipid-proteins interactions [ 1 ] 2) Spin-labeled and brominated lipid derivatives are able to quench the intrinsic tryptophan fluorescence from membrane proteins. The efficiency of quenching depends on the distance between the lipid derivative and the fluorescent tryptophans.
Most 2 H-NMR experiments with deuterated phospholipids demonstrate that the presence of proteins has little effect on either the order parameter of the lipids in the bilayer or the lipid dynamics , as measured by relaxation times. The overall view resulting from NMR experiments is 1) that the exchange rate between boundary and free lipids is rapid, (10 7 sec −1 ), 2) that the order parameters of the bound lipid are barely affected by being adjacent to proteins, 3) that the dynamics of the acyl chain reorientations are slowed only slightly in the frequency range of 10 9 sec −1 , and 4) that the orientation and the dynamics of the polar headgroups are similarly unaffected in any substantial manner by being adjacent to transmembrane proteins . 13C-NMR spectrum also gives information on specific lipid-protein interactions of biomembranes [ 2 ]
Recent results using non labeled optical methods such as Dual Polarisation Interferometry which measure the birefringence [ 3 ] (or order) within lipid bilayers have been used to show how peptide and protein interactions can influence bilayer order, specifically demonstrating the real time association to bilayer and critical peptide concentration after which the peptides penetrate and disrupt the bilayer order. [ 4 ]
Solid-state NMR techniques have the potential to yield detailed information about the dynamics of individual amino acid residues within a membrane protein. However, the techniques can require large amounts (100–200 mg) of isotopically labeled proteins and are most informative when applied to small proteins where spectroscopic assignments are possible.
Many peripheral membrane proteins bind to the membrane primarily through interactions with integral membrane proteins . But there is a diverse group of proteins which interact directly with the surface of the lipid bilayer . Some, such as myelin basic protein , and spectrin have mainly structural roles. A number of water-soluble proteins can bind to the bilayer surface transiently or under specific conditions.
Misfolding processes , typically exposing hydrophobic regions of proteins, often are associated with binding to lipid membranes and subsequent aggregation, for example, during neurodegenerative disorders , neuronal stress and apoptosis . [ 5 ] | https://en.wikipedia.org/wiki/Protein–lipid_interaction |
Protein–protein interactions ( PPIs ) are physical contacts of high specificity established between two or more protein molecules as a result of biochemical events steered by interactions that include electrostatic forces , hydrogen bonding and the hydrophobic effect . Many are physical contacts with molecular associations between chains that occur in a cell or in a living organism in a specific biomolecular context.
Proteins rarely act alone as their functions tend to be regulated. Many molecular processes within a cell are carried out by molecular machines that are built from numerous protein components organized by their PPIs. These physiological interactions make up the so-called interactomics of the organism, while aberrant PPIs are the basis of multiple aggregation-related diseases, such as Creutzfeldt–Jakob and Alzheimer's diseases .
PPIs have been studied with many methods and from different perspectives: biochemistry , quantum chemistry , molecular dynamics , signal transduction , among others. [ 1 ] [ 2 ] [ 3 ] All this information enables the creation of large protein interaction networks [ 4 ] – similar to metabolic or genetic/epigenetic networks – that empower the current knowledge on biochemical cascades and molecular etiology of disease, as well as the discovery of putative protein targets of therapeutic interest.
In many metabolic reactions, a protein that acts as an electron carrier binds to an enzyme that acts as its reductase . After it receives an electron, it dissociates and then binds to the next enzyme that acts as its oxidase (i.e. an acceptor of the electron). These interactions between proteins are dependent on highly specific binding between proteins to ensure efficient electron transfer. Examples: mitochondrial oxidative phosphorylation chain system components cytochrome c-reductase / cytochrome c / cytochrome c oxidase; microsomal and mitochondrial P450 systems. [ 5 ]
In the case of the mitochondrial P450 systems, the specific residues involved in the binding of the electron transfer protein adrenodoxin to its reductase were identified as two basic Arg residues on the surface of the reductase and two acidic Asp residues on the adrenodoxin. [ 6 ] More recent work on the phylogeny of the reductase has shown that these residues involved in protein–protein interactions have been conserved throughout the evolution of this enzyme. [ 7 ]
The activity of the cell is regulated by extracellular signals. Signal propagation inside and/or along the interior of cells depends on PPIs between the various signaling molecules. The recruitment of signaling pathways through PPIs is called signal transduction and plays a fundamental role in many biological processes and in many diseases including Parkinson's disease and cancer.
A protein may be carrying another protein (for example, from cytoplasm to nucleus or vice versa in the case of the nuclear pore importins). [ citation needed ]
In many biosynthetic processes enzymes interact with each other to produce small compounds or other macromolecules. [ citation needed ]
Physiology of muscle contraction involves several interactions. Myosin filaments act as molecular motors and by binding to actin enables filament sliding. [ 8 ] Furthermore, members of the skeletal muscle lipid droplet-associated proteins family associate with other proteins, as activator of adipose triglyceride lipase and its coactivator comparative gene identification-58, to regulate lipolysis in skeletal muscle
To describe the types of protein–protein interactions (PPIs) it is important to consider that proteins can interact in a "transient" way (to produce some specific effect in a short time, like signal transduction) or to interact with other proteins in a "stable" way to form complexes that become molecular machines within the living systems. A protein complex assembly can result in the formation of homo-oligomeric or hetero-oligomeric complexes . In addition to the conventional complexes, as enzyme-inhibitor and antibody-antigen, interactions can also be established between domain-domain and domain-peptide. Another important distinction to identify protein–protein interactions is the way they have been determined, since there are techniques that measure direct physical interactions between protein pairs, named “binary” methods, while there are other techniques that measure physical interactions among groups of proteins, without pairwise determination of protein partners, named “co-complex” methods.
Homo-oligomers are macromolecular complexes constituted by only one type of protein subunit . Protein subunits assembly is guided by the establishment of non-covalent interactions in the quaternary structure of the protein. Disruption of homo-oligomers in order to return to the initial individual monomers often requires denaturation of the complex. [ 9 ] Several enzymes , carrier proteins , scaffolding proteins, and transcriptional regulatory factors carry out their functions as homo-oligomers.
Distinct protein subunits interact in hetero-oligomers, which are essential to control several cellular functions. The importance of the communication between heterologous proteins is even more evident during cell signaling events and such interactions are only possible due to structural domains within the proteins (as described below).
Stable interactions involve proteins that interact for a long time, taking part of permanent complexes as subunits, in order to carry out functional roles. These are usually the case of homo-oligomers (e.g. cytochrome c ), and some hetero-oligomeric proteins, as the subunits of ATPase . On the other hand, a protein may interact briefly and in a reversible manner with other proteins in only certain cellular contexts – cell type , cell cycle stage , external factors, presence of other binding proteins, etc. – as it happens with most of the proteins involved in biochemical cascades . These are called transient interactions. For example, some G protein–coupled receptors only transiently bind to G i/o proteins when they are activated by extracellular ligands, [ 10 ] while some G q -coupled receptors, such as muscarinic receptor M3, pre-couple with G q proteins prior to the receptor-ligand binding. [ 11 ] Interactions between intrinsically disordered protein regions to globular protein domains (i.e. MoRFs ) are transient interactions. [ 12 ]
Covalent interactions are those with the strongest association and are formed by disulphide bonds or electron sharing . While rare, these interactions are determinant in some posttranslational modifications , as ubiquitination and SUMOylation . Non-covalent bonds are usually established during transient interactions by the combination of weaker bonds, such as hydrogen bonds , ionic interactions, Van der Waals forces , or hydrophobic bonds. [ 13 ]
Water molecules play a significant role in the interactions between proteins. [ 14 ] [ 15 ] The crystal structures of complexes, obtained at high resolution from different but homologous proteins, have shown that some interface water molecules are conserved between homologous complexes. The majority of the interface water molecules make hydrogen bonds with both partners of each complex. Some interface amino acid residues or atomic groups of one protein partner engage in both direct and water mediated interactions with the other protein partner. Doubly indirect interactions, mediated by two water molecules, are more numerous in the homologous complexes of low affinity. [ 16 ] Carefully conducted mutagenesis experiments, e.g. changing a tyrosine residue into a phenylalanine, have shown that water mediated interactions can contribute to the energy of interaction. [ 17 ] Thus, water molecules may facilitate the interactions and cross-recognitions between proteins.
The molecular structures of many protein complexes have been unlocked by the technique of X-ray crystallography . [ 18 ] [ 19 ] The first structure to be solved by this method was that of sperm whale myoglobin by Sir John Cowdery Kendrew . [ 20 ] In this technique the angles and intensities of a beam of X-rays diffracted by crystalline atoms are detected in a film, thus producing a three-dimensional picture of the density of electrons within the crystal. [ 21 ]
Later, nuclear magnetic resonance also started to be applied with the aim of unravelling the molecular structure of protein complexes. One of the first examples was the structure of calmodulin-binding domains bound to calmodulin . [ 19 ] [ 22 ] This technique is based on the study of magnetic properties of atomic nuclei, thus determining physical and chemical properties of the correspondent atoms or the molecules. Nuclear magnetic resonance is advantageous for characterizing weak PPIs. [ 23 ]
Some proteins have specific structural domains or sequence motifs that provide binding to other proteins. Here are some examples of such domains:
The study of the molecular structure can give fine details about the interface that enables the interaction between proteins. When characterizing PPI interfaces it is important to take into account the type of complex. [ 9 ]
Parameters evaluated include size (measured in absolute dimensions Å 2 or in solvent-accessible surface area (SASA) ), shape, complementarity between surfaces, residue interface propensities, hydrophobicity, segmentation and secondary structure, and conformational changes on complex formation. [ 9 ]
The great majority of PPI interfaces reflects the composition of protein surfaces, rather than the protein cores, in spite of being frequently enriched in hydrophobic residues, particularly in aromatic residues. [ 25 ] PPI interfaces are dynamic and frequently planar, although they can be globular and protruding as well. [ 26 ] Based on three structures – insulin dimer, trypsin -pancreatic trypsin inhibitor complex, and oxyhaemoglobin – Cyrus Chothia and Joel Janin found that between 1,130 and 1,720 Å 2 of surface area was removed from contact with water indicating that hydrophobicity is a major factor of stabilization of PPIs. [ 27 ] Later studies refined the buried surface area of the majority of interactions to 1,600±350 Å 2 . However, much larger interaction interfaces were also observed and were associated with significant changes in conformation of one of the interaction partners. [ 18 ] PPIs interfaces exhibit both shape and electrostatic complementarity. [ 9 ] [ 11 ]
There are a multitude of methods to detect them. [ 1 ] [ 28 ] Each of the approaches has its own strengths and weaknesses, especially with regard to the sensitivity and specificity of the method. The most conventional and widely used high-throughput methods are yeast two-hybrid screening and affinity purification coupled to mass spectrometry .
This system was firstly described in 1989 by Fields and Song using Saccharomyces cerevisiae as biological model. [ 29 ] [ 30 ] Yeast two hybrid allows the identification of pairwise PPIs (binary method) in vivo , in which the two proteins are tested for biophysically direct interaction. The Y2H is based on the functional reconstitution of the yeast transcription factor Gal4 and subsequent activation of a selective reporter such as His3. To test two proteins for interaction, two protein expression constructs are made: one protein (X) is fused to the Gal4 DNA-binding domain (DB) and a second protein (Y) is fused to the Gal4 activation domain (AD). In the assay, yeast cells are transformed with these constructs. Transcription of reporter genes does not occur unless bait (DB-X) and prey (AD-Y) interact with each other and form a functional Gal4 transcription factor. Thus, the interaction between proteins can be inferred by the presence of the products resultant of the reporter gene expression. [ 13 ] [ 31 ] In cases in which the reporter gene expresses enzymes that allow the yeast to synthesize essential amino acids or nucleotides, yeast growth under selective media conditions indicates that the two proteins tested are interacting. Recently, software to detect and prioritize protein interactions was published. [ 32 ] [ 33 ]
Despite its usefulness, the yeast two-hybrid system has limitations. It uses yeast as main host system, which can be a problem when studying proteins that contain mammalian-specific post-translational modifications. The number of PPIs identified is usually low because of a high false negative rate; [ 34 ] and, understates membrane proteins , for example. [ 35 ] [ 36 ]
In initial studies that utilized Y2H, proper controls for false positives (e.g. when DB-X activates the reporter gene without the presence of AD-Y) were frequently not done, leading to a higher than normal false positive rate. An empirical framework must be implemented to control for these false positives. [ 37 ] Limitations in lower coverage of membrane proteins have been overcoming by the emergence of yeast two-hybrid variants, such as the membrane yeast two-hybrid (MYTH) [ 36 ] and the split-ubiquitin system, [ 31 ] which are not limited to interactions that occur in the nucleus; and, the bacterial two-hybrid system, performed in bacteria; [ 38 ]
Affinity purification coupled to mass spectrometry mostly detects stable interactions and thus better indicates functional in vivo PPIs. [ 39 ] [ 31 ] This method starts by purification of the tagged protein, which is expressed in the cell usually at in vivo concentrations, and its interacting proteins (affinity purification). One of the most advantageous and widely used methods to purify proteins with very low contaminating background is the tandem affinity purification , developed by Bertrand Seraphin and Matthias Mann and respective colleagues. PPIs can then be analysed by mass spectrometry using different methods: chemical incorporation, biological or metabolic incorporation (SILAC), and label-free methods. [ 9 ] Furthermore, network theory has been used to study the whole set of identified protein–protein interactions in cells. [ 4 ]
This system was first developed by LaBaer and colleagues in 2004 by using in vitro transcription and translation system. They use DNA template encoding the gene of interest fused with GST protein, and it was immobilized in the solid surface. Anti-GST antibody and biotinylated plasmid DNA were bounded in aminopropyltriethoxysilane (APTES)-coated slide. BSA can improve the binding efficiency of DNA. Biotinylated plasmid DNA was bound by avidin. New protein was synthesized by using cell-free expression system i.e. rabbit reticulocyte lysate (RRL), and then the new protein was captured through anti-GST antibody bounded on the slide. To test protein–protein interaction, the targeted protein cDNA and query protein cDNA were immobilized in a same coated slide. By using in vitro transcription and translation system, targeted and query protein was synthesized by the same extract. The targeted protein was bound to array by antibody coated in the slide and query protein was used to probe the array. The query protein was tagged with hemagglutinin (HA) epitope. Thus, the interaction between the two proteins was visualized with the antibody against HA. [ 40 ] [ 41 ]
When multiple copies of a polypeptide encoded by a gene form a complex, this protein structure is referred to as a multimer. When a multimer is formed from polypeptides produced by two different mutant alleles of a particular gene, the mixed multimer may exhibit greater functional activity than the unmixed multimers formed by each of the mutants alone. In such a case, the phenomenon is referred to as intragenic complementation (also called inter-allelic complementation). Intragenic complementation has been demonstrated in many different genes in a variety of organisms including the fungi Neurospora crassa , Saccharomyces cerevisiae and Schizosaccharomyces pombe ; the bacterium Salmonella typhimurium ; the virus bacteriophage T4 , [ 42 ] an RNA virus [ 43 ] and humans. [ 44 ] In such studies, numerous mutations defective in the same gene were often isolated and mapped in a linear order on the basis of recombination frequencies to form a genetic map of the gene. Separately, the mutants were tested in pairwise combinations to measure complementation. An analysis of the results from such studies led to the conclusion that intragenic complementation, in general, arises from the interaction of differently defective polypeptide monomers to form a multimer. [ 45 ] Genes that encode multimer-forming polypeptides appear to be common. One interpretation of the data is that polypeptide monomers are often aligned in the multimer in such a way that mutant polypeptides defective at nearby sites in the genetic map tend to form a mixed multimer that functions poorly, whereas mutant polypeptides defective at distant sites tend to form a mixed multimer that functions more effectively. Direct interaction of two nascent proteins emerging from nearby ribosomes appears to be a general mechanism for homo-oligomer (multimer) formation. [ 46 ] Hundreds of protein oligomers were identified that assemble in human cells by such an interaction. [ 46 ] The most prevalent form of interaction is between the N-terminal regions of the interacting proteins. Dimer formation appears to be able to occur independently of dedicated assembly machines. The intermolecular forces likely responsible for self-recognition and multimer formation were discussed by Jehle. [ 47 ]
Diverse techniques to identify PPIs have been emerging along with technology progression. These include co-immunoprecipitation, protein microarrays , analytical ultracentrifugation , light scattering , fluorescence spectroscopy , luminescence-based mammalian interactome mapping (LUMIER), resonance-energy transfer systems, mammalian protein–protein interaction trap, electro-switchable biosurfaces , protein–fragment complementation assay , as well as real-time label-free measurements by surface plasmon resonance , and calorimetry . [ 35 ] [ 36 ]
The experimental detection and characterization of PPIs is labor-intensive and time-consuming. However, many PPIs can be also predicted computationally, usually using experimental data as a starting point. However, methods have also been developed that allow the prediction of PPI de novo, that is without prior evidence for these interactions.
The Rosetta Stone or Domain Fusion method is based on the hypothesis that interacting proteins are sometimes fused into a single protein in another genome. [ 48 ] Therefore, we can predict if two proteins may be interacting by determining if they each have non-overlapping sequence similarity to a region of a single protein sequence in another genome.
The Conserved Neighborhood method is based on the hypothesis that if genes encoding two proteins are neighbors on a chromosome in many genomes, then they are likely functionally related (and possibly physically interacting). [ 49 ]
The Phylogenetic Profile method is based on the hypothesis that if two or more proteins are concurrently present or absent across several genomes, then they are likely functionally related. [ 49 ] Therefore, potentially interacting proteins can be identified by determining the presence or absence of genes across many genomes and selecting those genes which are always present or absent together.
Publicly available information from biomedical documents is readily accessible through the internet and is becoming a powerful resource for collecting known protein–protein interactions (PPIs), PPI prediction and protein docking. Text mining is much less costly and time-consuming compared to other high-throughput techniques. Currently, text mining methods generally detect binary relations between interacting proteins from individual sentences using rule/pattern-based information extraction and machine learning approaches. [ 50 ] A wide variety of text mining applications for PPI extraction and/or prediction are available for public use, as well as repositories which often store manually validated and/or computationally predicted PPIs. Text mining can be implemented in two stages: information retrieval , where texts containing names of either or both interacting proteins are retrieved and information extraction, where targeted information (interacting proteins, implicated residues, interaction types, etc.) is extracted.
There are also studies using phylogenetic profiling , basing their functionalities on the theory that proteins involved in common pathways co-evolve in a correlated fashion across species. Some more complex text mining methodologies use advanced Natural Language Processing (NLP) techniques and build knowledge networks (for example, considering gene names as nodes and verbs as edges). Other developments involve kernel methods to predict protein interactions. [ 51 ]
Many computational methods have been suggested and reviewed for predicting protein–protein interactions. [ 52 ] [ 53 ] [ 54 ] Prediction approaches can be grouped into categories based on predictive evidence: protein sequence, comparative genomics , protein domains, protein tertiary structure, and interaction network topology. [ 52 ] The construction of a positive set (known interacting protein pairs) and a negative set (non-interacting protein pairs) is needed for the development of a computational prediction model. [ 53 ] Prediction models using machine learning techniques can be broadly classified into two main groups: supervised and unsupervised, based on the labeling of input variables according to the expected outcome. [ 54 ]
In 2005, integral membrane proteins of Saccharomyces cerevisiae were analyzed using the mating-based ubiquitin system (mbSUS). The system detects membrane proteins interactions with extracellular signaling proteins [ 55 ] Of the 705 integral membrane proteins 1,985 different interactions were traced that involved 536 proteins. To sort and classify interactions a support vector machine was used to define high medium and low confidence interactions. The split-ubiquitin membrane yeast two-hybrid system uses transcriptional reporters to identify yeast transformants that encode pairs of interacting proteins. [ 56 ] In 2006, random forest , an example of a supervised technique, was found to be the most-effective machine learning method for protein interaction prediction. [ 57 ] Such methods have been applied for discovering protein interactions on human interactome, specifically the interactome of Membrane proteins [ 58 ] and the interactome of Schizophrenia-associated proteins. [ 59 ]
As of 2020, a model using residue cluster classes (RCCs), constructed from the 3DID and Negatome databases, resulted in 96-99% correctly classified instances of protein–protein interactions. [ 60 ] RCCs are a computational vector space that mimics protein fold space and includes all simultaneously contacted residue sets, which can be used to analyze protein structure-function relation and evolution. [ 61 ]
Large scale identification of PPIs generated hundreds of thousands of interactions, which were collected together in specialized biological databases that are continuously updated in order to provide complete interactomes . The first of these databases was the Database of Interacting Proteins (DIP) . [ 62 ]
Primary databases collect information about published PPIs proven to exist via small-scale or large-scale experimental methods. Examples: DIP , Biomolecular Interaction Network Database (BIND), Biological General Repository for Interaction Datasets ( BioGRID ), Human Protein Reference Database (HPRD), IntAct Molecular Interaction Database, Molecular Interactions Database (MINT), MIPS Protein Interaction Resource on Yeast (MIPS-MPact), and MIPS Mammalian Protein–Protein Interaction Database (MIPS-MPPI).<
Meta-databases normally result from the integration of primary databases information, but can also collect some original data.
Prediction databases include many PPIs that are predicted using several techniques (main article). Examples: Human Protein–Protein Interaction Prediction Database (PIPs), [ 63 ] Interlogous Interaction Database (I2D), Known and Predicted Protein–Protein Interactions (STRING-db) , and Unified Human Interactive (UniHI).
The aforementioned computational methods all depend on source databases whose data can be extrapolated to predict novel protein–protein interactions . Coverage differs greatly between databases. In general, primary databases have the fewest total protein interactions recorded as they do not integrate data from multiple other databases, while prediction databases have the most because they include other forms of evidence in addition to experimental. For example, the primary database IntAct has 572,063 interactions, [ 64 ] the meta-database APID has 678,000 interactions, [ 65 ] and the predictive database STRING has 25,914,693 interactions. [ 66 ] However, it is important to note that some of the interactions in the STRING database are only predicted by computational methods such as Genomic Context and not experimentally verified.
Information found in PPIs databases supports the construction of interaction networks. Although the PPI network of a given query protein can be represented in textbooks, diagrams of whole cell PPIs are frankly complex and difficult to generate. [ 67 ]
One example of a manually produced molecular interaction map is the Kurt Kohn's 1999 map of cell cycle control. [ 68 ] Drawing on Kohn's map, Schwikowski et al. in 2000 published a paper on PPIs in yeast, linking 1,548 interacting proteins determined by two-hybrid screening. They used a layered graph drawing method to find an initial placement of the nodes and then improved the layout using a force-based algorithm. [ 69 ]
Bioinformatic tools have been developed to simplify the difficult task of visualizing molecular interaction networks and complement them with other types of data. For instance, Cytoscape is an open-source software widely used and many plugins are currently available. [ 70 ] Pajek software is advantageous for the visualization and analysis of very large networks. [ 49 ]
Identification of functional modules in PPI networks is an important challenge in bioinformatics. Functional modules means a set of proteins that are highly connected to each other in PPI network. It is almost similar problem as community detection in social networks . There are some methods such as Jactive [ 71 ] modules and MoBaS. [ 72 ] Jactive modules integrate PPI network and gene expression data where as MoBaS integrate PPI network and Genome Wide association Studies .
protein–protein relationships are often the result of multiple types of interactions or are deduced from different approaches, including co-localization, direct interaction, suppressive genetic interaction, additive genetic interaction, physical association, and other associations. [ 73 ]
Protein–protein interactions often result in one of the interacting proteins either being 'activated' or 'repressed'. Such effects can be indicated in a PPI network by "signs" (e.g. "activation" or "inhibition"). Although such attributes have been added to networks for a long time, [ 75 ] Vinayagam et al. (2014) coined the term Signed network for them. Signed networks are often expressed by labeling the interaction as either positive or negative. A positive interaction is one where the interaction results in one of the proteins being activated. Conversely, a negative interaction indicates that one of the proteins being inactivated. [ 76 ]
Protein–protein interaction networks are often constructed as a result of lab experiments such as yeast two-hybrid screens or 'affinity purification and subsequent mass spectrometry techniques. [ 77 ] However these methods do not provide the layer of information needed in order to determine what type of interaction is present in order to be able to attribute signs to the network diagrams.
RNA interference (RNAi) screens (repression of individual proteins between transcription and translation) are one method that can be utilized in the process of providing signs to the protein–protein interactions. Individual proteins are repressed and the resulting phenotypes are analyzed. A correlating phenotypic relationship (i.e. where the inhibition of either of two proteins results in the same phenotype) indicates a positive, or activating relationship. Phenotypes that do not correlate (i.e. where the inhibition of either of two proteins results in two different phenotypes) indicate a negative or inactivating relationship. If protein A is dependent on protein B for activation then the inhibition of either protein A or B will result in a cell losing the service that is provided by protein A and the phenotypes will be the same for the inhibition of either A or B. If, however, protein A is inactivated by protein B then the phenotypes will differ depending on which protein is inhibited (inhibit protein B and it can no longer inactivate protein A leaving A active however inactivate A and there is nothing for B to activate since A is inactive and the phenotype changes). Multiple RNAi screens need to be performed in order to reliably appoint a sign to a given protein–protein interaction. Vinayagam et al. who devised this technique state that a minimum of nine RNAi screens are required with confidence increasing as one carries out more screens. [ 76 ]
Modulation of PPI is challenging and is receiving increasing attention by the scientific community. [ 78 ] Several properties of PPI such as allosteric sites and hotspots, have been incorporated into drug-design strategies. [ 79 ] [ 80 ] Nevertheless, very few PPIs are directly targeted by FDA -approved small-molecule PPI inhibitors, emphasizing a huge untapped opportunity for drug discovery.
In 2014, Amit Jaiswal and others were able to develop 30 peptides to inhibit recruitment of telomerase towards telomeres by utilizing protein–protein interaction studies. [ 81 ] [ 82 ] Arkin and others were able to develop antibody fragment-based inhibitors to regulate specific protein-protein interactions. [ 83 ]
As the "modulation" of PPIs not only includes the inhibition, but also the stabilization of quaternary protein complexes , molecules with this mechanism of action (so called molecular glues ) are also intensively studied. [ 84 ] | https://en.wikipedia.org/wiki/Protein–protein_interaction |
Protein–protein interaction screening refers to the identification of Protein–protein interaction with high-throughput screening methods such as computer- and/or robot-assisted plate reading, flow cytometry analyzing.
The interactions between proteins are central to virtually every process in a living cell. Information about these interactions improves understanding of diseases and can provide the basis for new therapeutic approaches.
Though there are many methods to detect protein–protein interactions, [ citation needed ] the majority of these methods—such as co-immunoprecipitation , fluorescence resonance energy transfer (FRET) and dual polarisation interferometry —are not screening approaches.
Methods that screen protein–protein interactions in the living cells.
Bimolecular fluorescence complementation (BiFC) is a technique for observing the interactions of proteins. Combining it with other new techniques, dual expression recombinase based ( DERB ) methods can enable the screening of protein–protein interactions and their modulators. [ 1 ]
The yeast two-hybrid screen investigates the interaction between artificial fusion proteins inside the nucleus of yeast. This approach can identify the binding partners of a protein without bias. However, the method has a notoriously high false-positive rate, which makes it necessary to verify the identified interactions by co-immunoprecipitation . [ 2 ]
The tandem affinity purification (TAP) method allows the high-throughput identification of proteins interactions. In contrast with the Y2H approach, the accuracy of the method can be compared to those of small-scale experiments (Collins et al., 2007) and the interactions are detected within the correct cellular environment as by co-immunoprecipitation . However, the TAP tag method requires two successive steps of protein purification, and thus can not readily detect transient protein–protein interactions. Recent genome-wide TAP experiments were performed by Krogan et al., 2006, [ 3 ] and Gavin et al., 2006, [ 4 ] providing updated protein interaction data for yeast organisms.
Chemical crosslinking is often used to "fix" protein interactions in place before trying to isolate/identify interacting proteins. Common crosslinkers for this application include the non-cleavable [NHS-ester] crosslinker, [ bis -sulfosuccinimidyl suberate] (BS3); a cleavable version of BS3, [dithiobis(sulfosuccinimidyl propionate)](DTSSP); and the [imidoester] crosslinker [dimethyl dithiobispropionimidate] (DTBP) that is popular for fixing interactions in ChIP assays. [ 5 ] | https://en.wikipedia.org/wiki/Protein–protein_interaction_screening |
ProteoWizard is a set of open-source, cross-platform tools and libraries for proteomics data analyses. [ 1 ] [ 2 ] It provides a framework for unified mass spectrometry data file access and performs standard chemistry and LCMS dataset computations. Specifically, it is able to read many of the vendor-specific, proprietary formats and converting the data into an open data format.
On the application level, the software provides executables for data conversion (msConvert, msConvertGUI and idConvert), data visualization (msPicture and seeMS), data access (msAccess, msCat, idCat and msPicture), data analysis (peekaboo and msPrefix14) and basic proteomics utilities (chainsaw). In addition, the project also hosts the Skyline software which helps to create, acquire and analyze targeted proteomics experiments such as SRM experiments.
The main contributors to the project are the Tabb, MacCoss and Mallick research labs as well as Insilicos. [ 3 ] | https://en.wikipedia.org/wiki/ProteoWizard |
Proteoforms are the different forms of a protein produced from the genome with a variety of sequence variations , splice isoforms , and post-translational modifications . [ 1 ] [ 2 ] Proteoform captures the disparate sources of biological variation which alter primary sequence and composition at the whole-protein level. Protein characterization at the proteoform level has a crucial importance to fully understand biological processes since specific proteoforms can carry particular biological functions. [ 3 ] The proteoforms estimation in human can be in millions for around 20,000 proteins. [ 4 ]
This biology article is a stub . You can help Wikipedia by expanding it . | https://en.wikipedia.org/wiki/Proteoform |
A proteolipid is a protein covalently linked to lipid molecules, which can be fatty acids , isoprenoids or sterols . The process of such a linkage is known as protein lipidation , and falls into the wider category of acylation and post-translational modification . Proteolipids are abundant in brain tissue, and are also present in many other animal and plant tissues. They include ghrelin , a peptide hormone associated with feeding. Many proteolipids have bound fatty acid chains, [ 1 ] which often provide an interface for interacting with biological membranes [ 2 ] and act as lipidons that direct proteins to specific zones. [ 3 ]
Proteolipids were discovered serendipitously in 1951 by Jordi Folch Pi and Marjorie Lees while extracting sulfatides from brain lipids. [ 4 ]
They are not to be confused with lipoproteins , a kind of spherical assembly made up of many molecules of lipids and some apolipoproteins .
Depending on the type of fatty acid attached to the protein, a proteolipid can often contain myristoyl , palmitoyl , or prenyl groups . These groups each serve different functions and have different preferences as to which amino acid residue they attach to. The processes are respectively named myristoylation (usually at N-terminal Gly ), palmitoylation (to cysteine ), and prenylation (also to cysteine). Despite the seemingly specific names, N-myristoylation and S-palmitoylation can also involve some other fatty acids, most commonly in plants and viral proteolipids. [ 2 ] [ 5 ] The article on lipid-anchored proteins has more information on these canonical classes.
Lipidated peptides are a type of peptide amphiphile that incorporate one or more alkyl/lipid chains, attached to a peptide head group. As with peptide amphiphiles , they self-assemble depending on the hydrophilic/hydrophobic balance, as well interactions between the peptide units, which is dependent on the charge of the amino acid residues. [ 6 ] Lipidated peptides combine the structural features of amphiphilic surfactants with the functions of bioactive peptides , and they are known to assemble into a variety of nanostructures. [ 7 ] [ 8 ]
Due to the desirable properties of peptides such as high receptor affinity and bioactivity , and low toxicity, the use of peptides in therapeutics (i. e. as peptide therapeutics ) has great potential; shown by a fast growing market with over 100 approved peptide-based drugs. [ 9 ] The disadvantages are that peptides have low oral bioavailability and stability. Lipidation as a chemical modification tool in the development of therapeutic agents has proven to be useful in overcoming these issues, with four lipidized peptide drugs currently approved for use in humans, and various others in clinical trials. [ 10 ] Two of the approved drugs are long-acting anti-diabetic GLP-1 analogues liraglutide (Victoza®), and insulin detemir (Levemir®). The other two are the antibiotics daptomycin and polymyxin B .
Lipidated peptides also have applications in other areas, such as use in the cosmetic industry. [ 6 ] A commercially available lipidated peptide, Matrixyl , is used in anti-wrinkle creams. Matrixyl is a pentapeptide and has the sequence KTTKS, with an attached palmitoyl lipid chain , that is able to stimulate collagen and fibronectin production in fibroblasts. [ 11 ] Several studies have shown promising results of palmitoyl-KTTKS, and topical formulations have been found to significantly reduce fine lines and wrinkles, helping to delay the aging process in the skin. [ 12 ] The Hamley group have also carried out investigations of palmitoyl-KTTKS, and found it so self-assemble into nano tapes in the pH range 3-7, in addition to stimulating human dermal and corneal fibroblasts in a concentration dependant manner, suggesting that stimulation occurs above the critical aggregation concentration. [ 13 ]
There exist some rarer forms of protein acylation that may not have a membrane-related function. They include serine O-octanoylation in ghrelin , serine O- palmitoleoylation in Wnt proteins , and O-palmitoylation in histone H4 with LPCAT1 . Hedgehog proteins are double-modified by (N-)palmitate and cholesterol. Some skin ceramides are proteolipids. [ 2 ] The amino group on lysine can also be myristoylation via a poorly-understood mechanism. [ 14 ]
All bacteria use proteolipids, sometimes confusingly referred to as bacterial lipoproteins, in their cell membrane. A common modification consists of N-acyl- and S‑diacylglycerol attached to an N-terminal cystine residue. Braun's lipoprotein , found in gram-negative bacteria , is a representative of this group. In addition, Mycobacterium O- mycolate proteins destined for the outer membrane. [ 15 ] The plant chloroplast is capable of many of the same modifications that bacteria perform to proteolipids. [ 16 ] One database for such N-Acyl Diacyl Glycerylated cell wall proteolipids is DOLOP. [ 17 ]
Pathogenic spirochetes, including Borrelia burgdorferi and Treponema pallidum , use their proteolipid adhesins to stick to victim cells. [ 18 ] These proteins are also potent antigens , and are in fact the main immunogens of these two species. [ 19 ]
Proteolipids include bacterial antibiotics that aren't synthesised in the ribosome . [ 10 ] Products of nonribosomal peptide synthase may also involve a peptide structure linked to lipids. These are usually referred to as "lipopeptides". [ 15 ] Bacterial "lipoproteins" and "lipopeptides" (LP) are potent inducers of sepsis , second only to lipopolysaccharide (LPS) in its ability to cause an inflammation response. While LPS is detected by the toll-like receptor TLR4, LPs are detected by TLR2. [ 20 ]
Many proteolipids are produced by the Bacillus subtilis family, and are composed of a cyclic structure made up of 7-10 amino acids, and a β-hydroxy fatty acid chain of varying length ranging from 13-19 carbon atoms. [ 21 ] These can be divided into three families depending on the structure of the cyclic peptide sequence: surfactins, iturins, and fengycins. [ 22 ] [ 23 ] [ 24 ] Lipidated peptides produced by Bacillus strains have many useful bio-activities such as anti-bacterial, anti- viral, anti-fungal, and anti-tumour properties, [ 21 ] [ 22 ] making them very attractive for use in a wide range of industries.
As the name implies, surfactins are potent biosurfactants ( surfactants produced by bacteria , yeast , or fungi ), and they have been shown to reduce the surface tension of water from 72 to 27 mN/m at very low concentrations. [ 25 ] Furthermore, surfactins are also able to permeabilize lipid membranes , allowing them to have specific antimicrobial and antiviral activities. [ 22 ] [ 26 ] [ 27 ] Since surfactins are biosurfactants, they have diverse functional properties. These include low toxicity, biodegradability and a higher tolerance towards variation of temperature and pH, [ 22 ] making them very interesting for use in a wide range of applications.
Iturins are pore‐forming lipopeptides with antifungal activity, and this is dependent on the interaction with the cytoplasmic membrane of the target cells. [ 22 ] [ 23 ] [ 28 ] Mycosubtilin is an iturin isoform that can interact with membranes via its sterol alcohol group, to target ergosterol (a compound found in fungi) to give it antifungal properties. [ 21 ] [ 29 ]
Fengycins are another class of biosurfactant produced by Bacillus subtilis, with antifungal activity against filamentous fungi. [ 24 ] [ 28 ] [ 30 ] There are two classes of Fengycins, Fengycin A and Fengycin B, with the two only differing by one amino acid at position 6 in the peptide sequence, with the former having an alanine residue, and the latter having valine. [ 31 ]
Daptomycin is another naturally occurring lipidated peptide, produced by the Gram positive bacterium Streptomyces roseoporous . The structure of Daptomycin consists of a decanoyl lipid chain attached to a partially cyclised peptide head group. [ 6 ] It has very potent antimicrobial properties and is used as an antibiotic to treat life-threatening conditions caused by Gram positive bacteria including MRSA (methicillin-resistant Staphylococcus aureus) and vancomycin resistant Enterococci. [ 8 ] [ 32 ] [ 33 ] As with the Bacillus subtilis lipidated peptides, the permeation of the cell membrane is what gives it its properties, and the mechanism of action with daptomycin is thought to involve the insertion of the decanoyl chain into the bacterial membrane to cause disruption. This then causes a serious depolarization resulting in the inhibition of various synthesis processes including those of DNA, protein and RNA, leading to apoptosis . [ 34 ] [ 35 ] [ 36 ]
This article incorporates text by Jessica Hutchinson available under the CC BY-SA 3.0 license. | https://en.wikipedia.org/wiki/Proteolipid |
Proteolix, Inc. , was a private biotechnology company with headquarters in South San Francisco , California . Proteolix was founded in 2003 based on technology developed by co-founders Craig M. Crews (Yale University) and Raymond J. Deshaies (California Institute of Technology). Susan Molineaux and Phil Whitcome [ 1 ] joined as co-founders.
Proteolix was launched based on an $18.2 million A round comprising investments by Latterell Venture Partners, US Venture Partners, Advanced Technology Ventures, and The Vertical Group. Its lead product candidate, carfilzomib (PR-171), is a tetrapeptide epoxyketone for treating multiple myeloma , a blood cancer. The drug carfilzomib works by preventing proteasomes from breaking down other proteins. Proteolix focused primarily on the proteasome as a therapeutic target. [ 2 ]
At the time of its sale (see below), the company had two earlier-stage programs, an orally-bioavailable proteasome inhibitor for oncology (PR-047), and an agent preferentially targeting the immuno form of the proteasome (PR-957), with potential utility in areas such as rheumatoid arthritis. At the time of sale, carfilzomib's route of administration was intravenous , and the company was exploring its potential utility in multiple myeloma, Non-Hodgkin lymphoma (NHL) and other cancers .
Proteolix was acquired by Onyx Pharmaceuticals in 2009 for $810 million (nominal value). PR-171 is sold by Onyx Pharmaceuticals as Kyprolis . Onyx renamed PR-047 to "ONX 0912" and PR-957 to "ONX 0914". Onyx Pharmaceuticals is a subsidiary of Amgen .
This article about a medical , pharmaceutical or biotechnological corporation or company is a stub . You can help Wikipedia by expanding it . | https://en.wikipedia.org/wiki/Proteolix |
Proteolysis is the breakdown of proteins into smaller polypeptides or amino acids . Protein degradation is a major regulatory mechanism of gene expression [ 1 ] and contributes substantially to shaping mammalian proteomes. [ 2 ] Uncatalysed, the hydrolysis of peptide bonds is extremely slow, taking hundreds of years. Proteolysis is typically catalysed by cellular enzymes called proteases , but may also occur by intra-molecular digestion.
Proteolysis in organisms serves many purposes; for example, digestive enzymes break down proteins in food to provide amino acids for the organism, while proteolytic processing of a polypeptide chain after its synthesis may be necessary for the production of an active protein. It is also important in the regulation of some physiological and cellular processes including apoptosis , as well as preventing the accumulation of unwanted or misfolded proteins in cells. Consequently, abnormality in the regulation of proteolysis can cause diseases.
Proteolysis can also be used as an analytical tool for studying proteins in the laboratory, and it may also be used in industry, for example in food processing and stain removal.
Limited proteolysis of a polypeptide during or after translation in protein synthesis often occurs for many proteins. This may involve removal of the N-terminal methionine , signal peptide , and/or the conversion of an inactive or non-functional protein to an active one. The precursor to the final functional form of protein is termed proprotein , and these proproteins may be first synthesized as preproprotein. For example, albumin is first synthesized as preproalbumin and contains an uncleaved signal peptide. This forms the proalbumin after the signal peptide is cleaved, and a further processing to remove the N-terminal 6-residue propeptide yields the mature form of the protein. [ 3 ]
The initiating methionine (and, in bacteria, fMet ) may be removed during translation of the nascent protein. For E. coli , fMet is efficiently removed if the second residue is small and uncharged, but not if the second residue is bulky and charged. [ 4 ] In both prokaryotes and eukaryotes , the exposed N-terminal residue may determine the half-life of the protein according to the N-end rule .
Proteins that are to be targeted to a particular organelle or for secretion have an N-terminal signal peptide that directs the protein to its final destination. This signal peptide is removed by proteolysis after their transport through a membrane .
Some proteins and most eukaryotic polypeptide hormones are synthesized as a large precursor polypeptide known as a polyprotein that requires proteolytic cleavage into individual smaller polypeptide chains. The polyprotein pro-opiomelanocortin (POMC) contains many polypeptide hormones. The cleavage pattern of POMC, however, may vary between different tissues, yielding different sets of polypeptide hormones from the same polyprotein.
Many viruses also produce their proteins initially as a single polypeptide chain that were translated from a polycistronic mRNA. This polypeptide is subsequently cleaved into individual polypeptide chains. [ 3 ] Common names for the polyprotein include gag ( group-specific antigen ) in retroviruses and ORF1ab in Nidovirales . The latter name refers to the fact that a slippery sequence in the mRNA that codes for the polypeptide causes ribosomal frameshifting , leading to two different lengths of peptidic chains ( a and ab ) at an approximately fixed ratio.
Many proteins and hormones are synthesized in the form of their precursors - zymogens , proenzymes , and prehormones . These proteins are cleaved to form their final active structures. Insulin , for example, is synthesized as preproinsulin , which yields proinsulin after the signal peptide has been cleaved. The proinsulin is then cleaved at two positions to yield two polypeptide chains linked by two disulfide bonds . Removal of two C-terminal residues from the B-chain then yields the mature insulin. Protein folding occurs in the single-chain proinsulin form which facilitates formation of the ultimate inter-peptide disulfide bonds, and the ultimate intra-peptide disulfide bond, found in the native structure of insulin.
Proteases in particular are synthesized in the inactive form so that they may be safely stored in cells, and ready for release in sufficient quantity when required. This is to ensure that the protease is activated only in the correct location or context, as inappropriate activation of these proteases can be very destructive for an organism. Proteolysis of the zymogen yields an active protein; for example, when trypsinogen is cleaved to form trypsin , a slight rearrangement of the protein structure that completes the active site of the protease occurs, thereby activating the protein.
Proteolysis can, therefore, be a method of regulating biological processes by turning inactive proteins into active ones. A good example is the blood clotting cascade whereby an initial event triggers a cascade of sequential proteolytic activation of many specific proteases, resulting in blood coagulation. The complement system of the immune response also involves a complex sequential proteolytic activation and interaction that result in an attack on invading pathogens.
Protein degradation may take place intracellularly or extracellularly. In digestion of food, digestive enzymes may be released into the environment for extracellular digestion whereby proteolytic cleavage breaks proteins into smaller peptides and amino acids so that they may be absorbed and used. In animals the food may be processed extracellularly in specialized organs or guts , but in many bacteria the food may be internalized via phagocytosis . Microbial degradation of protein in the environment can be regulated by nutrient availability. For example, limitation for major elements in proteins (carbon, nitrogen, and sulfur) induces proteolytic activity in the fungus Neurospora crassa [ 5 ] as well as in of soil organism communities. [ 6 ]
Proteins in cells are broken into amino acids. This intracellular degradation of protein serves multiple functions: It removes damaged and abnormal proteins and prevents their accumulation. It also serves to regulate cellular processes by removing enzymes and regulatory proteins that are no longer needed. The amino acids may then be reused for protein synthesis.
The intracellular degradation of protein may be achieved in two ways—proteolysis in lysosome , or a ubiquitin -dependent process that targets unwanted proteins to proteasome . The autophagy -lysosomal pathway is normally a non-selective process, but it may become selective upon starvation whereby proteins with peptide sequence KFERQ or similar are selectively broken down. The lysosome contains a large number of proteases such as cathepsins .
The ubiquitin-mediated process is selective. Proteins marked for degradation are covalently linked to ubiquitin. Many molecules of ubiquitin may be linked in tandem to a protein destined for degradation. The polyubiquinated protein is targeted to an ATP-dependent protease complex, the proteasome. The ubiquitin is released and reused, while the targeted protein is degraded.
Different proteins are degraded at different rates. Abnormal proteins are quickly degraded, whereas the rate of degradation of normal proteins may vary widely depending on their functions. Enzymes at important metabolic control points may be degraded much faster than those enzymes whose activity is largely constant under all physiological conditions. One of the most rapidly degraded proteins is ornithine decarboxylase , which has a half-life of 11 minutes. In contrast, other proteins like actin and myosin have a half-life of a month or more, while, in essence, haemoglobin lasts for the entire life-time of an erythrocyte . [ 7 ]
The N-end rule may partially determine the half-life of a protein, and proteins with segments rich in proline , glutamic acid , serine , and threonine (the so-called PEST proteins ) have short half-life. [ 8 ] Other factors suspected to affect degradation rate include the rate deamination of glutamine and asparagine and oxidation of cystein , histidine , and methionine, the absence of stabilizing ligands, the presence of attached carbohydrate or phosphate groups, the presence of free α-amino group, the negative charge of protein, and the flexibility and stability of the protein. [ 7 ] Proteins with larger degrees of intrinsic disorder also tend to have short cellular half-life, [ 9 ] with disordered segments having been proposed to facilitate efficient initiation of degradation by the proteasome . [ 10 ] [ 11 ]
The rate of proteolysis may also depend on the physiological state of the organism, such as its hormonal state as well as nutritional status. In time of starvation, the rate of protein degradation increases.
In human digestion , proteins in food are broken down into smaller peptide chains by digestive enzymes such as pepsin , trypsin , chymotrypsin , and elastase , and into amino acids by various enzymes such as carboxypeptidase , aminopeptidase , and dipeptidase . It is necessary to break down proteins into small peptides (tripeptides and dipeptides) and amino acids so they can be absorbed by the intestines, and the absorbed tripeptides and dipeptides are also further broken into amino acids intracellularly before they enter the bloodstream. [ 12 ] Different enzymes have different specificity for their substrate; trypsin, for example, cleaves the peptide bond after a positively charged residue ( arginine and lysine ); chymotrypsin cleaves the bond after an aromatic residue ( phenylalanine , tyrosine , and tryptophan ); elastase cleaves the bond after a small non-polar residue such as alanine or glycine.
In order to prevent inappropriate or premature activation of the digestive enzymes (they may, for example, trigger pancreatic self-digestion causing pancreatitis ), these enzymes are secreted as inactive zymogen. The precursor of pepsin , pepsinogen , is secreted by the stomach, and is activated only in the acidic environment found in stomach. The pancreas secretes the precursors of a number of proteases such as trypsin and chymotrypsin . The zymogen of trypsin is trypsinogen , which is activated by a very specific protease, enterokinase , secreted by the mucosa of the duodenum . The trypsin, once activated, can also cleave other trypsinogens as well as the precursors of other proteases such as chymotrypsin and carboxypeptidase to activate them.
In bacteria, a similar strategy of employing an inactive zymogen or prezymogen is used. Subtilisin , which is produced by Bacillus subtilis , is produced as preprosubtilisin, and is released only if the signal peptide is cleaved and autocatalytic proteolytic activation has occurred.
Proteolysis is also involved in the regulation of many cellular processes by activating or deactivating enzymes, transcription factors, and receptors, for example in the biosynthesis of cholesterol, [ 13 ] or the mediation of thrombin signalling through protease-activated receptors . [ 14 ]
Some enzymes at important metabolic control points such as ornithine decarboxylase is regulated entirely by its rate of synthesis and its rate of degradation. Other rapidly degraded proteins include the protein products of proto-oncogenes, which play central roles in the regulation of cell growth.
Cyclins are a group of proteins that activate kinases involved in cell division. The degradation of cyclins is the key step that governs the exit from mitosis and progress into the next cell cycle . [ 15 ] Cyclins accumulate in the course the cell cycle, then abruptly disappear just before the anaphase of mitosis. The cyclins are removed via a ubiquitin-mediated proteolytic pathway.
Caspases are an important group of proteases involved in apoptosis or programmed cell death . The precursors of caspase, procaspase, may be activated by proteolysis through its association with a protein complex that forms apoptosome , or by granzyme B , or via the death receptor pathways.
Autoproteolysis takes place in some proteins, whereby the peptide bond is cleaved in a self-catalyzed intramolecular reaction . Unlike zymogens , these autoproteolytic proteins participate in a "single turnover" reaction and do not catalyze further reactions post-cleavage. Examples include cleavage of the Asp-Pro bond in a subset of von Willebrand factor type D (VWD) domains [ 16 ] [ 17 ] and Neisseria meningitidis FrpC self-processing domain, [ 18 ] cleavage of the Asn-Pro bond in Salmonella FlhB protein, [ 19 ] Yersinia YscU protein, [ 20 ] as well as cleavage of the Gly-Ser bond in a subset of sea urchin sperm protein, enterokinase, and agrin (SEA) domains. [ 21 ] In some cases, the autoproteolytic cleavage is promoted by conformational strain of the peptide bond. [ 21 ]
Abnormal proteolytic activity is associated with many diseases. [ 22 ] In pancreatitis , leakage of proteases and their premature activation in the pancreas results in the self-digestion of the pancreas . People with diabetes mellitus may have increased lysosomal activity and the degradation of some proteins can increase significantly. Chronic inflammatory diseases such as rheumatoid arthritis may involve the release of lysosomal enzymes into extracellular space that break down surrounding tissues. Abnormal proteolysis may result in age-related neurological diseases such as Alzheimer 's due to the generation and ineffective removal of peptides that aggregate in cells. [ 23 ]
Proteases may be regulated by antiproteases or protease inhibitors , and imbalance between proteases and antiproteases can result in diseases, for example, in the destruction of lung tissues in emphysema brought on by smoking tobacco. Smoking is thought to increase the neutrophils and macrophages in the lung which release excessive amount of proteolytic enzymes such as elastase , such that they can no longer be inhibited by serpins such as α 1 -antitrypsin , thereby resulting in the breaking down of connective tissues in the lung. Other proteases and their inhibitors may also be involved in this disease, for example matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs). [ 24 ]
Other diseases linked to aberrant proteolysis include muscular dystrophy , degenerative skin disorders, respiratory and gastrointestinal diseases, and malignancy .
Protein backbones are very stable in water at neutral pH and room temperature, although the rate of hydrolysis of different peptide bonds can vary. The half life of a peptide bond under normal conditions can range from 7 years to 350 years, even higher for peptides protected by modified terminus or within the protein interior. [ 25 ] [ 26 ] [ 27 ] The rate of hydrolysis however can be significantly increased by extremes of pH and heat. Spontaneous cleavage of proteins may also involve catalysis by zinc on serine and threonine. [ 28 ]
Strong mineral acids can readily hydrolyse the peptide bonds in a protein ( acid hydrolysis ). The standard way to hydrolyze a protein or peptide into its constituent amino acids for analysis is to heat it to 105 °C for around 24 hours in 6M hydrochloric acid . [ 29 ] However, some proteins are resistant to acid hydrolysis. One well-known example is ribonuclease A , which can be purified by treating crude extracts with hot sulfuric acid so that other proteins become degraded while ribonuclease A is left intact. [ 30 ]
Certain chemicals cause proteolysis only after specific residues, and these can be used to selectively break down a protein into smaller polypeptides for laboratory analysis. [ 31 ] For example, cyanogen bromide cleaves the peptide bond after a methionine . Similar methods may be used to specifically cleave tryptophanyl , aspartyl , cysteinyl , and asparaginyl peptide bonds. Acids such as trifluoroacetic acid and formic acid may be used for cleavage.
Like other biomolecules, proteins can also be broken down by high heat alone. At 250 °C, the peptide bond may be easily hydrolyzed, with its half-life dropping to about a minute. [ 29 ] [ 32 ] Protein may also be broken down without hydrolysis through pyrolysis ; small heterocyclic compounds may start to form upon degradation. Above 500 °C, polycyclic aromatic hydrocarbons may also form, [ 33 ] [ 34 ] which is of interest in the study of generation of carcinogens in tobacco smoke and cooking at high heat. [ 35 ] [ 36 ]
Proteolysis is also used in research and diagnostic applications:
Proteases may be classified according to the catalytic group involved in its active site. [ 41 ]
Certain types of venom, such as those produced by venomous snakes , can also cause proteolysis. These venoms are, in fact, complex digestive fluids that begin their work outside of the body. Proteolytic venoms cause a wide range of toxic effects, [ 42 ] including effects that are: | https://en.wikipedia.org/wiki/Proteolysis |
A proteolysis targeting chimera ( PROTAC ) [ 2 ] is a molecule that can remove specific unwanted proteins. Rather than acting as a conventional enzyme inhibitor , a PROTAC works by inducing selective intracellular proteolysis . A heterobifunctional molecule with two active domains and a linker, PROTACs consist of two covalently linked protein-binding molecules: one capable of engaging an E3 ubiquitin ligase , and another that binds to a target protein meant for degradation. Recruitment of the E3 ligase to the target protein results in ubiquitination and subsequent degradation of the target protein via the proteasome . Because PROTACs need only to bind their targets with high selectivity (rather than inhibit the target protein's enzymatic activity), there are currently many efforts to retool previously ineffective inhibitor molecules as PROTACs for next-generation drugs. [ 3 ] [ 4 ]
Initially described by Kathleen Sakamoto, Craig Crews and Ray Deshaies in 2001, [ 5 ] the PROTAC technology has been applied by a number of drug discovery labs using various E3 ligases, [ 6 ] including pVHL , [ 7 ] [ 8 ] [ 9 ] CRBN , [ 10 ] [ 11 ] Mdm2 , [ 12 ] beta-TrCP1 , [ 5 ] DCAF11 , [ 13 ] [ 14 ] DCAF15 , [ 15 ] DCAF16, [ 15 ] RNF114 , [ 15 ] and c-IAP1 . [ 16 ] Yale University licensed the PROTAC technology to Arvinas in 2013–14. [ 17 ] [ 18 ]
In 2019, Arvinas put two PROTACs into clinical trials: bavdegalutamide (ARV-110), an androgen receptor degrader, and vepdegestrant (ARV-471), an estrogen receptor degrader. [ 19 ] [ 20 ] In 2021, Arvinas put a second androgen receptor PROTAC, Luxdegalutamide (ARV-766), into the clinic. [ 21 ]
PROTACs achieve degradation through "hijacking" the cell's ubiquitin–proteasome system (UPS) by bringing together the target protein and an E3 ligase. [ 22 ]
First, the E1 activates and conjugates the ubiquitin to the E2. [ 15 ] The E2 then forms a complex with the E3 ligase. The E3 ligase targets proteins and covalently attaches the ubiquitin to the protein of interest. [ 22 ] Eventually, after a ubiquitin chain is formed, the protein is recognized and degraded by the 26S proteasome . [ 19 ] PROTACs take advantage of this cellular system by putting the protein of interest in close proximity to the E3 ligase to catalyze degradation. [ 19 ]
Unlike traditional inhibitors, PROTACs have a catalytic mechanism , with the PROTAC itself being recycled after the target protein is degraded. [ 19 ]
The protein targeting warhead, E3 ligase, and linker must all be considered for PROTAC development. Formation of a ternary complex between the protein of interest, PROTAC, and E3 ligase may be evaluated to characterize PROTAC activity because it often leads to ubiquitination and subsequent degradation of the targeted protein. [ 15 ] A hook effect is commonly observed with high concentrations of PROTACs due to the bifunctional nature of the degrader. [ 15 ]
Currently, pVHL and CRBN have been used in preclinical trials as E3 ligases. [ 15 ] However, there still remains hundreds of E3 ligases to be explored, with some giving the opportunity for cell specificity.
Additionally, there have been attempts to produce PROTACs which target bacteria (BacPROTAC) as a way of circumventing antibiotic resistance . These BacPROTACs target the ClpC:ClpP protease system, an analogue with similar function to E3 ubiquitin ligase in bacterial cells. [ 23 ]
Compared to traditional inhibitors, PROTACs display multiple benefits that make them desirable drug candidates. Due to their catalytic mechanism, PROTACs can be administered at lower doses compared to their inhibitor analogues, [ 20 ] though care needs to be taken in achieving oral bioavailability if administered by that route. [ 24 ] Some PROTACs have been shown to be more selective than their inhibitor analogues, reducing off-target effects. [ 20 ] PROTACs have the ability to target previously undruggable proteins, as they do not need to target catalytic pockets. [ 20 ] This also helps prevent mutation-driven drug resistance often found with enzymatic inhibitors. | https://en.wikipedia.org/wiki/Proteolysis_targeting_chimera |
Proteomimetics are molecules that mimic certain protein characteristics such as shape, binding properties or enzymatic activity. [ 1 ] While conceptually linked to peptidomimetics which mimic short peptide sequences or secondary structures, proteomimetics recapitulate tertiary structures. This can involve the mimicry of entire protein domains [ 2 ] or fragments thereof. [ 3 ] Proteomimetic approaches can range from entirely abiotic scaffolds [ 2 ] to specific main chain and side chain-modifications. [ 4 ] [ 5 ]
This science article is a stub . You can help Wikipedia by expanding it . | https://en.wikipedia.org/wiki/Proteomimetic |
Proteorhodopsin ( PR or pRhodopsin ) belongs to the family of bacterial transmembrane rhodopsins ( retinylidene proteins ). [ 1 ] In 1971, the first microbial transmembrane rhodopsin - Bacteriorhodopsin was discovered in archea domain by Dieter Oesterhelt and Walther Stoeckenius. [ 2 ] Later in 2000, the first bacterial transmembrane rhodopsins was discovered by Oded Béjà and Edward DeLong . [ 3 ] The Proteorhodopsin is widely expressed in various type of aquatic habitats . [ 1 ] It functions as light -driven proton pumps with the help of retinal chromophore at the active site . [ 1 ] [ 4 ] The light-driven proton pump gives bacteria energy in the form of adenosine triphosphate (ATP). [ 1 ] [ 4 ]
Efforts by Oded Béjà from Edward DeLong research group in pioneering bacterial artificial chromosome metagenomics analysis led the discovery of pRhodopsin in bacteria domain . [ 4 ] It was first detected in uncultured gammaproteobacteria ribotype group SAR86 at Monterey Bay water column in 2000. [ 4 ] Oded Béjà observed the sequence similarity between SAR86 pRhodopsin and bacteriorodopsin (a light driven proton pump in haloarchea ) open reading frame . [ 4 ] To further established pRhodopsin function as retinal -based light-driven proton pump , he expressed pRhodopsin open reading frame in Escherichia coli system . [ 4 ] Before the discovery of bacterial Proteorhodpsin, it was understood that light driven active transport only evolved in extreme halophilic archaea domain ( bacteriorodopsin , halorhodopsin , and sensory rhodopsin ) and animal kingdom (as a visual rhodopsin ). [ 1 ] [ 4 ]
pRhodopsin is not confined to a single species and single habitat . [ 1 ] It is distributed in many microorganisms from all over the world. [ 1 ] pRhodopsin containing microorganisms is distributed in Gammaproteobacteria , Alphaproteobacteria , Betaproteobacteria , Flavobacteria , Planctomycetes , Cyanobacteria , Actinobacteria , marine Archaea , and different eukaryotic groups, including fungi and dinoflagellates . [ 1 ] [ 4 ] pRhodopsin containing microorganisms are habited in marine environments , sea ice , brackish environments, fresh water lakes and on high mountains . [ 1 ] [ 4 ] In the marine environment , pRhodopsin containing microorganisms is primarily found in photic zone . [ 1 ] [ 4 ]
The topology and active site residues for proton transporting retinylidene proteins was first characterized in bacteriorhodopsin . [ 1 ] The pRhodopsin topology and active site residues are conserved to Bacteriorhodopsin . [ 1 ] pRhodopsin is a seven transmembrane α-helices that form a pocket in which retinal ( vitamin A aldehyde ) is covalently linked to ligand binding domain , as a protonated schiff base , to a lysine in the seventh transmembrane α-helix . [ 1 ] At ground state the retinal chromophore is all-trans configuration. [ 1 ] When visible light illuminates on pRhodopsin, the all-trans retinal molecule absorbs light energy and uses it to isomerize into 13-cis configuration. [ 1 ] This triggers a sequence of protein conformational changes including several proton transfer reactions against concentration gradient , generating a proton motive force . [ 1 ]
Light -activated proteorhodopsin pumps protons outwardly, increasing the proton motive force across the microbial cell membrane . [ 1 ] [ 4 ] Protons can then reenter the cell through the ATP synthase complex, powering the synthesis of ATP . Proteorhodopsin thus allows microbial cells to harvest light energy and convert it into usable chemical energy without the involvement of chlorophyll -based photosystems . [ 1 ] [ 4 ]
Microbes containing proteorhodopsin are considered phototrophs due to its functionality as a light-sensitive proton pump . [ 1 ] [ 4 ] Different variants of proteorhodopsin are spectrally tuned to absorb specific wavelengths of light , such as green or blue . [ 1 ] These adaptations allow organisms to occupy distinct ecological niches based on light availability at different water column depths. [ 1 ] These functional advantages make proteorhodopsin a key component in the marine microbial energy budget . [ 4 ]
If the gene for proteorhodopsin is inserted into E. coli and retinal is given to these modified bacteria , then they will incorporate the pigment into their cell membrane and will pump H+ in the presence of light energy . [ 5 ] This functionality can be used to acidify a vesicle type organelle . [ 5 ] | https://en.wikipedia.org/wiki/Proteorhodopsin |
Proteostasis is the dynamic regulation of a balanced, functional proteome . The proteostasis network includes competing and integrated biological pathways within cells that control the biogenesis , folding, trafficking, and degradation of proteins present within and outside the cell. [ 1 ] [ 2 ] Loss of proteostasis is central to understanding the cause of diseases associated with excessive protein misfolding and degradation leading to loss-of-function phenotypes , [ 3 ] as well as aggregation-associated degenerative disorders. [ 4 ] Therapeutic restoration of proteostasis may treat or resolve these pathologies. [ 5 ]
Cellular proteostasis is key to ensuring successful development, healthy aging , resistance to environmental stresses , and to minimize homeostatic perturbations from pathogens such as viruses . [ 2 ] Cellular mechanisms for maintaining proteostasis include regulated protein translation, chaperone assisted protein folding, and protein degradation pathways. Adjusting each of these mechanisms based on the need for specific proteins is essential to maintain all cellular functions relying on a correctly folded proteome .
One of the first points of regulation for proteostasis is during translation . This regulation is accomplished via the structure of the ribosome , a complex central to translation. Its characteristics shape the way the protein folds, and influence the protein's future interactions. The synthesis of a new peptide chain using the ribosome is very slow; the ribosome can even be stalled when it encounters a rare codon , a codon found at low concentrations in the cell. [ 6 ] The slow synthesis rate and any such pauses provide an individual protein domain with the necessary time to become folded before the production of subsequent domains. This facilitates the correct folding of multi-domain proteins. [ 6 ]
The newly synthesized peptide chain exits the ribosome into the cellular environment through the narrow ribosome exit channel (width: 10Å to 20Å, length 80Å). [ 6 ] Characteristics of the exit channel control the formation of secondary and limited tertiary structures in the nascent chain. For example, an alpha helix is a structural property that is commonly induced in this exit channel. [ 7 ] At the same time, the exit channel prevents premature folding by impeding large scale interactions within the peptide chain that would require more space.
In order to maintain protein homeostasis post-translationally, the cell makes use of molecular chaperones sometimes including chaperonins , which aid in the assembly or disassembly of proteins. [ 8 ] They recognize exposed segments of hydrophobic amino acids in the nascent peptide chain and then work to promote the proper formation of noncovalent interactions that lead to the desired folded state. [ 8 ] Chaperones begin to assist in protein folding as soon as a nascent chain longer than 60 amino acids emerges from the ribosome exit channel. [ 9 ]
One of the most studied ribosome binding chaperones is trigger factor. Trigger factor works to stabilize the peptide, promotes its folding, prevents aggregation, and promotes refolding of denatured model substrates. [ 10 ] Ribosome profiling experiments have shown that TF predominantly targets ribosomes translating outer membrane proteins in vivo, and moreover are underrepresented on ribosomes translating inner membrane proteins. [ 11 ] Trigger factor not only directly works to properly fold the protein but also recruits other chaperones to the ribosome, such as Hsp70. Hsp70 surrounds an unfolded peptide chain, thereby preventing aggregation and promoting folding. [ 8 ] [ 9 ]
Chaperonins are a special class of chaperones that promote native state folding by cyclically encapsulating the peptide chain. [ 9 ] Chaperonins are divided into two groups. Group 1 chaperonins are commonly found in bacteria, chloroplasts, and mitochondria. Group 2 chaperonins are found in the cytosol of eukaryotic cells as well as in archaea. [ 12 ] Group 2 chaperonins also contain an additional helical component which acts as a lid for the cylindrical protein chamber, unlike Group 1 which instead relies on an extra cochaperone to act as a lid. All chaperonins exhibit two states (open and closed), between which they can cycle. This cycling process is important during the folding of an individual polypeptide chain as it helps to avoid undesired interactions as well as to prevent the peptide from entering into kinetically trapped states. [ 12 ]
The third component of the proteostasis network is the protein degradation machinery. Protein degradation occurs in proteostasis when the cellular signals indicate the need to decrease overall cellular protein levels. The effects of protein degradation can be local, with the cell only experiencing effects from the loss of the degraded protein itself or widespread, with the entire protein landscape changing due to loss of other proteins’ interactions with the degraded protein. [ 7 ]
Multiple substrates are targets for proteostatic degradation. These degradable substrates include nonfunctional protein fragments produced from ribosomal stalling during translation, misfolded or unfolded proteins, aggregated proteins, and proteins that are no longer needed to carry out cellular function.
Several different pathways exist for carrying out these degradation processes. When proteins are determined to be unfolded or misfolded, they are typically degraded via the unfolded protein response (UPR) or endoplasmic-reticulum-associated protein degradation (ERAD). Substrates that are unfolded, misfolded, or no longer required for cellular function can also be ubiquitin tagged for degradation by ATP dependent proteases, such as the proteasome in eukaryotes or ClpXP in prokaryotes. Autophagy , or self engulfment, lysosomal targeting, and phagocytosis (engulfment of waste products by other cells) can also be used as proteostatic degradation mechanisms. [ 7 ]
Protein misfolding is detected by mechanisms that are specific for the cellular compartment in which they occur. Distinct surveillance mechanisms that respond to unfolded protein have been characterized in the cytoplasm, ER and mitochondria. This response acts locally in a cell autonomous fashion but can also extend to intercellular signaling to protect the organism from anticipated proteotoxic stress.
Cellular stress response pathways detect and alleviate proteotoxic stress which is triggered by imbalances in proteostasis. The cell-autonomous regulation occurs through direct detection of misfolded proteins or inhibition of pathway activation by sequestering activating components in response to heat shock. Cellular responses to this stress signaling include transcriptional activation of chaperone expression, increased efficiency in protein trafficking and protein degradation and translational reduction.
The cytosolic HSR is mainly mediated by the transcription factor family HSF (heat shock family). HSF is constitutively bound by Hsp90. Upon a proteotoxic stimulus Hsp90 is recruited away from HSF, which can then bind to heat response elements in the DNA and upregulate gene expression of proteins involved in the maintenance of proteostasis.
The unfolded protein response in the endoplasmatic reticulum (ER) is activated by imbalances of unfolded proteins inside the ER and the proteins mediating protein homeostasis. Different “detectors” - such as IRE1, ATF6 and PERK - can recognize misfolded proteins in the ER and mediate transcriptional responses which help alleviate the effects of ER stress.
The mitochondrial unfolded protein response detects imbalances in protein stoichiometry of mitochondrial proteins and misfolded proteins. The expression of mitochondrial chaperones is upregulated by the activation of the transcription factors ATF-1 and/or DVE-1 with UBL-5.
Stress responses can also be triggered in a non-cell autonomous fashion by intercellular communication. The stress that is sensed in one tissue could thereby be communicated to other tissues to protect the proteome of the organism or to regulate proteostasis systemically. Cell non-autonomous activation can occur for all three stress responses.
Work on the model organism C. elegans has shown that neurons play a role in this intercellular communication of cytosolic HSR. Stress induced in the neurons of the worm can in the long run protect other tissues such as muscle and intestinal cells from chronic proteotoxicity . Similarly ER and mitochondrial UPR in neurons are relayed to intestinal cells . These systemic responses have been implicated in mediating systemic proteostasis; they also influence organismal aging. [ 13 ]
Dysfunction in proteostasis can arise from errors in or misregulation of protein folding. The classic examples are missense mutations and deletions that change the thermodynamic and kinetic parameters for the protein folding process. [ 1 ] These mutations are often inherited and range in phenotypic severity from having no noticeable effect to embryonic lethality. Disease develops when these mutations render a protein significantly more susceptible to misfolding, aggregation, and degradation.
If these effects only alter the mutated protein, the negative consequences will only be local loss of function. However, if these mutations occur in a chaperone or a protein that interacts with many other proteins, dramatic global alterations in the proteostasis boundary will occur. Examples of diseases resulting from proteostatic changes from errors in protein folding include cystic fibrosis, Huntington's disease, Alzheimer's disease, lysosomal storage disorders, and others. [ 14 ]
Small animal model systems have been and continue to be instrumental in the identification of functional mechanisms that safeguard proteostasis. Model systems of diverse misfolding-prone disease proteins have so far revealed numerous chaperone and co-chaperone modifiers of proteotoxicity . [ 15 ]
The unregulated cell division that marks cancer development requires increased protein synthesis for cancer cell function and survival. This increased protein synthesis is typically seen in proteins that modulate cell metabolism and growth processes. Cancer cells are sometimes susceptible to drugs that inhibit chaperones and disrupt proteostasis, such as Hsp90 inhibitors or proteasome inhibitors . [ 1 ]
Furthermore, cancer cells tend to produce misfolded proteins, which are removed mainly by proteolysis. [ 16 ] Inhibitors of proteolysis allow accumulation of both misfolded protein aggregates, as well as apoptosis signaling proteins in cancer cells. [ 17 ] [ 18 ] This can change the sensitivity of cancer cells to antineoplastic drugs; cancer cells either die at a lower drug concentration, or survive, depending on the type of proteins that accumulate, and the function these proteins have. [ 19 ] Proteasome inhibitor bortezomib was the first drug of this type to receive approval for treatment of multiple myeloma. [ 20 ]
A hallmark of cellular proteostatic networks is their ability to adapt to stress via protein regulation. Metabolic disease, such as that associated with obesity, alters the ability of cellular proteostasis networks adapt to stress, often with detrimental health effects. For example, when insulin production exceeds the cell's insulin secretion capacity, proteostatic collapse occurs and chaperone production is severely impaired. This disruption leads to the disease symptoms exhibited in individuals with diabetes. [ 1 ]
Over time, the proteostasis network becomes burdened with proteins modified by reactive oxygen species and metabolites that induce oxidative damage. [ 1 ] These byproducts can react with cellular proteins to cause misfolding and aggregation (especially in nondividing cells like neurons). This risk is particularly high for intrinsically disordered proteins. The IGFR-1 pathway has been shown in C. elegans to protect against these harmful aggregates, and some experimental work has suggested that upregulation of insulin growth factor receptor 1 (IGFR-1) may stabilize proteostatic network and prevent detrimental effects of aging. [ 1 ]
Expression of the chaperome , the ensemble of chaperones and co-chaperones that interact in a complex network of molecular folding machines to regulate proteome function, is dramatically repressed in human aging brains and in the brains of patients with neurodegenerative diseases. Functional assays in C. elegans and human cells have identified a conserved chaperome sub-network of 16 chaperone genes, corresponding to 28 human orthologs as a proteostasis safeguard in aging and age-onset neurodegenerative disease. [ 21 ]
There are two main approaches that have been used for therapeutic development targeting the proteostatic network: pharmacologic chaperones and proteostasis regulators. The principle behind designing pharmacologic chaperones for intervention in diseases of proteostasis is to design small molecules that stabilize proteins exhibiting borderline stability.
Previously, this approach has been used to target and stabilize G-protein coupled receptors, neurotransmitter receptors, glycosidases, lysosomal storage proteins, and the mutant CFTR protein that causes cystic fibrosis and transthyretin, which can misfiled and aggregate leading to amyloidoses. [ 1 ] Vertex Pharmaceuticals and Pfizer sell regulatory agency approved pharmacologic chaperones for ameliorating cystic fibrosis and the transthyretin amyloidoses, respectively. [ 22 ] Amicus sells a regulatory agency approved pharmacologic chaperone for Fabry disease–a lysosomal storage disease.
The principle behind proteostasis regulators is different. These molecules alter the biology of protein folding and/or degradation by altering the stoichiometry of the proteostasis network components in a given sub cellular compartment. For example, some proteostasis regulators initiate stress responsive signaling, such as the unfolded protein response, which transcriptionally reprograms the endoplasmic reticulum proteostasis network. [ 23 ]
It has been suggested that this approach could even be applied prophylactically, such as upregulating certain protective pathways before experiencing an anticipated severe cellular stress. One theoretical mechanism for this approach includes upregulating the heat shock response response to rescue proteins from degradation during cellular stress. [ 1 ] | https://en.wikipedia.org/wiki/Proteostasis |
PROTEUS (acronym for Reconfigurable Platform for Observation, Telecommunications and Scientific Uses) is a 3-axis stabilized platform designed for mini-satellites weighing approximately 500 kg operating in low Earth orbit. The platform is used by six scientific satellites developed as part of the space program of the National Center for Space Studies (CNES) for the European Space Agency : Jason-1 , 2 and 3, CALIPSO , CoRoT , and SMOS . The platform is developed by the satellite division of Aérospatiale (in 2016 Thales Alenia Space ).
In 1993, CNES decided to launch the development of the PROTEUS mini-satellite program in parallel and jointly with the Jason-1 satellite, the first user for the platform. [ 1 ] Program goals including meeting recurring requirements for satellite solutions in the 500 kg-700 kg class intended for operation in low orbits as platforms for various science and applications. After an industrial consultation with the national prime contractors of the time, Aérospatiale (Cannes) was selected, in May 1996, as industrial prime contractor, with the system to be built in the Cannes-Mandelieu space center. [ 2 ]
By 2010, the PROTEUS platform accumulated 20 years of on-orbit success, with the five satellites that had been orbited: Jason-1, CALIPSO, CoRoT, Jason-2, and SMOS. [ 3 ]
In the same multi-mission platform perspective, the Myriade program to support mission objectives achievable with microsatellites weighing less than 200 kg. [ 4 ]
PROTEUS is a 3-axis stabilized platform designed for missions in low earth orbit for satellites with a total mass of approximately 500 kg, including 270 kg for the platform excluding propellants. Its main features are as follows: [ 5 ]
The PROTEUS platform and command and control segment has been developed based on a partnership between CNES and Aérospatiale (now Thales). The integrated team carries out the design of the PROTEUS multi-mission bus, the industrial production of the platform and associated satellites of which is the responsibility of Thales Alenia Space. In accordance with the partnership agreements, CNES remains in contro of workfor its own missions.
Six satellites use this platform: | https://en.wikipedia.org/wiki/Proteus_(satellite) |
Prothrombin fragment 1+2 ( F1+2 ), also written as prothrombin fragment 1.2 ( F1.2 ), is a polypeptide fragment of prothrombin (factor II) generated by the in vivo cleavage of prothrombin into thrombin (factor IIa) by the enzyme prothrombinase (a complex of factor Xa and factor Va ). [ 1 ] [ 2 ] [ 3 ] It is released from the N-terminus of prothrombin. [ 3 ] F1+2 is a marker of thrombin generation and hence of coagulation activation. [ 4 ] [ 3 ] [ 1 ] It is considered the best marker of in vivo thrombin generation. [ 1 ]
F1+2 levels can be quantified with blood tests and is used in the diagnosis of hyper- and hypocoagulable states and in the monitoring of anticoagulant therapy. [ 4 ] [ 1 ] It was initially determined with a radioimmunoassay , but is now measured with several enzyme-linked immunosorbent assays . [ 1 ]
The molecular weight of F1+2 is around 41 to 43 kDa. [ 4 ] [ 1 ] Its biological half-life is 90 minutes and it persists in blood for a few hours after formation. [ 4 ] [ 3 ] [ 1 ] The half-life of F1+2 is relatively long, which makes it more reliable for measuring ongoing coagulation than other markers like thrombin–antithrombin complexes and fibrinopeptide A . [ 1 ] [ 3 ] Concentrations of F1+2 in healthy individuals range from 0.44 to 1.11 nM. [ 4 ]
F1+2 levels increase with age . [ 3 ] Levels of F1+2 have been reported to be elevated in venous thromboembolism , protein C deficiency , protein S deficiency , atrial fibrillation , unstable angina , acute myocardial infarction , acute stroke , atherosclerosis , peripheral arterial disease , and in smokers . [ 3 ] [ 1 ] Anticoagulants have been found to reduce F1+2 levels. [ 1 ] F1+2 levels are increased with pregnancy [ 5 ] and by ethinylestradiol -containing birth control pills . [ 6 ] Conversely, they do not appear to be increased with estetrol - or estradiol-containing birth control pills . [ 6 ] However, F1+2 levels have been reported to be increased with oral estrogen -based menopausal hormone therapy , whereas transdermal estradiol-based menpausal hormone therapy appears to result in less or no consistent increase. [ 7 ]
This biochemistry article is a stub . You can help Wikipedia by expanding it .
This hematology article is a stub . You can help Wikipedia by expanding it . | https://en.wikipedia.org/wiki/Prothrombin_fragment_1+2 |
The ProTide technology is a prodrug approach used in molecular biology and drug design . It is designed to deliver nucleotide analogues (as monophosphate) into the cell (ProTide: PROdrug + nucleoTIDE). This technology was invented by Professor Chris McGuigan from the School of Pharmacy and Pharmaceutical Sciences at Cardiff University in the early 1990s. ProTides form a critical part of antiviral drugs such as sofosbuvir , tenofovir alafenamide , and remdesivir . [ 1 ]
The first demonstration of the ProTide approach was made in 1992, when the efficiency of aryloxy phosphates and phosphoramidates was noted. [ 2 ] In particular, diaryl phosphates were prepared from zidovudine (AZT) using simple phosphorochloridate chemistry. For the first time, the anti-HIV activity of these phosphate derivatives of AZT exceeded that of the parent nucleoside in some cases. Moreover, while AZT was almost inactive ( EC 50 100μM) in the JM cell line, the substituted diaryl phosphate was 10 times more active (EC 50 10μM). At the time, JM was considered AZT-insensitive due to poor phosphorylation. It later emerged that an AZT-efflux pump was the source of this poor AZT sensitivity. However, the conclusion remains valid that the diaryl phosphate was more able to retain activity in the JM cell line and that this may imply a (small) degree of intracellular phosphate delivery. The electron-withdrawing power of the p-nitro groups and putative enhancements in aryl leaving group ability were suggested as the major driving force of this SAR.
Subsequently, a series of aryloxy phosphoramidates of AZT were prepared with various p-aryl substituents and several amino acids . [ 3 ] These compounds were studied exclusively in the AZT-resistant JM cell line to explore potential (implied) AZT-monophosphate release, with the alanine phosphoramidate proving to be exceptionally effective. In all HIV-1 infected JM cultures, AZT was inhibited at a concentration of 100μM, while the phenyl methoxy alaninyl phosphoramidate was active at 0.8μM. This was taken as the first evidence of a successful nucleotide delivery. It was also noted that in other series, there was a marked preference for alanine over leucine (10-fold) and glycine (>100-fold). Furthermore, although electron-withdrawing aryl substitution had proven highly effective in diaryl systems, it was detrimental in this context. Para fluoro substitution had a slight adventitious effect, but not significantly so, while para-nitro substitution led to a 100-fold loss of activity. In a subsequent study, the range of aryl substituents was expanded, and compounds were tested in both TK+ ( thymidine kinase competent) and TK- (thymidine kinase deficient) cell lines. None of the phosphoramidates retained the high (2–4 nM) potency of AZT in TK-competent cell lines (CEM and MT-4) against either HIV-1 or HIV-2 . [ 4 ] However, while AZT lost all of its activity in the TK- deficient cell line CEM/TK-, most of the phosphoramidates retained antiviral activity, thus being ca >10–35-fold more active than AZT in this assay. Again, alanine emerged as an important component, with the glycine analogue being inactive in HIV-infected CEM/TK- all cultures. In this assay, leucine and phenylalanine were as effective as alanine, although they were less so in CEM/TK+ assays. Thus, the parent phenyl methoxy alanyl phosphoramidate emerged as an important lead compound.
Stavudine (d4T) was an early application of the ProTide approach. [ 5 ] This was a rational choice based on the known kinetics of phosphorylation of d4T. Thus, while the second phosphorylation (AZT-monophosphate to AZT-diphosphate) but not the first phosphorylation (AZT to AZT-monophosphate) is regarded as rate limiting for AZT activation to the triphosphate, the first step (d4T to d4T monophosphate) is thought in general to be the slow step for d4T. Thus, an intracellular (mono)nucleotide delivery should have a maximal impact for d4T and similar nucleosides. In the first instance (halo)alkyloxy phosphoramidates of d4T were prepared and found to retain activity in d4T-resistant JM cells. The activity was dependent on the haloalkyl group; the parent propyl system was poorly active. Subsequent studies in HIV-infected CEM/TK- cell cultures revealed the aryloxy phosphoramidates of d4T to be highly effective and, notably, to retain their full activity in CEM/TK- cells. In this study the benzyl ester emerged as slightly more potent than the parent methyl compound, being almost 10-times more active than d4T in CEM/TK+ assays and thus ca 300-500 fold more active than d4T, in CEM/TK- assays.
The ProTide pro-drugs are useful for delivering phosphonate containing drugs to cell types with high expression of CTSA and CES1 , such as immune cells. Tenofovir alafenamide is a successful example of this iteration. ProTides are also useful for nucleoside analogues that do not get phosphorylated efficiently by endogenous nucleoside kinases. For the nucleoside GS-334750, the parent of sofosbuvir, phosphorylation by nucleoside kinases is effectively nilled, and the only way to deliver active nucleotide is through ProTide. A major limitation of ProTides is that they require an expression of esterases like CTSA and CES1, which is very high in some cell types like hepatocytes and plays to an advantage for the treatment of Hepatitis C of Sofosbuvir.
Extensive studies followed on these promising d4T derivatives and the ProTide technology was successfully applied to a wide range of nucleoside analogues. [ 6 ] [ 7 ] In particular, the ProTide approach has been used on several clinically evaluated anti- HCV nucleoside analogues, including the 2013 FDA approved compound sofosbuvir , and the 2016 FDA approved compound, Tenofovir alafenamide . Remdesivir also uses the ProTide prodrug technology (self-immolation is the key principle of ProTide nucleotide prodrugs [ 8 ] ). Because GS-441524 nucleoside can be phosphorylated and activated, some researchers have argued that the Protide application is an unnecessary complication in Remdesivir's design and that the parent nucleoside would be a cheaper and more effective COVID-19 drug. [ 9 ] [ 10 ]
ProTides have been tested to deliver key phosphorylated metabolites in inborn errors of metabolism, such as phosphopantothenate for PANK2 deficiency, however these were a clinical failure. [ 11 ] | https://en.wikipedia.org/wiki/Protide |
Protistology is a scientific discipline devoted to the study of protists , a highly diverse group of eukaryotic organisms. All eukaryotes apart from animals, plants and fungi are considered protists. [ 1 ] Its field of study therefore overlaps with the more traditional disciplines of phycology , mycology , and protozoology , just as protists embrace mostly unicellular organisms described as algae , some organisms regarded previously as primitive fungi , and protozoa ("animal" motile protists lacking chloroplasts). [ 1 ]
They are a paraphyletic group with very diverse morphologies and lifestyles. Their sizes range from unicellular picoeukaryotes only a few micrometres in diameter to multicellular marine algae several metres long. [ 1 ]
The history of the study of protists has its origins in the 17th century . Since the beginning, the study of protists has been intimately linked to developments in microscopy , which have allowed important advances in the understanding of these organisms due to their generally microscopic nature. Among the pioneers was Anton van Leeuwenhoek , who observed a variety of free-living protists and in 1674 named them “very little animalcules ”. [ 2 ]
During the 18th century studies on the Infusoria were dominated by Christian Gottfried Ehrenberg and Félix Dujardin . [ 3 ]
The term " protozoology " has become dated as understanding of the evolutionary relationships of the eukaryotes has improved, and is frequently replaced by the term "protistology". For example, the Society of Protozoologists, founded in 1947, was renamed International Society of Protistologists in 2005. However, the older term is retained in some cases (e.g., the Polish journal Acta Protozoologica ). [ 4 ]
Dedicated academic journals include: [ 5 ]
Other less specialized journals, important to protistology before the appearance of the more specialized:
Some societies:
The field of protistology was idealized by Haeckel, but its widespread recognition is more recent. In fact, many of the researchers cited below considered themselves as protozoologists, phycologists, mycologists, microbiologists, microscopists, parasitologists, limnologists, biologists, naturalists, zoologists, botanists, etc., but made significant contributions to the field. | https://en.wikipedia.org/wiki/Protistologist |
Protistology is a scientific discipline devoted to the study of protists , a highly diverse group of eukaryotic organisms. All eukaryotes apart from animals, plants and fungi are considered protists. [ 1 ] Its field of study therefore overlaps with the more traditional disciplines of phycology , mycology , and protozoology , just as protists embrace mostly unicellular organisms described as algae , some organisms regarded previously as primitive fungi , and protozoa ("animal" motile protists lacking chloroplasts). [ 1 ]
They are a paraphyletic group with very diverse morphologies and lifestyles. Their sizes range from unicellular picoeukaryotes only a few micrometres in diameter to multicellular marine algae several metres long. [ 1 ]
The history of the study of protists has its origins in the 17th century . Since the beginning, the study of protists has been intimately linked to developments in microscopy , which have allowed important advances in the understanding of these organisms due to their generally microscopic nature. Among the pioneers was Anton van Leeuwenhoek , who observed a variety of free-living protists and in 1674 named them “very little animalcules ”. [ 2 ]
During the 18th century studies on the Infusoria were dominated by Christian Gottfried Ehrenberg and Félix Dujardin . [ 3 ]
The term " protozoology " has become dated as understanding of the evolutionary relationships of the eukaryotes has improved, and is frequently replaced by the term "protistology". For example, the Society of Protozoologists, founded in 1947, was renamed International Society of Protistologists in 2005. However, the older term is retained in some cases (e.g., the Polish journal Acta Protozoologica ). [ 4 ]
Dedicated academic journals include: [ 5 ]
Other less specialized journals, important to protistology before the appearance of the more specialized:
Some societies:
The field of protistology was idealized by Haeckel, but its widespread recognition is more recent. In fact, many of the researchers cited below considered themselves as protozoologists, phycologists, mycologists, microbiologists, microscopists, parasitologists, limnologists, biologists, naturalists, zoologists, botanists, etc., but made significant contributions to the field. | https://en.wikipedia.org/wiki/Protistology |
The proto-mitochondrion is the hypothetical ancestral bacterial endosymbiont from which all mitochondria in eukaryotes are thought to descend, after an episode of symbiogenesis which created the aerobic eukaryotes.
The phylogenetic analyses of the few genes that are still encoded in the genomes of modern mitochondria suggest an alphaproteobacterial origin for this endosymbiont , in an ancient episode of symbiogenesis early in the history of the eukaryotes .
Protomitochondrion Martijn et al 2018
Rhodospirillales,Sphingomonadales, Rhodobacteraceae, Hyphomicrobiales, Holosporales, etc
Iodidimonas
Protomitochondrion Geiger et al 2023
Protomitochondrion Emelyanov 2001
Rickettsia
Midichloria
Neorickettsia
Pelagibacterales
Magnetococcales, Mariprofundales
Zetaproteobacteria
Gammaproteobacteria , Betaproteobacteria , Hydrogenophilalia
Although the order Rickettsiales has been proposed as the alphaproteobacterial sister-group of mitochondria , [ 1 ] [ 2 ] [ 3 ] no definitive evidence pinpoints the alphaproteobacterial group from which the proto-mitochondrion emerged, and some contradictory evidence, especially in the early, sparse genome samplings. Martijn et al found mitochondria are a possible sister-group to all other alphaproteobacteria. [ 4 ] The phylogenetic tree of the Rickettsidae has been inferred by Ferla et al. from the comparison of 16S + 23S ribosomal RNA sequences. [ 5 ] Geiger et alii (2023) propose placing the recently-discovered (2016) genus Iodidimonas – found in a sister clade to Rickettsidae : the Caulobacteridae [ 5 ] – as the closest free-living relative of mitochondria, as it possesses more metabolic products matching that of mitochondria today, such as cardiolipins and sphingolipids , and important genetic markers such as the COX operon and the bc 1 complex (Complex IV and Complex III in mitochondria, respectively). [ 6 ]
Gabaldón & Huynen (2003) reconstructed the proteome (the entire set of proteins expressed by a genome ) and corresponding metabolism of the proto-mitochondrion by comparing extant alpha-proteobacterial and eukaryotic genomes. They concluded that this organism was an aerobic alpha-proteobacterium respiring lipids , glycerol and other compounds provided by the host. At least 630 gene families derived from this organism can still be found in the 9 eukaryotic genomes analyzed in the study. [ 7 ] | https://en.wikipedia.org/wiki/Proto-mitochondrion |
Proto.io is an application prototyping platform launched in 2011 and developed by PROTOIO Inc. [ 1 ] [ 2 ] Originally designed to prototype on mobile devices, Proto.io has expanded to allow users to prototype apps for anything with a screen interface, including Smart TVs , digital camera interfaces, cars , airplanes , and gaming consoles . Proto.io utilizes a drag and drop user interface (UI) and does not require coding . [ 1 ] [ 3 ]
Since its launch in 2011, there have been six versions of Proto.io released.
In 2011, the 100% web-based Proto.io tool became available online. [ 1 ] The web-based environment allowed users to create a project for either the iPad or iPhone . After a user created a few screens for a developing app, Proto.io could then link those pages together with interactive actions that are custom to hand held devices, such as clicks, taps, tap and holds, and swipes. [ 4 ] [ 5 ] [ 6 ] With the platform, users could also create reusable templates into which prepackaged and editable elements could be dragged. Once the user had completed the prototype, Proto.io could then publish and preview the finished product not only on the web browser but also on the actual mobile device. [ 4 ]
Proto.io V2 was released in early 2012 and expanded the supported mobile devices to accommodate for the Android platform, to include the Android Smartphone and Tablet. The platform also came with a newer user interface. Proto.io V2 also added collaboration features like comments and annotations as well as export to HTML functionality. [ 7 ]
On September 28, 2012, with the release of version 3 of the platform, Proto.io became the first prototyping tool to allow users to prototype on almost any device with a screen interface, and the first mobile prototyping tool to support full feature animations of user interface items within a prototype screen. [ 8 ] The included icon gallery contains thousands of SVG icons for use as buttons, lists and tab bars. [ 6 ] Proto.io V3 also supports web fonts, which allows the user to access all available online fonts. [ 8 ]
The fourth version of Proto.io was launched in April 2013. This version was not as heavily focused on introducing new individual features, but rather aimed to improve the tool’s user interface and overall efficiency with a completely revamped editor. [ 9 ]
October 2013 brought one of the major releases of Proto.io. With Version 5 users gained the capability to build HTML5 interactive animations more intuitively with the new Animation States & Timelines feature. This release introduced a wide variety of other new functionalities as well, such as easier Drag & Rotate, Variables and Item property interactions and new touch events. [ 10 ]
The most recent release of Proto.io was launched in July 2016. The entire interface was redesigned, making the most used tools easily accessible to users. Additionally, animations became for the first time replayable directly on the editor, making it easier to finalize the motion design process. Adding and editing interactions was also simplified, with the introduction of an Interaction Wizard and Interaction Design Patterns. Single-click sharing and exporting also became available in the same release. [ 11 ] | https://en.wikipedia.org/wiki/Proto.io |
A protocell (or protobiont ) is a self-organized , endogenously ordered, spherical collection of lipids proposed as a rudimentary precursor to cells during the origin of life . [ 1 ] [ 2 ] A central question in evolution is how simple protocells first arose and how their progeny could diversify, thus enabling the accumulation of novel biological emergences over time (i.e. biological evolution ). Although a functional protocell has not yet been achieved in a laboratory setting , the goal to understand the process appears well within reach. [ 3 ] [ 4 ] [ 5 ] [ 6 ]
A protocell is a pre-cell in abiogenesis , and was a contained system consisting of simple biologically relevant molecules like ribozymes , and encapsulated in a simple membrane structure – isolating the entity from the environment and other individuals – thought to consist of simple fatty acids, mineral structures, or rock-pore structures.
Compartmentalization was important in the origin of life. [ 7 ] Membranes form enclosed compartments that are separate from the external environment, thus providing the cell with functionally specialized aqueous spaces. As the lipid bilayer of membranes is impermeable to most hydrophilic molecules (dissolved by water), modern cells have membrane transport-systems that achieve nutrient uptake as well as the export of waste. [ 8 ] Prior to the development of these molecular assemblies, protocells likely employed vesicle dynamics that are relevant to cellular functions, such as membrane trafficking and self-reproduction, using amphiphilic molecules. On the primitive Earth, numerous chemical reactions of organic compounds produced the ingredients of life. [ 9 ] Of these substances, amphiphilic molecules might be the first player in the evolution from molecular assembly to cellular life. [ 10 ] [ 11 ] Vesicle dynamics could progress towards protocells with the development of self-replication coupled with early metabolism. [ 12 ] It is possible that protocells might have had a primitive metabolic system ( Wood-Ljungdahl pathway ) at alkaline hydrothermal vents or other geological environments like impact crater lakes from meteorites, [ 13 ] which are known to be composed of elements found in the Wood-Ljungdahl pathway. [ 14 ]
Another conceptual model of a protocell relates to the term " chemoton " (short for 'chemical automaton ') which refers to the fundamental unit of life introduced by Hungarian theoretical biologist Tibor Gánti . [ 15 ] It is the oldest known computational abstract of a protocell. Gánti conceived the basic idea in 1952 and formulated the concept in 1971 in his book The Principles of Life (originally written in Hungarian, and translated to English only in 2003). He surmised the chemoton as the original ancestor of all organisms, or the last universal common ancestor . [ 16 ]
The basic assumption of the chemoton model is that life should fundamentally and essentially have three properties: metabolism , self-replication , and a bilipid membrane . [ 17 ] The metabolic and replication functions together form an autocatalytic subsystem necessary for the basic functions of life, and a membrane encloses this subsystem to separate it from the surrounding environment. Therefore, any system having such properties may be regarded as alive, and will contain self sustaining cellular information that is subject to natural selection . Some consider this model a significant contribution to origin of life as it provides a philosophy of evolutionary units . [ 18 ]
Self-assembled vesicles are essential components of primitive cells. [ 19 ] The second law of thermodynamics requires that the universe becomes increasingly disordered ( entropy ), yet life is distinguished by its great degree of organization. Therefore, a boundary is needed to separate life processes from non-living matter. [ 20 ] This fundamental necessity is underpinned by the universality of the cell membrane which is the only cellular structure found in all organisms on Earth. [ 21 ]
In the aqueous environment in which all known cells function, a non-aqueous barrier is required to surround a cell and separate it from its surroundings. [ 22 ] This non-aqueous membrane establishes a barrier to free diffusion, allowing for regulation of the internal environment within the barrier. The necessity of thermodynamically isolating a subsystem is an irreducible condition of life. [ 22 ] In modern biology, such isolation is ordinarily accomplished by amphiphilic bilayers of a thickness of around 10 −8 meters.
Researchers including Irene A. Chen and Jack W. Szostak have demonstrated that simple physicochemical properties of elementary protocells can give rise to simpler conceptual analogues of essential cellular behaviors, including primitive forms of Darwinian competition and energy storage. Such cooperative interactions between the membrane and encapsulated contents could greatly simplify the transition from replicating molecules to true cells. [ 23 ] Competition for membrane molecules would favor stabilized membranes, suggesting a selective advantage for the evolution of cross-linked fatty acids and even the phospholipids of today. [ 23 ] This micro-encapsulation allowed for metabolism within the membrane, exchange of small molecules and prevention of passage of large substances across it. [ 24 ] The main advantages of encapsulation include increased solubility of the cargo and creating energy in the form of chemical gradients. Energy is thus often said to be stored by cells in molecular structures such as carbohydrates (including sugars), lipids , and proteins , which release energy when chemically combined with oxygen during cellular respiration . [ 25 ] [ 26 ]
When phospholipids or simple lipids like fatty acids are placed in water, the molecules spontaneously arrange such that the hydrophobic tails are shielded from the water, resulting in the formation of membrane structures such as bilayers , vesicles , and micelles . [ 27 ] In modern cells, vesicles are involved in metabolism , transport, buoyancy control, [ 28 ] and enzyme storage. They can also act as natural chemical reaction chambers. A typical vesicle or micelle in aqueous solution forms an aggregate with the hydrophilic "head" regions in contact with surrounding solvent , sequestering the hydrophobic single-tail regions in the micelle center. This phase is caused by the packing behavior of single-tail lipids in a bilayer . Although the spontaneous self-assembly process that form lipid monolayer vesicles and micelles in nature resemble the kinds of primordial vesicles or protocells that might have existed at the beginning of evolution, they are not as sophisticated as the bilayer membranes of today's living organisms. [ 29 ] However, in a prebiotic context, electrostatic interactions induced by short, positively charged, hydrophobic peptides containing seven amino acids in length or fewer, can attach RNA to a vesicle membrane, the basic cell membrane. [ 30 ] [ 31 ]
Rather than being made up of phospholipids, early membranes may have formed from monolayers or bilayers of simple fatty acids , which may have formed more readily in a prebiotic environment. [ 32 ] Fatty acids have been synthesized in laboratories under a variety of prebiotic conditions and have been found on meteorites, suggesting their natural synthesis in nature. [ 33 ] Oleic acid vesicles represent good models of membrane protocells [ 34 ]
Cohen et al. (2022) suggest that plausible prebiotic production of fatty acids — leading to the development of early protocell membranes — is enriched on metal-rich mineral surfaces, possibly from impact craters, increasing the prebiotic environmental mass of lipids by 10 2 times. [ 13 ] They evaluate three different possible synthesis pathways of fatty acids in the Hadean, and found that these metal surfaces could produce 10 11 - 10 15 kg of 6-18 carbon fatty acids. Of these products, the 8-18C fatty acids are compatible with membrane formation. They also propose that alternative amphiphiles like alcohols are co-synthesized with fatty acid, and can help improve membrane stability. However, despite this production, the authors state that net fatty acid synthesis would not yield sufficient concentrations for spontaneous membrane formation without significant evaporation of Earth's aqueous environments.
For cellular organisms, the transport of specific molecules across compartmentalizing membrane barriers is essential in order to exchange content with their environment and with other individuals. For example, content exchange between individuals enables the exchange of genes between individuals ( horizontal gene transfer ), an important factor in the evolution of cellular life. [ 35 ] While modern cells can rely on complicated protein machineries to catalyze these crucial processes, protocells must have accomplished this using more simple mechanisms.
Protocells composed of fatty acids [ 36 ] would have been able to easily exchange small molecules and ions with their environment. [ 37 ] Modern phospholipid bilayer cell membranes exhibit low permeability, but contain complex molecular assemblies which both actively and passively transport relevant molecules across the membrane in a highly specific manner. In the absence of these complex assemblies, simple fatty acid based protocell membranes would be more permeable and allow for greater non-specific transport across membranes. [ 7 ] Molecules that would be highly permeable across protocell membranes include nucleoside monophosphate (NMP), nucleoside diphosphate (NDP), and nucleoside triphosphate (NTP), and may withstand millimolar concentrations of Mg 2+ . [ 38 ] Osmotic pressure can also play a significant role regarding this passive membrane transport. [ 37 ]
Environmental effects have been suggested to trigger conditions under which a transport of larger molecules, such as DNA and RNA , across the membranes of protocells is possible. For example, it has been proposed that electroporation resulting from lightning strikes could enable such transport. [ 39 ] Electroporation is the rapid increase in bilayer permeability induced by the application of a large artificial electric field across the membrane. During electroporation, the lipid molecules in the membrane shift position, opening up a pore (hole) that acts as a conductive pathway through which hydrophobic molecules like nucleic acids can pass the lipid bilayer. [ 40 ] A similar transfer of content across protocells and with the surrounding solution can be caused by freezing and subsequent thawing. This could, for instance, occur in an environment in which day and night cycles cause recurrent freezing. Laboratory experiments have shown that such conditions allow an exchange of genetic information between populations of protocells. [ 41 ] This can be explained by the fact that membranes are highly permeable at temperatures slightly below their phase transition temperature. If this point is reached during the freeze-thaw cycle, even large and highly charged molecules can temporarily pass the protocell membrane.
Some molecules or particles are too large or too hydrophilic to pass through a lipid bilayer even under these conditions, but can be moved across the membrane through fusion or budding of vesicles , [ 42 ] events which have also been observed for freeze-thaw cycles. [ 43 ] This may eventually have led to mechanisms that facilitate movement of molecules to the inside of the protocell ( endocytosis ) or to release its contents into the extracellular space ( exocytosis ). [ 42 ]
See also: Abiogenesis: Suitable Geologic Environment , RNA World: Prebiotic RNA Synthesis
It has been proposed that life began in hydrothermal vents in the deep sea, but a 2012 study suggests that hot springs have the ideal characteristics for the origin of life. [ 44 ] The conclusion is based mainly on the chemistry of modern cells, where the cytoplasm is rich in potassium, zinc, manganese, and phosphate ions, not widespread in marine environments. Such conditions, the researchers argue, are found only where hot hydrothermal fluid brings the ions to the surface—places such as geysers , mud pots , fumaroles and other geothermal features. Within these fuming and bubbling basins, water laden with zinc and manganese ions could have collected, cooled and condensed in shallow pools. [ 44 ] However, a recent discovery of alkaline hydrothermal vents with an ionic concentration of sodium lower than in seawater suggests that high concentrations of potassium can be found at marine environments. [ 45 ]
A study in the 1990s showed that montmorillonite clay can help create RNA chains of as many as 50 nucleotides joined together spontaneously into a single RNA molecule. [ 46 ] Later, in 2002, it was discovered that by adding montmorillonite to a solution of fatty acid micelles (lipid spheres), the clay sped up the rate of vesicle formation 100-fold. [ 46 ]
Some minerals can catalyze the stepwise formation of hydrocarbon tails of fatty acids from hydrogen and carbon monoxide gases—gases that may have been released from hydrothermal vents or geysers . Fatty acids of various lengths are eventually released into the surrounding water, [ 47 ] but vesicle formation requires a higher concentration of fatty acids, so it is suggested that protocell formation started at land-bound hydrothermal freshwater environments such as geysers , mud pots, fumaroles and other geothermal features where water evaporates and concentrates the solute. [ 46 ] [ 48 ] [ 49 ]
In 2019, Nick Lane and colleagues show that vesicles form readily in seawater conditions at pH between 6.5 and >12 and temperatures 70 °C, meant to mimic the conditions of alkaline hydrothermal vents, with the presence of lipid mixtures, [ 50 ] however a prebiotic source to such mixtures is unclear in those environments. Simple amphiphilic compounds in seawater do not assemble into vesicles because of the high concentration of ionic solutes. Research has shown that vesicles can be bound and stabilized by prebiotic amino acids even while in the presence of salt ions and magnesium ions. [ 51 ]
In hot spring conditions, self-assembly of vesicles occurs, which have a lower concentration of ionic solutes. [ 52 ] Scientists oligomerized RNA in alkaline hydrothermal vent conditions in the laboratory. Although they were estimated to be 4 units in length, it implies RNA polymers possibly were synthesized at such environments. [ 53 ] Experimental research at hot springs gave higher yields of RNA-like polymers than in the laboratory. The polymers were encapsulated in fatty acid vesicles when rehydrated, further supporting the hot spring hypothesis of abiogenesis. [ 54 ] These wet-dry cycles also improved vesicle stability and binding. [ 51 ] UV exposure has also been shown to promote the synthesis of stable biomolecules like nucleotides. [ 55 ] [ 56 ]
In the origin of chemiosmosis , if early cells originated at alkaline hydrothermal vents, proton gradients can be maintained by the acidic ocean and alkaline water from white smokers while an inorganic membranous structure is in a rock cavity. [ 57 ] [ 51 ] If early cells originated in terrestrial pools such as hot springs, quinones present in meteorites like the Murchison meteorite would promote the development of proton gradients by coupled redox reactions if the ferricyanide, the electron acceptor, was within the vesicle and an electron donor like a sulfur compound was outside of the lipid membrane. [ 52 ] [ 58 ] Because of the "water problem", a primitive ATP synthase and other biomolecules would go through hydrolysis due to the absence of wet-dry cycles at hydrothermal vents, unlike at terrestrial pools. [ 52 ] Other researchers propose hydrothermal pore systems coated in mineral gels at deep sea hydrothermal vents to an alternative compartment of membranous structures, promote biochemical reactions of biopolymers, and could solve the "water problem". [ 59 ] [ 57 ] David Deamer and Bruce Damer argue that biomolecules would become trapped within these pore systems upon polymerization and would not undergo combinatorial selection. [ 52 ] Catalytic FeS and NiS walls at alkaline hydrothermal vents has also been suggested to have promoted polymerization. [ 60 ]
However, Jackson (2016) evaluates how the pH gradient between alkaline hydrothermal vents and acidic Hadean seawater might influence prebiotic synthesis. [ 61 ] Three main criticisms emerge from this evaluation. Firstly, the maintenance and stability of membranes positioned suitably between turbulent pH gradients seemed implausible. They claim that the proposition of CaCO 3 and Mg(OH) 2 precipitates interacting with fluid mixing in subsurface pores do not produce satisfactory environments. Secondly, they suggest that the molecular assemblies required to utilize key energetic gradients available at hydrothermal systems were too complex to have been relevant at the origin of life. Lastly, they argue that even if a molecular assembly could have harvested available hydrothermal energy, those assemblies would have been too large to operate within the proposed membrane thicknesses accepted by proponents of the hydrothermal vent hypothesis. In 2017, Jackson takes a further stance, suggesting that even if an organism successfully originated in alkaline hydrothermal pores, exploiting natural pH gradients for energy, it would not be able to withstand the drastic change of environment after emergence from the vent environment in which it had solely evolved. [ 62 ] This emergence, however, is essential to the niche differentiation of life, allowing for the diversification of habitats and energetic strategies. Counters to these arguments suggest that the close resemblance between biochemical pathways and geochemical systems at alkaline hydrothermal vents gives merit to the hypothesis, and that selection on these protocells would improve resilience to environmental change, allowing for emergence and distribution. [ 63 ]
It has been considered by other researchers that life originating in hydrothermal volcanic ponds exposed to UV radiation, zinc sulfide photocatalysis, and occurrence of continuous wet-dry cycling would not resemble modern biochemistry. [ 64 ] [ 65 ] [ 66 ] Maximal ATP synthesis is shown to occur at high water activity and low ion concentrations. Despite this, hydrothermal vents are still considered to be a feasible environment as some shallow hydrothermal vents emit freshwater and the concentration of divalent cations in Hadean oceans were likely lower than in modern oceans. Nick Lane and coauthors state that "alkaline hydrothermal systems tend to precipitate Ca 2+ and Mg 2+ ions as aragonite and brucite, so their concentrations are typically much lower than mean ocean values. Modelling work in relation to Hadean systems indicates that hydrothermal concentrations of Ca 2+ and Mg 2+ would likely have been <1 mM, which is in the range that enhanced phosphorylation here. Other conditions considered here, including salinity and high pressure, would have only limited effects on ATP synthesis in submarine hydrothermal systems (which typically have pressures in the range of 100 to 300 Bars). Alkaline hydrothermal systems might also have generated Fe 3+ in situ for ADP phosphorylation. Thermodynamic modelling shows that the mixing of alkaline hydrothermal fluids with seawater in submarine systems can promote continuous cycling between ferrous and ferric iron, potentially forming soluble hydrous ferric chlorides, which our experiments show have the same effect as ferric sulphate". [ 67 ]
Another group suggests that primitive cells might have formed inside inorganic clay microcompartments, which can provide an ideal container for the synthesis and compartmentalization of complex organic molecules. [ 68 ] Clay-armored bubbles form naturally when particles of montmorillonite clay collect on the outer surface of air bubbles under water. This creates a semi permeable vesicle from materials that are readily available in the environment. The authors remark that montmorillonite is known to serve as a chemical catalyst, encouraging lipids to form membranes and single nucleotides to join into strands of RNA. Primitive reproduction can be envisioned when the clay bubbles burst, releasing the lipid membrane-bound product into the surrounding medium. [ 68 ]
Another way to form primitive compartments that may lead to the formation of a protocell is polyesters membraneless structures that have the ability to host biochemicals (proteins and RNA) and/or scaffold the assemblies of lipids around them. [ 69 ] [ 70 ] While these droplets are leaky towards genetic materials, this leakiness could have facilitated the progenote hypothesis. [ 71 ]
Researchers have also proposed early encapsulation in aqueous phase-separated droplets called coacervates . These droplets are driven by the accumulation of macromolecules, producing a distinct dense phase liquid droplet within a more dilute liquid medium. [ 7 ] These droplets can propagate, retaining their internal composition, through shear forces and turbulence in the medium, and could have acted as a means of replicating encapsulation for an early protocell. However, replication was highly disordered and droplet fusion is common, calling into question coacervates true potential for distinct compartmentalization leading to competition and early Darwinian-selection. [ citation needed ]
Eigen et al . [ 72 ] and Woese [ 73 ] proposed that the genomes of early protocells were composed of single-stranded RNA , and that individual genes corresponded to separate RNA segments, rather than being linked end-to-end as in present-day DNA genomes . A protocell that was haploid (one copy of each RNA gene) would be vulnerable to damage, since a single lesion in any RNA segment would be potentially lethal to the protocell (e.g. by blocking replication or inhibiting the function of an essential gene).
Vulnerability to damage could be reduced by maintaining two or more copies of each RNA segment in each protocell, i.e. by maintaining diploidy or polyploidy. Genome redundancy would allow a damaged RNA segment to be replaced by an additional replication of its homolog . For such a simple organism, the proportion of available resources tied up in the genetic material would be a large fraction of the total resource budget. Under limited resource conditions, the protocell reproductive rate would likely be inversely related to ploidy number, and the protocell's fitness would be reduced by the costs of redundancy. Consequently, coping with damaged RNA genes while minimizing the costs of redundancy would likely have been a fundamental problem for early protocells.
A cost-benefit analysis was carried out in which the costs of maintaining redundancy were balanced against the costs of genome damage. [ 74 ] This analysis led to the conclusion that, under a wide range of circumstances, the selected strategy would be for each protocell to be haploid, but to periodically fuse with another haploid protocell to form a transient diploid. The retention of the haploid state maximizes the growth rate. The periodic fusions permit mutual reactivation of otherwise lethally damaged protocells. If at least one damage-free copy of each RNA gene is present in the transient diploid, viable progeny can be formed. For two, rather than one, viable daughter cells to be produced would require an extra replication of the intact RNA gene homologous to any RNA gene that had been damaged prior to the division of the fused protocell. The cycle of haploid reproduction, with occasional fusion to a transient diploid state, followed by splitting to the haploid state, can be considered to be the sexual cycle in its most primitive form. [ 74 ] [ 75 ] In the absence of this sexual cycle, haploid protocells with damage in an essential RNA gene would simply die.
This model for the early sexual cycle is hypothetical, but it is very similar to the known sexual behavior of the segmented RNA viruses, which are among the simplest organisms known. Influenza virus , whose genome consists of 8 physically separated single-stranded RNA segments, [ 76 ] is an example of this type of virus. In segmented RNA viruses, "mating" can occur when a host cell is infected by at least two virus particles. If these viruses each contain an RNA segment with a lethal damage, multiple infection can lead to reactivation providing that at least one undamaged copy of each virus gene is present in the infected cell. This phenomenon is known as "multiplicity reactivation". Multiplicity reactivation has been reported to occur in influenza virus infections after induction of RNA damage by UV-irradiation , [ 77 ] and ionizing radiation. [ 78 ]
Starting with a technique commonly used to deposit molecules on a solid surface, Langmuir–Blodgett deposition, scientists are able to assemble phospholipid membranes of arbitrary complexity layer by layer. [ 79 ] [ 80 ] These artificial phospholipid membranes support functional insertion both of purified and of in situ expressed membrane proteins . [ 80 ] The technique could help astrobiologists understand how the first living cells originated. [ 79 ]
Jeewanu protocells are synthetic chemical particles that possess cell -like structure and seem to have some functional living properties. [ 81 ] First synthesized in 1963 from simple minerals and basic organics while exposed to sunlight , it is still reported to have some metabolic capabilities, the presence of semipermeable membrane , amino acids , phospholipids , carbohydrates and RNA-like molecules. [ 81 ] [ 82 ] The nature and properties of the Jeewanu remains to be clarified. [ 81 ] [ 82 ] [ 83 ]
In a similar synthesis experiment a frozen mixture of water, methanol , ammonia and carbon monoxide was exposed to ultraviolet (UV) radiation. This combination yielded large amounts of organic material that self-organised to form globules or vesicles when immersed in water. [ 84 ] The investigating scientist considered these globules to resemble cell membranes that enclose and concentrate the chemistry of life, separating their interior from the outside world. The globules were between 10 and 40 micrometres (0.00039 and 0.00157 in), or about the size of red blood cells. Remarkably, the globules fluoresced , or glowed, when exposed to UV light. Absorbing UV and converting it into visible light in this way was considered one possible way of providing energy to a primitive cell. If such globules played a role in the origin of life, the fluorescence could have been a precursor to primitive photosynthesis . Such fluorescence also provides the benefit of acting as a sunscreen, diffusing any damage that otherwise would be inflicted by UV radiation. Such a protective function would have been vital for life on the early Earth, since the ozone layer , which blocks out the sun's most destructive UV rays, did not form until after photosynthetic life began to produce oxygen . [ 85 ]
The synthesis of three kinds of "jeewanu" have been reported; two of them were organic, and the other was inorganic. Other similar inorganic structures have also been produced. The investigating scientist (V. O. Kalinenko) referred to them as "bio-like structures" and "artificial cells". Formed in distilled water (as well as on agar gel) under the influence of an electric field, they lack protein, amino acids, purine or pyrimidine bases, and certain enzyme activities. According to NASA researchers, "presently known scientific principles of biology and biochemistry cannot account for living inorganic units" and "the postulated existence of these living units has not been proved". [ 86 ]
In March 2014, NASA's Jet Propulsion Laboratory demonstrated a unique way to study the origins of life: fuel cells. [ 87 ] Fuel cells are similar to biological cells in that electrons are also transferred to and from molecules. In both cases, this results in electricity and power. The study of fuel cells suggest that an important factor in protocell development was that the Earth provides electrical energy at the seafloor. "This energy could have kick-started life and could have sustained life after it arose. Now, we have a way of testing different materials and environments that could have helped life arise not just on Earth, but possibly on Mars , Europa and other places in the Solar System ." [ 87 ]
Protocell research has created controversy and opposing opinions, including criticism of vague definitions of "artificial life". [ 88 ] The creation of a basic unit of life is the most pressing ethical concern, although the most widespread worry about protocells is their potential threat to human health and the environment through uncontrolled replication. [ 89 ]
Additionally, postulation into the conditions for protocellular origins of life on Earth remain debated. Scientists in the field emphasize the importance of further hypothesis based experimentation over theoretical conjecture to more concretely constrain the prebiotic plausibility of different protocell morphologies, geologic conditions, and synthetic schemes. [ 90 ] | https://en.wikipedia.org/wiki/Protocell |
Protocol engineering is the application of systematic methods to the development of communication protocols . It uses many of the principles of software engineering , but it is specific to the development of distributed systems.
When the first experimental and commercial computer networks were developed in the 1970s, the concept of protocols was not yet well developed. These were the first distributed systems . In the context of the newly adopted layered protocol architecture (see OSI model ), the definition of the protocol of a specific layer should be such that any entity implementing that specification in one computer would be compatible with any other computer containing an entity implementing the same specification, and their interactions should be such that the desired communication service would be obtained. On the other hand, the protocol specification should be abstract enough to allow different choices for the implementation on different computers.
It was recognized that a precise specification of the expected service provided by the given layer was important. [ 1 ] It is important for the verification of the protocol, which should demonstrate that the communication service is provided if both protocol entities implement the protocol specification correctly. This principle was later followed during the standardization of the OSI protocol stack , in particular for the transport layer .
It was also recognized that some kind of formalized protocol specification would be useful for the verification of the protocol and for developing implementations, as well as test cases for checking the conformance of an implementation against the specification. [ 2 ] While initially mainly finite-state machine were used as (simplified) models of a protocol entity, [ 3 ] in the 1980s three formal specification languages were standardized, two by ISO [ 4 ] and one by ITU. [ 5 ] The latter, called SDL , was later used in industry and has been merged with UML state machines .
The following are the most important principles for the development of protocols: [ 1 ]
Tools for the activities of protocol verification, entity implementation and test suite development can be developed when the protocol specification is written in a formalized language which can be understood by the tool. As mentioned, formal specification languages have been proposed for protocol specification, and the first methods and tools where based on finite-state machine models. Reachability analysis was proposed to understand all possible behaviors of a distributed system, which is essential for protocol verification. This was later complemented with model checking . However, finite-state descriptions are not powerful enough to describe constraints between message parameters and the local variables in the entities. Such constraints can be described by the standardized formal specification languages mentioned above, for which powerful tools have been developed.
It is in the field of protocol engineering that model-based development was used very early. These methods and tools have later been used for software engineering as well as hardware design, especially for distributed and real-time systems. On the other hand, many methods and tools developed in the more general context of software engineering can also be used of the development of protocols, for instance model checking for protocol verification, and agile methods for entity implementations.
Most protocols are designed by human intuition and discussions during the standardization process. However, some methods have been proposed for using constructive methods possibly supported by tools to automatically derive protocols that satisfy certain properties. The following are a few examples: | https://en.wikipedia.org/wiki/Protocol_engineering |
Mutualism describes the ecological interaction between two or more species where each species has a net benefit. [ 1 ] Mutualism is a common type of ecological interaction. Prominent examples are:
Mutualism can be contrasted with interspecific competition , in which each species experiences reduced fitness, and exploitation , and with parasitism , in which one species benefits at the expense of the other. [ 2 ] However, mutualism may evolve from interactions that began with imbalanced benefits, such as parasitism. [ 3 ]
The term mutualism was introduced by Pierre-Joseph van Beneden in his 1876 book Animal Parasites and Messmates to mean "mutual aid among species". [ 4 ] [ 5 ]
Mutualism is often conflated with two other types of ecological phenomena: cooperation and symbiosis . Cooperation most commonly refers to increases in fitness through within-species (intraspecific) interactions, although it has been used (especially in the past) to refer to mutualistic interactions, and it is sometimes used to refer to mutualistic interactions that are not obligate. [ 1 ] Symbiosis involves two species living in close physical contact over a long period of their existence and may be mutualistic, parasitic, or commensal , so symbiotic relationships are not always mutualistic, and mutualistic interactions are not always symbiotic. Despite a different definition between mutualism and symbiosis, they have been largely used interchangeably in the past, and confusion on their use has persisted. [ 6 ]
Mutualism plays a key part in ecology and evolution . For example, mutualistic interactions are vital for terrestrial ecosystem function as:
A prominent example of pollination mutualism is with bees and flowering plants. Bees use these plants as their food source with pollen and nectar. In turn, they transfer pollen to other nearby flowers, inadvertently allowing for cross-pollination. Cross-pollination has become essential in plant reproduction and fruit/seed production. The bees get their nutrients from the plants, and allow for successful fertilization of plants, demonstrating a mutualistic relationship between two seemingly-unlike species.
Mutualism has also been linked to major evolutionary events, such as the evolution of the eukaryotic cell ( symbiogenesis ) and the colonization of land by plants in association with mycorrhizal fungi.
Mutualistic relationships can be thought of as a form of " biological barter" [ 10 ] in mycorrhizal associations between plant roots and fungi , with the plant providing carbohydrates to the fungus in return for primarily phosphate but also nitrogenous compounds. Other examples include rhizobia bacteria that fix nitrogen for leguminous plants (family Fabaceae) in return for energy-containing carbohydrates . [ 11 ] Metabolite exchange between multiple mutualistic species of bacteria has also been observed in a process known as cross-feeding . [ 12 ] [ 13 ]
Service-resource relationships are common. Three important types are pollination , cleaning symbiosis, and zoochory .
In pollination, a plant trades food resources in the form of nectar or pollen for the service of pollen dispersal. However, daciniphilous Bulbophyllum orchid species trade sex pheromone precursor or booster components via floral synomones /attractants in a true mutualistic interactions with males of Dacini fruit flies (Diptera: Tephritidae: Dacinae). [ 14 ] [ 15 ]
Phagophiles feed (resource) on ectoparasites , thereby providing anti-pest service, as in cleaning symbiosis . Elacatinus and Gobiosoma , genera of gobies , feed on ectoparasites of their clients while cleaning them. [ 16 ]
Zoochory is the dispersal of the seeds of plants by animals. This is similar to pollination in that the plant produces food resources (for example, fleshy fruit, overabundance of seeds) for animals that disperse the seeds (service). Plants may advertise these resources using colour [ 17 ] and a variety of other fruit characteristics, e.g., scent. Fruit of the aardvark cucumber (Cucumis humifructus) is buried so deeply that the plant is solely reliant upon the aardvark's keen sense of smell to detect its ripened fruit, extract, consume and then scatter its seeds; [ 18 ] C. humifructus ' s geographical range is thus restricted to that of the aardvark.
Another type is ant protection of aphids , where the aphids trade sugar -rich honeydew (a by-product of their mode of feeding on plant sap ) in return for defense against predators such as ladybugs . [ citation needed ]
Strict service-service interactions are very rare, for reasons that are far from clear. [ 10 ] One example is the relationship between sea anemones and anemone fish in the family Pomacentridae : the anemones provide the fish with protection from predators (which cannot tolerate the stings of the anemone's tentacles) and the fish defend the anemones against butterflyfish (family Chaetodontidae ), which eat anemones. However, in common with many mutualisms, there is more than one aspect to it: in the anemonefish-anemone mutualism, waste ammonia from the fish feeds the symbiotic algae that are found in the anemone's tentacles. [ 19 ] [ 20 ] Therefore, what appears to be a service-service mutualism in fact has a service-resource component. A second example is that of the relationship between some ants in the genus Pseudomyrmex and trees in the genus Acacia , such as the whistling thorn and bullhorn acacia . The ants nest inside the plant's thorns. In exchange for shelter, the ants protect acacias from attack by herbivores (which they frequently eat when those are small enough, introducing a resource component to this service-service relationship) and competition from other plants by trimming back vegetation that would shade the acacia. In addition, another service-resource component is present, as the ants regularly feed on lipid -rich food-bodies called Beltian bodies that are on the Acacia plant. [ 21 ]
In the neotropics , the ant Myrmelachista schumanni makes its nest in special cavities in Duroia hirsuta . Plants in the vicinity that belong to other species are killed with formic acid . This selective gardening can be so aggressive that small areas of the rainforest are dominated by Duroia hirsute . These peculiar patches are known by local people as " devil's gardens ". [ 22 ]
In some of these relationships, the cost of the ant's protection can be quite expensive. Cordia sp. trees in the Amazon rainforest have a kind of partnership with Allomerus sp. ants, which make their nests in modified leaves. To increase the amount of living space available, the ants will destroy the tree's flower buds. The flowers die and leaves develop instead, providing the ants with more dwellings. Another type of Allomerus sp. ant lives with the Hirtella sp. tree in the same forests, but in this relationship, the tree has turned the tables on the ants. When the tree is ready to produce flowers, the ant abodes on certain branches begin to wither and shrink, forcing the occupants to flee, leaving the tree's flowers to develop free from ant attack. [ 22 ]
The term "species group" can be used to describe the manner in which individual organisms group together. In this non-taxonomic context one can refer to "same-species groups" and "mixed-species groups." While same-species groups are the norm, examples of mixed-species groups abound. For example, zebra ( Equus burchelli ) and wildebeest ( Connochaetes taurinus ) can remain in association during periods of long distance migration across the Serengeti as a strategy for thwarting predators. Cercopithecus mitis and Cercopithecus ascanius , species of monkey in the Kakamega Forest of Kenya , can stay in close proximity and travel along exactly the same routes through the forest for periods of up to 12 hours. These mixed-species groups cannot be explained by the coincidence of sharing the same habitat. Rather, they are created by the active behavioural choice of at least one of the species in question. [ 23 ]
Protocooperation is a form of mutualism, but the cooperating species do not depend on each other for survival. The term, initially used for intraspecific interactions, was popularized by Eugene Odum (1953), although it is now rarely used. [ 24 ]
Mutualistic symbiosis can sometimes evolve from parasitism or commensalism . Symbiogenesis , a leading theory on the evolution of Eukaryotes states the origin of the mitochondria and cell nucleus emerged from a parasitic relationship of ancient Archaea and Bacteria . Fungi's relationship to plants in the form of mycelium evolved from parasitism and commensalism. Under certain conditions species of fungi previously in a state of mutualism can turn parasitic on weak or dying plants. [ 25 ] Likewise the symbiotic relationship of clown fish and sea anemones emerged from a commensalist relationship. [ 26 ] [ 27 ] [ 28 ] Once a mutualistic relationship emerges both symbionts are pushed towards co-evolution with each other. [ 29 ] [ 30 ]
Mathematical treatments of mutualisms, like the study of mutualisms in general, have lagged behind those for predation , or predator-prey, consumer-resource, interactions. In models of mutualisms, the terms "type I" and "type II" functional responses refer to the linear and saturating relationships, respectively, between the benefit provided to an individual of species 1 ( dependent variable ) and the density of species 2 (independent variable). [ citation needed ]
One of the simplest frameworks for modeling species interactions is the Lotka–Volterra equations . [ 31 ] In this model, the changes in population densities of the two mutualists are quantified as:
where
Mutualism is in essence the logistic growth equation modified for mutualistic interaction. The mutualistic interaction term represents the increase in population growth of one species as a result of the presence of greater numbers of another species. As the mutualistic interactive term β is always positive, this simple model may lead to unrealistic unbounded growth. [ 32 ] So it may be more realistic to include a further term in the formula, representing a saturation mechanism, to avoid this occurring.
In 1989, David Hamilton Wright modified the above Lotka–Volterra equations by adding a new term, βM / K , to represent a mutualistic relationship. [ 33 ] Wright also considered the concept of saturation, which means that with higher densities, there is a decrease in the benefits of further increases of the mutualist population. Without saturation, depending on the size of parameter α, species densities would increase indefinitely. Because that is not possible due to environmental constraints and carrying capacity, a model that includes saturation would be more accurate. Wright's mathematical theory is based on the premise of a simple two-species mutualism model in which the benefits of mutualism become saturated due to limits posed by handling time. Wright defines handling time as the time needed to process a food item, from the initial interaction to the start of a search for new food items and assumes that processing of food and searching for food are mutually exclusive. Mutualists that display foraging behavior are exposed to the restrictions on handling time. Mutualism can be associated with symbiosis. [ citation needed ]
In 1959, C. S. Holling performed his classic disc experiment that assumed that
where
The equation that incorporates Type II functional response and mutualism is:
where
or, equivalently,
where
This model is most effectively applied to free-living species that encounter a number of individuals of the mutualist part in the course of their existences. Wright notes that models of biological mutualism tend to be similar qualitatively, in that the featured isoclines generally have a positive decreasing slope, and by and large similar isocline diagrams. Mutualistic interactions are best visualized as positively sloped isoclines, which can be explained by the fact that the saturation of benefits accorded to mutualism or restrictions posed by outside factors contribute to a decreasing slope.
The type II functional response is visualized as the graph of b a M 1 + a T H M {\displaystyle {\cfrac {baM}{1+aT_{H}M}}} vs. M .
Mutualistic networks made up out of the interaction between plants and pollinators were found to have a similar structure in very different ecosystems on different continents, consisting of entirely different species. [ 34 ] The structure of these mutualistic networks may have large consequences for the way in which pollinator communities respond to increasingly harsh conditions and on the community carrying capacity. [ 35 ]
Mathematical models that examine the consequences of this network structure for the stability of pollinator communities suggest that the specific way in which plant-pollinator networks are organized minimizes competition between pollinators, [ 36 ] reduce the spread of indirect effects and thus enhance ecosystem stability [ 37 ] and may even lead to strong indirect facilitation between pollinators when conditions are harsh. [ 38 ] This means that pollinator species together can survive under harsh conditions. But it also means that pollinator species collapse simultaneously when conditions pass a critical point. [ 39 ] This simultaneous collapse occurs, because pollinator species depend on each other when surviving under difficult conditions. [ 38 ]
Such a community-wide collapse, involving many pollinator species, can occur suddenly when increasingly harsh conditions pass a critical point and recovery from such a collapse might not be easy. The improvement in conditions needed for pollinators to recover could be substantially larger than the improvement needed to return to conditions at which the pollinator community collapsed. [ 38 ]
Humans are involved in mutualisms with other species: their gut flora is essential for efficient digestion . [ 40 ] Infestations of head lice might have been beneficial for humans by fostering an immune response that helps to reduce the threat of body louse borne lethal diseases. [ 41 ]
Some relationships between humans and domesticated animals and plants are to different degrees mutualistic. [ citation needed ] For example, domesticated cereals that provide food for humans have lost the ability to spread seeds by shattering , a strategy that wild grains use to spread their seeds. [ 42 ]
In traditional agriculture , some plants have mutualistic relationships as companion plants , providing each other with shelter, soil fertility or natural pest control . For example, beans may grow up cornstalks as a trellis, while fixing nitrogen in the soil for the corn, a phenomenon that is used in Three Sisters farming . [ 43 ]
One researcher has proposed that the key advantage Homo sapiens had over Neanderthals in competing over similar habitats was the former's mutualism with dogs. [ 44 ]
The microbiota in the human intestine coevolved with the human species, and this relationship is considered to be a mutualism that is beneficial both to the human host and the bacteria in the gut population. [ 45 ] The mucous layer of the intestine contains commensal bacteria that produce bacteriocins , modify the pH of the intestinal contents, and compete for nutrition to inhibit colonization by pathogens. [ 46 ] The gut microbiota, containing trillions of microorganisms , possesses the metabolic capacity to produce and regulate multiple compounds that reach the circulation and act to influence the function of distal organs and systems. [ 47 ] Breakdown of the protective mucosal barrier of the gut can contribute to the development of colon cancer . [ 46 ]
Every generation of every organism needs nutrients – and similar nutrients – more than they need particular defensive characteristics, as the fitness benefit of these vary heavily especially by environment. This may be the reason that hosts are more likely to evolve to become dependent on vertically transmitted bacterial mutualists which provide nutrients than those providing defensive benefits. This pattern is generalized beyond bacteria by Yamada et al. 2015's demonstration that undernourished Drosophila are heavily dependent on their fungal symbiont Issatchenkia orientalis for amino acids. [ 48 ]
Mutualisms are not static, and can be lost by evolution. [ 49 ] Sachs and Simms (2006) suggest that this can occur via four main pathways:
There are many examples of mutualism breakdown. For example, plant lineages inhabiting nutrient-rich environments have evolutionarily abandoned mycorrhizal mutualisms many times independently. [ 52 ] Evolutionarily, headlice may have been mutualistic as they allow for early immunity to various body-louse borne disease; however, as these diseases became eradicated, the relationship has become less mutualistic and more parasitic. [ 50 ]
Measuring the exact fitness benefit to the individuals in a mutualistic relationship is not always straightforward, particularly when the individuals can receive benefits from a variety of species, for example most plant- pollinator mutualisms. It is therefore common to categorise mutualisms according to the closeness of the association, using terms such as obligate and facultative . Defining "closeness", however, is also problematic. It can refer to mutual dependency (the species cannot live without one another) or the biological intimacy of the relationship in relation to physical closeness ( e.g. , one species living within the tissues of the other species). [ 10 ] | https://en.wikipedia.org/wiki/Protocooperation |
A protocrystalline phase is a distinct phase occurring during crystal growth , which evolves into a microcrystalline form. The term is typically associated with silicon films in optical applications such as solar cells . [ 1 ]
Amorphous silicon (a-Si) is a popular solar cell material owing to its low cost and ease of production. Owing to its disordered structure ( Urbach tail ), its absorption extends to the energies below the band gap, resulting in a wide-range spectral response; however, it has a relatively low solar cell efficiency . Protocrystalline Si (pc-Si:H) also has a relatively low absorption near the band gap, owing to its more ordered crystalline structure. Thus, protocrystalline and amorphous silicon can be combined in a tandem solar cell, where the top thin layer of a-Si:H absorbs short-wavelength light whereas the underlying protocrystalline silicon layer absorbs the longer wavelengths [ 2 ]
This crystallography -related article is a stub . You can help Wikipedia by expanding it . | https://en.wikipedia.org/wiki/Protocrystalline |
Protodeboronation , or protodeborylation is a chemical reaction involving the protonolysis of a boronic acid (or other organoborane compound) in which a carbon-boron bond is broken and replaced with a carbon-hydrogen bond. Protodeboronation is a well-known undesired side reaction , and frequently associated with metal-catalysed coupling reactions that utilise boronic acids (see Suzuki reaction ). [ 1 ] For a given boronic acid, the propensity to undergo protodeboronation is highly variable and dependent on various factors, such as the reaction conditions employed and the organic substituent of the boronic acid.
The deliberate protodeboronation of boronic acids (and derivatives) have been applied to some synthetic procedures, such as the installation of a stereospecific proton at chiral centers, [ 2 ] and also in purification procedures, such as the removal of unwanted regioisomeric boronic acid by-products. [ 3 ]
Recent mechanistic studies have revealed a variety of protodeboronation pathways in aqueous media, and have demonstrated the reaction pH (and subsequently the boronic acid speciation) to be an important factor in understanding the modes of protodeboronation. [ 4 ] [ 5 ]
One of the earliest reports of protodeboronation was made by Ainley and Challenger, who were the first researchers to explore the reactivity of boronic acids with common chemical reagents. [ 6 ] They reported the reaction of phenylboronic acid in water (140-150 °C) to afford the protodeboronated product, benzene , after 40 hours.
Initial synthetic applications of protodeboronation were found alongside the discovery of the hydroboration reaction, in which sequential hydroboration-protodeboronation reactions were used to convert alkynes or alkenes into the corresponding saturated compounds. [ 7 ] Beyond this synthetic application, protodeboronation was rarely noted or valued in other chemical processes throughout the early 20th century. However, in more recent years, protodeboronation has emerged as a problematic side reaction with many chemical processes that utilise boronic acids. In particular, boronic acids have become increasingly important reagents for the facile construction of carbon-carbon and carbon-heteroatom bonds via metal-catalysed cross-coupling reactions. This has resulted in an increased usage of boronic acids, and subsequently followed by an increased number of reports concerning problematic protodeboronation. Many boronic acids are now commercially available and many novel boronic acids and derivatives are constantly in pursuit.
Many efforts have been put towards mitigating undesired protodeboronation in cross-coupling reactions. Catalyst design and optimisation has led the way for very efficient systems that can undergo rapid catalytic turnover. [ 8 ] This increases the rate of productive reaction and thus subdues unwanted decomposition pathways such as protodeboronation. Cross-coupling reactions have also been accelerated with metal additives such as silver [ 9 ] [ 10 ] [ 11 ] [ 12 ] and copper. [ 13 ] [ 14 ]
Boronic acid derivatives have also been used to suppress protodeboronation. [ 15 ] MIDA boronate esters and organotrifluoroborates have both been utilised in "slow release" strategies, in which the reaction conditions are optimised to provide a slow release of boronic acid. This protocol has proved useful in the cross-coupling of some notoriously unstable boronic acids, such as the 2-pyridine boronic acid. [ 16 ] [ 17 ] This ensures that the boronic acid concentration is low during the cross-coupling reaction, which in turn minimises the potential for side reactions.
The mechanism of protodeboronation was initially investigated by Kuivila in the 1960s, long before the discovery of the Suzuki reaction and the popularisation of boronic acids . Their studies focused on the protodeboronation of some substituted aromatic boronic acids in aqueous conditions, and they reported the presence of two distinct mechanisms; a general acid-catalysed and a specific base-catalysed mechanism. [ 18 ] [ 19 ] The acid-catalysed process is dependent on a reaction between boronic acid and an acid, such as sulfuric acid . On the other hand, the base-catalysed process arises from a pre-equilibrium between boronic acid and hydroxide to form the corresponding boronate, this is usually followed by a rate-limiting reaction between boronate and water (acting as the proton source). Substrates that display only these two modes of protodeboronation (typically simple aromatic and alkyl boronic acids) are generally very stable in neutral pH solution, where both acid- and base-catalysed processes are minimised. For aromatic boronic acids bearing electron-withdrawing substituents, there is a competing dissociative mechanism involving generation of a transient aryl anion. These substrates are stabilized by acidic conditions. [ 5 ]
Basic heteroaromatic boronic acids (boronic acids that contain a basic nitrogen atom, such as 2-pyridine boronic acid) display additional protodeboronation mechanisms. [ 4 ] A key finding shows the speciation of basic heteroaromatic boronic acids to be analogous to that of simple amino acids , with zwitterionic species forming under neutral pH conditions. For the 2-pyridine boronic acid, the zwitterionic compound is responsible for its rapid protodeboronation under neutral pH, through a unimolecular fragmentation of the C-B bond. In fact, the addition of acid (H+) or hydroxide (OH-) acts to attenuate protodeboronation by shifting the speciation away from the reactive zwitterion.
It is important to note that not all basic heteroaromatic boronic acids are reactive through a zwitterionic intermediate. | https://en.wikipedia.org/wiki/Protodeboronation |
Protofection is a protein-mediated transfection of foreign mitochondrial DNA (mtDNA) into the mitochondria of cells in a tissue to supplement or replace the native mitochondrial DNA already present. The complete mtDNA genome or just fragments of mtDNA generated by polymerase chain reaction can be transferred into the target mitochondria through the technique. [ 1 ]
Scientists have hypothesized for the last couple of decades that protofection can be beneficial for patients with mitochondrial diseases. This technique is a recent development and is continuously being improved. As mitochondrial DNA becomes progressively more damaged with age, this may provide a method of at least partially rejuvenating mitochondria in old tissue, restoring them to their original, youthful function. [ 1 ] [ 2 ]
Protofection is a developing technique and is continuously being improved. A specific protein transduction system has been created that is complexed with mtDNA, which enables the mtDNA to move across the targeted cell's membrane and specifically target mitochondria. The transduction system used consists of a protein transduction domain , mitochondrial localization sequences, and mitochondrial transcription factor A . Each of these play a specific role in protofection:
This process can lead to an increase in the amount of mtDNA present in the mitochondria of the target cells. [ 3 ]
The transduction system has been tweaked and modified, since the first use of protofection. To shorten the name of the complex, which was previously called PTD-MLS-TFAM complex, it is now named MTD-TFAM. MTD stands for mitochondrial transduction domain and it includes the protein transduction domain and the mitochondrial localization sequences. [ 4 ]
One hypothesis for mitochondrial diseases is that mitochondrial damage and dysfunction play an important role in aging . Protofection is being researched as a possibly viable laboratory technique for constructing gene therapies for inherited mitochondrial diseases, such as Leber's hereditary optic neuropathy . Studies have shown that protofection can lead to improved mitochondrial function in targeted cells. [ 5 ] [ 6 ]
Protofection could be applied to modified or artificial mitochondria. Mitochondria could be modified to produce few or no free radicals without compromising energy production. Recent studies have demonstrated that mitochondrial transplants may be useful to rejuvenate dead or dying tissue, such as in heart attacks, for which the mitochondria is the first part of the cell that dies. [ 7 ]
This cell biology article is a stub . You can help Wikipedia by expanding it . | https://en.wikipedia.org/wiki/Protofection |
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