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10_1002_pro_3406 | methodology | analysis methods. The ProtaBank database, together with its analysis and visualization tools, will
help scientists gain insights into sequencewactivity and structurewactivity relationships, improve
our understanding of how proteins function, and ultimately facilitate the design of proteins with
new and improved prop... |
10_1002_pro_3406 | discussion | web interface and API for data searching and analysis. The design and workflow for ProtaBank
is summarized in Figure 1. Users can submit data into the database through the web interface;
access to external databases such as PubMed,20 the PDB, and UniProt are provided to facilitate
the entry of publication informatio... |
10_1002_pro_3406 | discussion | information. More advanced analysis and comparison tools are also available via the web Page 6 of 39
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7interface. For example, users can do a sequence search with the Basic Local Alignment Search
Tool (BLAST)21 and use visual... |
10_1002_pro_3406 | results | results), and the units associated with the numerical value (Fig. 2). ProtaBank also has separate
corresponding tables to represent computational protocols and derived quantities, and to store
qualitative data (e.g., folded/unfolded) or data expressed in terms of a range or limit (e.g., 20–30,
>100). In addition to ... |
10_1002_pro_3406 | experiments | data on the protein that was engineered (i.e., the PDB file, if available), and experimental gene
construct information. This information adds context and additional query and filter parameters
to the PE data. Nonwpublished PE studies can also be input in a similar fashion. In these cases,
the researchers and organi... |
10_1002_pro_3406 | results | Depositors of nonwpublished results may embargo the release of the data until publication.
The ProtaBank schema design incorporates two crucial elements: (1) the full amino acid
sequence of the protein is stored to facilitate comparison of mutants across different assays and
studies, and (2) for each assay, informat... |
10_1002_pro_3406 | results | 8we believe they are necessary to enable useful comparisons of results across different studies.
Our reasoning is as follows.
First, PE studies and databases13,16 typically describe a mutant by listing the changes to its
protein sequence relative to a specified starting sequence. However, the starting sequences used... |
10_1002_pro_3406 | results | each mutant confounds comparisons, as any differences in the reported results could be due to
differences in the background residues. ProtaBank addresses this issue by providing web forms
and an API that parses the input mutant information to return the full sequence so it can be stored
as such, allowing for a strai... |
10_1002_pro_3406 | results | techniques, which can greatly affect the results.22w24 The ProtaBank schema takes these issues
into account. As outlined above, the database uses the assay_expassay table to describe the Page 8 of 39
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9procedure that was used... |
10_1002_pro_3406 | methodology | relationships with a series of other tables (category, property, technique, units) that help
categorize and describe the many ways these properties can be measured. The category table
provides the general type of protein property that was engineered or studied (e.g., stability,
activity, binding). The property table... |
10_1002_pro_3406 | experiments | ProtaBank are listed in Supporting Information, Table S1. Commonly used experimental or
computational techniques are also provided to indicate how the property was assayed (e.g.,
circular dichroism, surface plasmon resonance). Note that the properties and techniques supplied
are not comprehensive, and users can ente... |
10_1002_pro_3406 | abstract | details (e.g., authors, title, journal, date, abstract) can be fetched from PubMed, and the protein
sequence can be retrieved from the PDB or UniProt. If available, structural data for the protein
can be fetched from the PDB. |
10_1002_pro_3406 | methodology | was engineered or studied, the specific property measured, the technique employed, and the units
used. All items except the assay name are specified by selecting from options in a dropwdown
menu. Additional details can be included if desired. By entering this information, assays can be
clearly defined and compared. ... |
10_1002_pro_3406 | abstract | for relevant studies queried by publication/study details (title, abstract, author), protein name,
PDB ID, UniProt accession number, or protein sequence, (2) identify data and mutants related to
a given protein sequence by BLAST search, (3) visualize mutational data mapped onto a threew
dimensional (3D) protein struc... |
10_1002_pro_3406 | discussion | screenshot in which study analysis tools were used to visualize mutational data on a 3D protein
structure. The visualizer is based on PV, an openwsource javascript protein viewer
(https://biasmv.github.io/pv/index.html) that was extended to allow mutations to be represented Page 11 of 39
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10_1002_pro_3406 | references | gradient, minimum, maximum, median, mean, proportion above a reference value, and median
deviation from a reference value. In the study depicted here, Jacquier et al. investigated the
effects of mutations on TEMw1 βwlactamase activity by computing the amoxicillin minimum
inhibitory concentration (MIC) score for ~990... |
10_1002_pro_3406 | discussion | Utility and Discussion
The following case studies demonstrate how ProtaBank search and analysis tools can aid in
analyzing and interpreting PE data.
Case study 1: Identify and compare data for a protein sequence
Before beginning any PE study, a review of existing literature on the protein of interest provides |
10_1002_pro_3406 | references | a useful reference point. Therefore, a simple but important application of ProtaBank is to identify
and compare previously measured properties of a given sequence. Because ProtaBank stores the
full sequence information for each mutant, a simple query on a specified protein sequence
retrieves all the relevant data fo... |
10_1002_pro_3406 | results | similar results when the temperature and pH were similar. These results suggest that the pH
and/or temperature can have a notable effect on ∆Gu. Thus, in order to make meaningful
comparisons of engineered mutants relative to the wild type, it is clearly important to select the |
10_1002_pro_3406 | experiments | assay parameters that can impact the results, and enable an informed evaluation of results
obtained under different assay conditions.
For theoretical and computational scientists, ProtaBank provides another valuable service— |
10_1002_pro_3406 | methodology | easy access to data sets that can be used to benchmark, test, and improve predictive methods. For
example, the experimental results provided in this case study could be used to test theoretical Page 13 of 39
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14methods aimed ... |
10_1002_pro_3406 | results | in the data reported for closely related sequences. By comparing results between a sequence and
its mutants, the effects of mutation at a given position can be determined. The knowledge gained
can then be used to guide the selection of positions and mutations in future engineering efforts.
In this case study, we use... |
10_1002_pro_3406 | abstract | that are closely related to wildwtype G β1. Summary information is displayed in a mutant
distribution heat map and a histogram showing the distribution of the number of mismatches
(Fig. 4). The heat map [Fig. 4(A)] shows the number of sequences containing a mutation to a
given amino acid at a given position; the wil... |
10_1002_pro_3406 | results | results
In this case study, we use additional features of ProtaBank's "Identify and analyze sequence
mutations" tool to perform further analyses on the closely related G β1 sequences retrieved in
Case study 2 above.
Plot one property vs. another
For any two measured properties, users can plot one property vs. anot... |
10_1002_pro_3406 | results | Compare assay results
A recently published study by Olson et al.38 used mRNA display and deep mutational scanning
to determine the fitness of all single and double mutants of G β1. The authors further calculated a
∆∆G predictor (∆∆ Gscreen), which used their fitness values to predict the ∆ G change in protein
stabi... |
10_1002_pro_3406 | results | comparing the predicted results to experimentally obtained ∆∆ Gs reported in the literature
(∆∆Gliterature ). ProtaBank provides a feature that allows this type of comparison to be done
quickly and easily. The “Compare assay to others by mutation” feature allows all the input
mutants for one assay to be searched for... |
10_1002_pro_3406 | related_work | information. All the results can then be further sorted and filtered by background sequence,
mutation, or study. We used this feature to reproduce the comparison of ∆∆ Gscreen to existing
biochemical measurements of ∆∆ G as shown in the Olson et al. study.38 First, we did a
"Compare assay to others by mutation" on t... |
10_1002_pro_3406 | related_work | these results to the set of 10 background sequences and single point mutants listed in the Olson et
al. study [Fig. 6(B)]. Our filtered results match the data in their paper exactly except for one Page 16 of 39
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17point—the m... |
10_1002_pro_3406 | results | This feature makes it easy to compare the results for the set of mutants in a given assay to
those from any other group of assays (the properties measured can be the same or different). This
allows one to see if new assay data is consistent with previously observed trends. It can also be
used to identify protein pro... |
10_1002_pro_3406 | experiments | study, we look at experimental data from the Olson et al. Gβ1 study described above38 by
mapping the effect of single mutations onto the crystal structure of the protein. By visualizing the
data in this way, trends associated with structural features become more obvious than when
viewed in a table or chart.
In the ... |
10_1002_pro_3406 | discussion | view this data in 3D with the protein visualizer, which is accessible via the study analysis page.
We could map the fitness data onto the G β1 backbone using the median deviation from the wildw
type value color scheme to help identify residue positions that are sensitive to mutation, as we
did for the βwlactamase stu... |
10_1002_pro_3406 | discussion | Further analysis and visualization capabilities are therefore provided. ProtaBank allows you
to save the data values from the selected color scheme in the occupancy column of the PDB file
so that other modeling or visualization software can be used. In this case study, we used visual
molecular dynamics (VMD)43 softw... |
10_1002_pro_3406 | discussion | structural analysis thus helps explain why these residues are particularly sensitive to mutation
and suggests that the observed sensitivity is likely due to disruption of the binding site rather
than a destabilization of the G β1 fold. |
10_1002_pro_3406 | conclusion | Concluding Remarks and Future Development
ProtaBank offers an easily accessible cloudwbased modern database for PE data. It emphasizes
the specification of detailed assay information and full protein sequences in an effort to ensure
that all collected data is not just stored, but that data from diverse studies are c... |
10_1002_pro_3406 | results | 19submissions directly from researchers, ProtaBank can incorporate the most recent results and be
managed with fewer resources. Although this requires some effort on the part of the individual
researcher, ProtaBank offers many benefits to submitters, including storing their data in an |
10_1002_pro_3406 | results | data, create plots and charts, and view results on the 3D structure. We have also started more
advanced integration with protein structural data to allow for data selection and filtering on
structural properties and to allow for computational predictions based on structural and sequence |
10_1002_pro_3406 | methodology | information. Future tools include incorporating computational methods to predict the effect of
mutations on protein properties such as stability, binding, and activity.
ProtaBank will provide a central location and valuable entry point for researchers to store,
retrieve, compare, and analyze PE data. It will make it... |
10_1002_pro_3406 | results | results to guide their designs and provide valuable data sets that theoreticians can use as
benchmarking cases in developing better predictive algorithms. We expect that ProtaBank will
serve a pivotal role in centralizing PE data and leveraging the increasingly large amount of |
10_1002_pro_3406 | discussion | mutational data being generated. ProtaBank and its analysis tools will accelerate our ability to
understand sequencewfunction relationships and greatly facilitate future protein design and
engineering efforts. Page 19 of 39
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... |
10_1002_pro_3406 | results | results obtained from a sequence search.
Acknowledgments
This work was supported by the National Institute of General Medical Sciences of the National
Institutes of Health under Award Number R44GM117961. The content is solely the
responsibility of the authors and does not necessarily represent the official views of... |
10_1002_pro_3406 | references | References
1. Goodwin S, McPherson JD, McCombie WR (2016) Coming of age: ten years of nextw
generation sequencing technologies. Nat Rev Genet 17:333w351.
2. Romero PA, Tran TM, Abate AR (2015) Dissecting enzyme function with microfluidicw
based deep mutational scanning. Proc Natl Acad Sci USA 112:7159w7164.
3. Chen ... |
10_1002_pro_3406 | discussion | Cochran JR (2016) Highwthroughput analysis and protein engineering using microcapillary
arrays. Nat Chem Biol 12:76w81.
4. Quan J, Saaem I, Tang N, Ma S, Negre N, Gong H, White KP, Tian J (2011) Parallel onw
chip gene synthesis and application to optimization of protein expression. Nat Biotechnol
29:449w452. Page 20... |
10_1002_pro_3406 | methodology | Highwresolution mapping of protein sequencewfunction relationships. Nat Methods 7:741w
746.
6. Hietpas RT, Jensen JD, Bolon DNA (2011) Experimental illumination of a fitness
landscape. Proc Natl Acad Sci USA 108:7896w7901.
7. Whitehead TA, Chevalier A, Song Y, Dreyfus C, Fleishman SJ, De Mattos C, Myers CA,
Kamiset... |
10_1002_pro_3406 | methodology | Methods 11:801w807.
9. Wrenbeck EE, Faber MS, Whitehead TA (2017) Deep sequencing methods for protein
engineering and design. Curr Opin Struct Biol 45:36w44.
10. Rose PW, Prlić A, Altunkaya A, Bi C, Bradley AR, Christie CH, Costanzo LD, Duarte JM,
Dutta S, Feng Z, Green RK, Goodsell DS, Hudson B, Kalro T, Lowe R, P... |
10_1002_pro_3406 | discussion | 21. Madden T. The BLAST Sequence Analysis Tool. In: Hoeppner M, Ostell J, Eds. (2013)
The NCBI Handbook [Internet]. National Center for Biotechnology Information, Bethesda,
MD, https://www.ncbi.nlm.nih.gov/books/NBK153387/ . Page 22 of 39
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This article is protected by copyright. All ri... |
10_1002_pro_3406 | discussion | 32. Choi EJ, Mayo SL (2006) Generation and analysis of proline mutants in protein G. Protein
Eng Des Sel 19:285w289.
33. Davey JA, Damry AM, Goto NK, Chica RA (2017) Rational design of proteins that
exchange on functional timescales. Nat Chem Biol 13:1280w1285.
34. Schaefer M, Sommer M, Karplus M (1997) pHwdependen... |
10_1002_pro_3406 | results | 26Table I. Assay Details Help Explain Differences in ∆Gu Results for wild-type G β1
Study Reference ∆Gu (kcal/mol)a Techniqueb T (°C)c pH
57 Choi and Mayo, 200632 5.9d Thermal denaturation, circular dichroism 25 5.5
61 Gronenborn et al., 199629 5.6 GdmCl denaturation, fluorescence 25 5.4
72 Frank et al. , 199530 4.... |
10_1002_pro_3406 | experiments | Figure 2. ProtaBank database schema showing the table for experimental data represented by a
number (data_expfdatum, blue header) and all tables with foreign key relationships to it. Each
table shows the field name (left) and the variable type for the field (right). Each datum in the
data_expfdatum table has a forei... |
10_1002_pro_3406 | experiments | (study_study) that organizes the context in which the experiments were performed, an assay table
(assay_expassay) that describes the procedure used to obtain the measurement, a sequence table
(sequence_complex) that holds the protein sequence of the mutant, and a units table (data_unit)
that describes the units of t... |
10_1002_pro_3406 | discussion | Figure 3. Screenshots of the web interface when using ProtaBank search and analysis tools. (A)
A textwbased search for "ubiquitin" returns a sortable table containing all studies with ubiquitin in
the protein name or study title. Clicking on the study ID at the left brings up the analysis page for |
10_1002_pro_3406 | discussion | that study. (B) The analysis page for a study on βwlactamase27 includes a protein visualizer in
which mutational results are mapped onto the protein structure according to the selected color
scheme. Here, Leu57 is highlighted in yellow and the single mutant data for that residue is Page 27 of 39
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10_1002_pro_3406 | references | than (blue) the value of the reference at that position (white).
Figure 4. Identifying and analyzing closely related mutants of G β1 in ProtaBank. (A) A BLAST
search of the ProtaBank database finds ~1.3 million sequences that are closely related to wildw
type G β1. The heat map shows the frequency of each residue at ... |
10_1002_pro_3406 | experiments | predictor values (∆∆ Gscreen) were plotted against experimental ∆∆ G values reported in the
literature (∆∆ Gliterature ). (A) Unfiltered search of ProtaBank database identifies 343 mutant
sequence pairs with both predicted and experimental ∆∆ G values. (B) Search filtered by the Page 28 of 39
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10_1002_pro_3406 | related_work | 29mutations and background sequences from the Olson et al. study yields 82 pairs, reproducing
their data. Note that ProtaBank identifies ~260 additional data points.
Figure 7. Comparing fitness and proximity to the binding site for G β1 point mutants. The
ProtaBank visualizer was used to map the Olson et al.38 fitne... |
10_1002_pro_3406 | discussion | red if more than 3.5 Å away. The structural analysis shows that most of the G β1 residues near
the binding interface are particularly sensitive to mutation. Page 29 of 39
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Figure 1. ProtaBank design. Users can interact wit... |
10_1002_pro_3406 | experiments | Figure 2. ProtaBank database schema showing the table for experimental data represented by a number
(data_expfdatum, blue header) and all tables with foreign key relationships to it. Each table shows the field
name (left) and the variable type for the field (right). Each datum in the data_expfdatum table has a foreig... |
10_1002_pro_3406 | references | greater than (blue) the value of the reference at that position (white).
193x256mm (300 x 300 DPI)
Page 32 of 39
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Figure 4. Identifying and analyzing closely related mutants of Gβ1 in ProtaBank. (A) A BLAST search of ... |
10_1002_pro_3406 | experiments | database identifies 343 mutant sequence pairs with both predicted and experimental ∆∆G values. (B)
Search filtered by the mutations and background sequences from the Olson et al. study yields 82 pairs,
reproducing their data. Note that ProtaBank identifies ~260 additional data points.
139x73mm (150 x 150 DPI)
P... |
verma-et-al-2024-investigation-of-rare-earth-element-binding-to-a-surface-bound-affinity-peptide-derived-from-ef-hand | introduction | ■INTRODUCTION
Rareearthelements (REEs)areessential inseveralemerging
technologies suchaswindturbines, electricvehicles, computer
memory, autocatalytic converters, magnetic resonance imaging,
andsmartphones.1BecauseREEsarevaluableandhavelimited
availability, therecovery oftheseelements fromindustrial waste
streamsisanat... |
verma-et-al-2024-investigation-of-rare-earth-element-binding-to-a-surface-bound-affinity-peptide-derived-from-ef-hand | methodology | Thereisanurgentneedtoidentifyefficientseparation methods
thatrecycleREEsfromwastestreamsandprovideasteady,
domestic sourceoftheseelements. Varioustechniques have
beenappliedtorecoverREEsfromthewastestreamssuchassolventextraction,3filtration,4ionexchange,5chemical precip-
itation,6adsorption,7andelectrochemical processe... |
verma-et-al-2024-investigation-of-rare-earth-element-binding-to-a-surface-bound-affinity-peptide-derived-from-ef-hand | methodology | biobased approach withselective adsorption ofREEsrequires
deepacquaintance withthesupportmaterials andtheligandof
choicesuchaspeptides thatcanbegraftedonthedesiredsurface
forselective separation ofREEs.Peptides aredesirable asthey
areshortchainsofaminoacids(lessthan50)andarehighly
tunableligandsthatareselective forions... |
verma-et-al-2024-investigation-of-rare-earth-element-binding-to-a-surface-bound-affinity-peptide-derived-from-ef-hand | experiments | Through isothermal titration calorimetry (ITC)experiments,
theyfoundthatthethermodynamic properties ofioncomplex-
ationstrongly varywithlanthanide ionsize,andmolecular
dynamics (MD)simulations revealed thatthehighbinding
affinityisachieved through complete iondehydration. In
anotherinvestigation, Xuetal.16derivedapepti... |
verma-et-al-2024-investigation-of-rare-earth-element-binding-to-a-surface-bound-affinity-peptide-derived-from-ef-hand | experiments | (ITC)experiments wereperformed todetermine the
thermodynamic parameters (KD,ΔH, ΔS,and ΔG)ofthe
interaction between thepeptides andionsinsolutions. QCM-D
wasconducted toshowthatsurface-bound peptides derived
fromlanmodulin canbindREEsandtocharacterize thesurface-
boundaffinityusingtheLangmuir adsorption modeltoestimate... |
verma-et-al-2024-investigation-of-rare-earth-element-binding-to-a-surface-bound-affinity-peptide-derived-from-ef-hand | experiments | particles wasdetermined inaproof-of-concept experiment.
Overall,thisstudycharacterizes theproperties ofsurface-bound
LanM1peptideforthefirsttimeanddemonstrates itspotential
utilityinREEseparation forfurther optimization and
exploration inthefuture.
■MATERIALS ANDMETHODS
Materials. Thepeptidesequences inthisstudywereder... |
verma-et-al-2024-investigation-of-rare-earth-element-binding-to-a-surface-bound-affinity-peptide-derived-from-ef-hand | experiments | experiments wasprocured fromAirgas.Goldnanoparticles withsize
lessthan100nm(powder), 99.9%trace,wereobtained fromSigma-
Aldrich.
PeptideDesign. Thepeptidesequences forbindingstudieswere
designed fromEF-hand loopIoflanmodulin. Thesequence intended
forREEbinding iscalledLanM1(Ac-CGGGDPDKDGTIDLKE-ACSAppliedMaterials &Inte... |
verma-et-al-2024-investigation-of-rare-earth-element-binding-to-a-surface-bound-affinity-peptide-derived-from-ef-hand | experiments | experiments, thesolutions weredegassed. Thefirstinjection pointwas
removed duetothedilution/mixing effectandthebaseline was
corrected priortofurtheranalysis. Thedataanalysiswasdonewith
Origin7.0usingtheonesetofsite-fitting modeltocalculate thebindingconstant (Kd),enthalpy (ΔH),andentropy (ΔS).Adjustments to
solutionpHw... |
verma-et-al-2024-investigation-of-rare-earth-element-binding-to-a-surface-bound-affinity-peptide-derived-from-ef-hand | experiments | experiments wereoperated inaflowmoduleat18°Candataflowrate
of150 μL/min. Thefrequency decrease andincrease reflectmass
accumulation andremovalonthesurface,respectively, anddissipation
changesshowtheviscoelastic characteristics oftheadsorbed material.
Afteracquiring astablebaseline withultrapure water,lanmodulin
peptide... |
verma-et-al-2024-investigation-of-rare-earth-element-binding-to-a-surface-bound-affinity-peptide-derived-from-ef-hand | experiments | Adsorption Experiments onGoldNanoparticles (GNPs). A
protocol wasprepared andusedtoestimate theadsorption ofREEson
LanM1-functionalized GNPs.Inallexperiments, roughly8to12mgof
GNPpowerwasaddedtoa2.0mLEppendorf tubeandtheexactweight
ofGNPswascalculated bytakingtheweightofthetubebeforeandafter
addingGNPs.Later,1mLofLanM1... |
verma-et-al-2024-investigation-of-rare-earth-element-binding-to-a-surface-bound-affinity-peptide-derived-from-ef-hand | methodology | usingthecolorimetric technique, theconcentration oftheREEwas
estimated. Control experiments without GNPwereperformed to
ensurethatnometalleachedfromthetubematerialintothesolution
andnoREEabsorbed onthetubesurface.Apictorialrepresentation of
thestep-by-step procedure usedforREEbindingongoldnanoparticles
(GNPs) isshownin... |
verma-et-al-2024-investigation-of-rare-earth-element-binding-to-a-surface-bound-affinity-peptide-derived-from-ef-hand | results | between 5.0and5.5throughout theexperiment.■RESULTS ANDDISCUSSION
Conformational Analysis andBinding SiteInvestiga-
tion.Boththepeptides, LanM1andscrambled LanM1, are
newlydesigned peptides, andaninitialbindinginvestigation was
neededtoidentifytheirabilitytobindREEionsinsolution.
Therefore, theabilitytobindtocerium(III)... |
verma-et-al-2024-investigation-of-rare-earth-element-binding-to-a-surface-bound-affinity-peptide-derived-from-ef-hand | experiments | usingcirculardichroism (CD)experiments, aswehavedone
withotherEF-hand peptidesequences previously.16Severalexperiments wereperformed bykeepingthepeptideconcen-
trationat100μMandvaryingthecerium(III) ionconcentration
withintherangeof100to1000 μM.Theeffectofion
concentration onthesecondary structure ofthepeptides was
obs... |
verma-et-al-2024-investigation-of-rare-earth-element-binding-to-a-surface-bound-affinity-peptide-derived-from-ef-hand | experiments | additional control experiment conducted onpolyproline,
whichdoesnotbindtocerium(III), showslittletonochange
in1HNMRpeaksandchemical shiftsascerium(III) isadded
(FigureS2).
Ascerium(III) isaddedtoLanM1,weobservebroadening of
peaksintherangesof0.75to1ppm,2.95to3.05,and3.75to
3.85ppm(gray-highlighted regionsinFigure4a,c,e... |
verma-et-al-2024-investigation-of-rare-earth-element-binding-to-a-surface-bound-affinity-peptide-derived-from-ef-hand | results | cerium(III), confirming CDandMDsimulation results.In
contrast, scrambled LanM1hasthesamesidechainsasLanM1
butdoesnotbindcerium(III), indicating theimportance ofthe
aminoacidsequence.
Figure5.ITCrawdata,Wiseman plot,andone-sitebindingmodelfitfor(a)LanM1withcerium(III) ions,(b)scrambled LanM1withcerium(III)
ions,(c)LanM1... |
verma-et-al-2024-investigation-of-rare-earth-element-binding-to-a-surface-bound-affinity-peptide-derived-from-ef-hand | experiments | Eu(III),andY(III))at303.15K.Inaddition, experiments on
scrambled LanM1withcerium(III) ionswereconducted to
serveasacomparison. Figure5a,bshowstheceriumtitration
curves(rawdata),isotherm, andone-sitebindingmodelfitfor
LanM1andscrambled LanM1,respectively. Thetitrationresults
forneodymium andeuropium ionswithLanM1areprov... |
verma-et-al-2024-investigation-of-rare-earth-element-binding-to-a-surface-bound-affinity-peptide-derived-from-ef-hand | results | findings. Theresultsarealsoinagreement withWeiandco-
workers23whofoundthatlanmodulin proteinimmobilized to
magnetic nanoparticles (MNP-LanM) didnotadsorbnon-
REEs.
WealsotestedLanM1bindingaffinitywithcerium(III) at
low-pH conditions. Thestudiesatlow-pH conditions are
relevanttocoalminedrainages wherethedischarges areat... |
verma-et-al-2024-investigation-of-rare-earth-element-binding-to-a-surface-bound-affinity-peptide-derived-from-ef-hand | discussion | Binding Analysis ofImmobilized Peptides. Tounder-
standthebindingaffinityoftheimmobilized peptidewithsome
REEsandcompeting non-REEs, aquartzcrystalmicrobalance
withdissipation (QCM-D) analysiswasperformed. Inatypical |
verma-et-al-2024-investigation-of-rare-earth-element-binding-to-a-surface-bound-affinity-peptide-derived-from-ef-hand | experiments | QCM-D experiment, ultrapure waterisintroduced onthegold
sensortogenerate abaseline. Onceastablebaseline is
established, peptide solution ofknownconcentration is
introduced, andanegative shiftinfrequency isobserved,
whichisproportional tothehydrated massloadingofthe
peptideonthesurface.Rinsesareoftenemployed toremove
we... |
verma-et-al-2024-investigation-of-rare-earth-element-binding-to-a-surface-bound-affinity-peptide-derived-from-ef-hand | experiments | peptides, control experiments wereconducted, whereno
bindingwasobserved, asshowninFigureS8.Ourprevious
studyshowsthatwhennopeptidewaspresentontheQCM
sensor,noirreversible bindingwasobserved forphosphate,17
indicating thatallphosphate bindingseenisduetointeractions
withpeptide−cerium films.Wealsoobserved thathigher
LanM... |
verma-et-al-2024-investigation-of-rare-earth-element-binding-to-a-surface-bound-affinity-peptide-derived-from-ef-hand | experiments | respectively. Figure6displays experiments performed atthe
lowestREEconcentrations wherebindingwasobserved, where
thebluelinecorresponds tothefrequency shift,whilethered
linecorresponds tothedissipation shift.Ultrapure waterserved
asthebaseline fortheexperiment, whichwasimmediately
followed bya6.0μMpeptidesolution. Anex... |
verma-et-al-2024-investigation-of-rare-earth-element-binding-to-a-surface-bound-affinity-peptide-derived-from-ef-hand | experiments | cerium(III).57Therefore, similarQCM-D experiments were
performed withtwonon-REE Ca(II)andCu(II)competitor
ions.Theresultsofthesurface-bound LanM1peptidewiththese
ionsareprovided inFigureS11.Likethebulk,theLanM1
peptidedoesnotshowaffinityforeithercalciumorcopperions.
Evenatahighercompetitor ionconcentration of29.0 μM,no... |
verma-et-al-2024-investigation-of-rare-earth-element-binding-to-a-surface-bound-affinity-peptide-derived-from-ef-hand | experiments | QCM-D experiments wereperformed atvariousREEion
concentrations toestimate surface-bound LanM1dissociation
constants forCe(III)andNd(III). Inacidminedrainage, the
concentration ofCe(III)andNd(III)isthehighestamongother
REEs,58whichmakesthispairaparticular interestforthat
application. TheLangmuir adsorption isotherm mode... |
verma-et-al-2024-investigation-of-rare-earth-element-binding-to-a-surface-bound-affinity-peptide-derived-from-ef-hand | experiments | QCM-D experiments todescribe theadsorption process.59,60
Although, itisanidealmodelthatmaynotfullycapturethe
complexity ofadsorption, itcanstillprovidevaluable insights
intotheadsorption behavior. Themodelisgivenas
q
RC
C Ke
T e
e d =
+
(2)
whereqeisthephosphate loading(mol/cm2),RT(mol/cm2)is
theLangmuir constant rela... |
verma-et-al-2024-investigation-of-rare-earth-element-binding-to-a-surface-bound-affinity-peptide-derived-from-ef-hand | experiments | experiment. Becauseofthehigherror,itisdifficulttodetermine
ifthereisashiftforNd(III) uponpeptideimmobilization.
However, theseresultsshowforthefirsttimethatsurface-bound
LanM1peptides bindREEs,whichmotivates futurestudiesto
explorehowimmobilization impactsthetrendsinKd. |
verma-et-al-2024-investigation-of-rare-earth-element-binding-to-a-surface-bound-affinity-peptide-derived-from-ef-hand | discussion | Selectivity Analysis ofImmobilized Peptides. Ina
practical separation scheme,immobilized peptides areexposed
toamixtureofREEs.Separation occursifthepeptidehasa
higheraffinity(lowerKd)foroneREEoveranotherduetothe
preferential partitioning oftheREEionontothesolidpeptide-
containing phase.Fordilutesolutions, selectivity c... |
verma-et-al-2024-investigation-of-rare-earth-element-binding-to-a-surface-bound-affinity-peptide-derived-from-ef-hand | discussion | discussion hereinwillrefertogeneraltrendsandorderof
magnitude differences, recognizing thecaveatthatthe
lanmodulin proteindatainTable2represent amorechallenging
separation ofneighbors Nd/PrthanNd/Ce�immobilized
LanM1peptides exhibitselectivity thatisonparwiththeLanM
proteinat0.5.Thisisinteresting becausetheKdoftheLanM
... |
verma-et-al-2024-investigation-of-rare-earth-element-binding-to-a-surface-bound-affinity-peptide-derived-from-ef-hand | experiments | Severalequilibrium adsorption experiments ofLanM1-GNPs
atdifferent concentrations wereperformed forREEsCe(III),
Nd(III), Eu(III), andY(III)andnon-REEs Ca(II)andthe
estimated adsorption capacity fortheionsatdifferent
concentrations isreported inTableS5.Acomparison ofaverage
equilibrium adsorption capacities ofREEsandcom... |
verma-et-al-2024-investigation-of-rare-earth-element-binding-to-a-surface-bound-affinity-peptide-derived-from-ef-hand | experiments | experiment wasperformed using20mM2-(N-morpholino)-
ethanesulfonic acid(MES)bufferatpH5.0with2.5mg/mL
MNP-LanM and ∼62.93 μMTb(III),23whichhassome
similarities toourexperiment inDIwateratpH5.0−5.5with
5−8mgGNP/mL and20−125 μMREE,servingasauseful
comparison. Intheirstudy,theloadingonMNPswas2.67 ±
0.15 μmolLanMSpyCatcher/... |
verma-et-al-2024-investigation-of-rare-earth-element-binding-to-a-surface-bound-affinity-peptide-derived-from-ef-hand | experiments | TheresultsintheQCM-D experiment showaminimum
dissipation duringpeptideloading,whichindicates arigidlayer
formation andlowwatercontent; however, thedegreeof
hydration ofthelayerisunknown. Nonetheless, themolar
peptideloadingonGNPsisofsimilarorderofmagnitude tothat
oftheSpyCatcher MNPs.Whenadsorbing inamonolayer, the
mol... |
verma-et-al-2024-investigation-of-rare-earth-element-binding-to-a-surface-bound-affinity-peptide-derived-from-ef-hand | conclusion | ■CONCLUSIONS
Insummary, LanM1peptidederivedfromtheEF-hand loop1of
lanmodulin wastestedforitsbindingaffinityfordifferent REEs,
Ce(III),Nd(III), Eu(III),andY(III),bothinthebulkandwhen
boundtoagoldsurface usingbothexperimental and
computational techniques. TheabilityofCe(III)ionstobind
withLanM1andscrambled LanM1wasconfir... |
verma-et-al-2024-investigation-of-rare-earth-element-binding-to-a-surface-bound-affinity-peptide-derived-from-ef-hand | discussion | andnuclearmagnetic resonance spectroscopy (NMR)analysis.
Isothermal titration calorimetry (ITC)resultsshowedsponta-
neousbindingofallREEstotheLanM1peptide.Thebinding
wasenthalpically disfavored butwasdrivenbyapositivechange
inentropy.Thedissociation constants fromITCwerecorrelated
totheionicradiusofREEswithaKdof3.84±1.... |
verma-et-al-2024-investigation-of-rare-earth-element-binding-to-a-surface-bound-affinity-peptide-derived-from-ef-hand | discussion | Quartzcrystalmicrobalance withdissipation (QCM-D) analysis
wasperformed tocharacterize thebindingofREEsandnon-
REEstosurface-immobilized LanM1. Thedissociation con-
stantsobtained fromLangmuir modelfittingsuggested thatthe
bindingaffinities forCe(III)andNd(III) withsurface-bound
LanM1 wereestimated toberoughly ∼0.9and1... |
verma-et-al-2024-investigation-of-rare-earth-element-binding-to-a-surface-bound-affinity-peptide-derived-from-ef-hand | results | ITCresults,QCM-D results,Scatchard plot,Langmuir
modelregression data,summary ofadsorption metrics
usedintheselectivity analysis, estimated adsorption
capacities forREEsusingGNPs,andderivation foratwo- |
verma-et-al-2024-investigation-of-rare-earth-element-binding-to-a-surface-bound-affinity-peptide-derived-from-ef-hand | discussion | stepScatchard analysis. Arepository oftheGROMCAS
inputfilesiscreatedonGitHub: https://github.com/
gev28/Gromacs_input_file (PDF)
■AUTHOR INFORMATION
Corresponding Author
JulieRenner −Department ofChemicalandBiomolecular
Engineering, CaseWesternReserveUniversity,Cleveland,Ohio
44106,UnitedStates;
orcid.org/0000-0002-61... |
verma-et-al-2024-investigation-of-rare-earth-element-binding-to-a-surface-bound-affinity-peptide-derived-from-ef-hand | acknowledgment | Theauthorsdeclarenocompeting financial interest.■ACKNOWLEDGMENTS
TheauthorsthankMolecular Biotechnology CoreatCleveland
ClinicandthankDr.Smarajit Bandyopadhyay, Director,
Molecular Biotechnology Core,forhishelpwithCDandITC |
verma-et-al-2024-investigation-of-rare-earth-element-binding-to-a-surface-bound-affinity-peptide-derived-from-ef-hand | experiments | experiments. AspecialthankstoSayaniBiswas,agraduate
student intheDepartment ofChemical andBiomolecular
Engineering atOhioStateUniversity, forvalidating MD
calculations. TheauthorsalsothankDr.JeffreyCapadona, aprofessor intheDepartment ofBiomedical Engineering atCase
Western Reserve University, forallowing ustouseQCM-D
... |
verma-et-al-2024-investigation-of-rare-earth-element-binding-to-a-surface-bound-affinity-peptide-derived-from-ef-hand | references | Technology.■REFERENCES
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