Electrostatic Tethering of the Intrinsically Disordered Domain with - Singh et al. - 2021 - Unknown

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pubs.acs.org/JPCLLetterUltrasensitiveCharacterizationofthePrionProteinbySurface-EnhancedRamanScattering:SelectiveEnhancementviaElectrostaticTetheringoftheIntrinsicallyDisorderedDomainwithFunctionalizedSilverNanoparticles∥∥SwapnilSingh,AishwaryaAgarwal,AnamikaAvni,andSamratMukhopadhyay*CiteThis:J.Phys.Chem.Lett.2021,12,3187−3194ReadOnlineACCESSMetrics&MoreArticleRecommendations*sıSupportingInformationABSTRACT:Surface-enhancedRamanscattering(SERS)circumventstheinherentinsensitivityofRamanspectroscopyandoffersapowerfultoolfortheultrasensitivedetectionandcharacterizationofbiomoleculesatlowconcentrations.HereweshowthatSERSviaelectrostatictetheringbetweensurface-modifiednegativelychargedsilvernanoparticlesandhighlypositivelychargedintrinsicallydisorderedN-terminaldomainoftheprionproteinallowshighlysensitiveandreproducibleproteindetectionandcharacterizationataslowashundredsofnanomolarproteinconcentrations.Thesemeasurementspreferentiallyilluminateaselectivepartoftheproteinduetoasharpdependenceofthenear-fieldintensityonthedistancebetweenthenanoparticlesurfaceandtheprotein.Wealsodemonstratethatbyshorteningthelengthofthedisorderedtailitispossibletoachieveadomain-selectiveRamanenhancementtostudytheC-terminalglobulardomain.Ourtether-length-dependentSERSmethodologywillserveasapotent,noninvasive,andlabel-freestrategytodetectandcharacterizeawiderangeofproteinspossessingdisorderedsegments.onformationalconversionoftheprionprotein(PrP)VibrationalRamanspectroscopyisapowerfultooltoCfromanα-helix-richcellularform(PrPc)toamisfolded,characterizethevibrationalmodesthatcanprovideawealthofaggregation-prone,β-richscrapieform(PrPSc)isassociatedconformationalinformationonproteinandotherbiomole-14−18withaclassofdeadlytransmissibleneurodegenerativecules.However,theinherentinsensitivityofRaman1,2scatteringposesasignificantchallengeincharacterizingthediseases.Creutzfeldt-Jakobdisease,kuru,fatalfamilial19insomnia,andGerstmann−Straussler̈−Scheinker(GSS)syn-structuralstatesofproteinatlowconcentrations.Todromearehumanpriondiseases,whereasscrapieandbovinecircumventthisissue,surface-enhancedRamanscatteringDownloadedviaUNIVOFNEWMEXICOonMay16,2021at08:41:32(UTC).spongiformencephalopathyor“madcowdisease”arefoundin(SERS)hasemergedasapromisingtoolforitsremarkableanimals.1−4RecentstudieshaveprovidedkeystructuralchemicalandelectromagneticenhancementofRamansignalsinsightsintothesetransmissibleprionaggregates.5−8Inbycreatinghotspotsinthevicinityofmetalnanoparticles20−26Seehttps://pubs.acs.org/sharingguidelinesforoptionsonhowtolegitimatelysharepublishedarticles.additiontohigh-resolutionstructuralstudiesofinfectious(NPs)enablingevensingle-moleculemeasurements.Theprions,itisimportanttodetectPrPinbiologicalfluids.directlabel-freeSERStechniqueisasimple,sensitive,cost-AlthoughPrPisprimarilyconfinedtothebrainandthecentralefficient,robust,andnonperturbativeapproachtodetectandnervoussystem,aminusculequantityofPrPisshowntobecharacterizeproteinsinanaqueousmedium.However,sincepresentinmanytissuesandbiologicalfluidsevenattheearlythemetallicsubstratesarerequiredforSERSmeasurements,presymptomaticstageofthedisease.9,10Therefore,thereisaeffortshavebeendirectedtoprepareSERSsubstrateswithpotentialriskofpriontransmissionviabloodtransfusion.11increasingenhancementandreproducibility.PreviousSERSstudieshavedetectedthePrPsignalwithconcentrationaslowHowever,detectionorconfirmationofitspresenceintheas100pM,butinsuchcases,theexperimentswereperformedbloodisdifficultevenbyadvancedmethodssuchaseitherusingalabel-basedapproachorwerecarriedinadriedfluorescenceimmunoassay,radioimmunoassay,enzyme-linkedimmunosorbentassay,andproteinmisfoldingcyclicamplifi-cation,anditstillreliesonpost-mortemimageanalysisofReceived:January23,2021degeneratetissues.12,13Moreover,todate,thereisnospecificAccepted:March16,2021biochemicalassayforearly,sensitive,andnoninvasivePublished:March24,2021diagnosisofthedisease.Therefore,thereisapressingneedtoutilizesensitivetechniquesfortheultrasensitive-detectionofPrPinvariousbiologicalfluids.©2021AmericanChemicalSocietyhttps://doi.org/10.1021/acs.jpclett.1c002403187J.Phys.Chem.Lett.2021,12,3187−3194

1TheJournalofPhysicalChemistryLetterspubs.acs.org/JPCLLetterFigure1.(A)Schematicrepresentationofthehumanprionprotein(PrP)indicatingvariousdomainsandsegments.(B)Aminoacidsequenceofthefull-lengthprion,PrP(23−231).Thepositivelyandnegativelychargedaminoacidsarehighlightedinblueandred,respectively.(C)NMRstructureofhumanPrP(90−231)(PDBID:2LSB)generatedusingPyMOL(Schrödinger,LLC,NewYork).Figure2.(A)SEMimageofsilvernanoparticles(AgNPs).(B)HistogramforNPssize-distributionderivedfromtheSEManalysis.(C)UV−visspectrumforiodide-modifiedsilvernanoparticles(AgIMNPs)(olive)andAgIMNPsinthepresenceofPrP(23−231)(blue).(D)AFMimagesofAgIMNPsandAgIMNPs+PrPindicatingnanoparticleaggregation.Thecorrespondingheightprofilesarealsoshown.27−30stateoftheprotein.Additionally,thereareafewreportssolution-basedSERSdetectionofPrPremainelusive.Inthis31onthedetectionofPrPindifferentcelllines;however,thework,wehaveutilizedSERStodetectandcharacterizethemoleculardesignandthemechanisticunderstandingforthehumanPrPinsolutionmaintainingitsnativestructureunder3188https://doi.org/10.1021/acs.jpclett.1c00240J.Phys.Chem.Lett.2021,12,3187−3194

2TheJournalofPhysicalChemistryLetterspubs.acs.org/JPCLLetterphysiologicallyrelevantconditions.Whileachievingaproteinmonolayercoatingandsuggestedtheabsenceofany39,42RamansignalenhancementbymodifyingtheshapeandsizeofaggregatedNPspostfunctionalization(FigureS3A).23,32,33NPsconstitutesamajorongoingeffort,theinfluenceofWhilerecordingSERSspectra,weonlyobservedintensedifferentstructuralelementsofproteinsintheadsorptiononpeakscorrespondingtometalhalidesat116,163,and241thenanoparticlesurfaceandtheproteinSERSspectraremaincm−1forsilveriodide(AgI),silverbromide(AgBr),andsilver42poorlyunderstood.chloride(AgCl),respectively.TheabsenceofpeaksHumanPrPisaC-terminallyglycophosphatidylinositol-correspondingtocitrateindicatedasuccessfulsurface-anchoredcell-surfaceproteinthatispredominantlyα-helicalmodificationofNPs(FigureS3B).Next,wecentrifugedtheandiscomposedoftwodistinctregions(Figure1).Initshalide-modifiedNPstoremoveanyresidualimpuritiesornativeform,afterthecleavageofthesignalpeptide,theN-citratepresentinthecolloidalsolutiontominimizeanyterminaldomain(23−127)isintrinsicallydisorderedandinterferencewiththeproteinRamansignals.Aftercentrifuga-carriesanetpositivecharge.Onthecontrary,theC-terminaltion,theUV−visabsorbancebandofanyofthefunctionalizeddomainiswell-structuredwiththreeα-helices(144−154,NPsdidnotshowanychange;however,theSERSspectrafor173−194,and200−228),ashortantiparallelβ-sheet,andabromide-andchloride-coatedNPsstartedshowingtracesofsingledisulfidelinkagebetweentwocysteinesat179and214citratepeaks,whichweremaskedearlier,indicatingthe34,35(Figure1).AcloserlookattheaminoacidsequenceofunstablehalidecoatingontheNPs.ThispointstowardthePrPrevealedalargenumberofpositivelychargedaminoacidstronginteractionbetweensilverandiodidewhichareasoftresiduesintheN-terminaltailwithachargeof∼+14atneutralLewisacid−basepair,asaresultofwhichiodide-modifiedAgpH.We,therefore,hypothesizedthatthepositivelychargedNPsarebettercoatedascomparedtobromideandchloride.intrinsicallydisorderedN-terminaldomainofPrPcanactasanTherefore,wechoseiodide-modifiedAgNPs(AgIMNPs)anchortonegativelychargedsurface-modifiedmetalnano-forourfurtherstudies.WethenrecordedtheZeta(ζ)particlesenablingapotentdetectionandcharacterizationofpotentialforAgIMNPswhichwasfoundtobe−33mVtheproteinusingSERSspectroscopy.Wefurtherpostulated(FigureS4).Also,theSEMimagingofhalide-modifiedNPsthat,duetoasharpdistancedependenceinthefielddidnotshowanymorphologicaldifferenceascomparedtothatenhancementbymetalnanoparticles,wewouldbeabletoofthenonmodifiedAgNPs(FigureS5).AgIMNPswerethenselectivelymonitordifferentsegmentsoftheproteinbyusingamixedwithdifferentconcentrationsofPrP(23−231)atroomvariedlengthofthedisorderedN-terminaltailtetheredtothetemperature,followingwhichthecolorofthecolloidalsolutionnanoparticles.Thisisbecausetheintensityofthenear-fieldchangedimmediatelyfromolivegreentoarmygreen,intensitydecayssharplyasafunctionofthe12thpoweroftheindicatingtheaggregationofNPs.Also,werecordeddistancebetweenthesurfaceofthenanoparticleandtheabsorptionspectraforthiscolloidalsolution,whichshowed36vibrationalprobe.anadditionalbroadbandat700−800nmreflectingcollectiveInordertotesttheaforesaidhypothesis,theveryfirstsurfaceplasmonoscillationcausedbytheaggregationofNPsconsiderationwastochooseasensitiveSERSsubstrate.WemediatedbyPrP(Figure2C).Ingeneral,mostofthepracticalchosequasi-sphericalsilvernanoparticles(AgNPs)asaSERSmeasurementsrequiretheadditionofcertainagentsto37plasmonicsubstrateduetotheirhighestSERSactivity.AsainduceaggregationofNPs.Interestingly,weobservedthattheprelude,AgNPsweresynthesizedbychemicalreductionusingproteinitselfwasabletoinducetheaggregationoftheNPs,38,39citrate.Citrateactsasbothareducingagentandaresultingintheformationofalargenumberofhotspotswithin40stabilizingagentarrestingthecolloidgrowth.TheUV−viswhichproteinmoleculesgettrapped.Therefore,wewereableabsorptionspectrawererecordedtoevaluatetheshapeandtoperformSERSmeasurementsonourproteinofinterestsizeofAgNPs.Weobservedasingleabsorptionbandlocatedwithoutanyexternalagentstoinducenanoparticleaggregationat∼412nm,suggestingsmallsilvernanospheres(∼40−50ashasbeenalsoobservedpreviouslyforotherpositively4139,43nm)(FigureS1).Wefurtherverifiedthesizeandshapeofchargedproteins.AstrongelectrostaticinteractionoftheAgNPsusingscanningelectronmicroscopy(SEM)(FigurepositivelychargedN-terminaltailoftheproteinwithnegatively2A,B).Theaveragesizeofquasi-sphericalAgNPsobservedchargedNPsdrivesproteinadsorption,resultinginthewas∼49±12nm,whichisinagreementwiththesizeaggregationofNP.Toconfirmprotein-mediatednanoparticleobtainedfromabsorptionstudies.Furthermore,toassesstheaggregation,wecarriedoutatomicforcemicroscopy(AFM)stabilityofNPs,werecordedtheζpotentialofAgNPs,whichimagingtovisualizetheNPsinthepresenceandabsenceofwasfoundtobe−40±1mV,corroboratingthepreviousprotein.TheAFMimagesclearlyshowedaggregatesofthe39,42results(FigureS2).NPsinthepresenceofPrP(Figure2D).SincethedirectinteractionoftheproteinwiththemetalOurmaingoalwastodetectandcharacterizePrPinitssubstratesmightalterthenativestructureoftheprotein,wenativeformusinglabel-freeSERS.InordertomaintainthenextfunctionalizedthesurfaceofAgNPswithhalidesthatcannativestructuralintegrityoftheprotein,weperformedsolutionhaveseveraladvantages.ThehalidemonolayermaskstheSERSmeasurementstominimizehigh-energylaser-inducedoverwhelmingcitratepeaksandotherinterferencesbydamagebydissipatingtheexcessivelocalizedheatgenerated39displacingthecitrateandotherimpuritiespresentonthenearhotspotstotheentiresolution.Additionally,toavoidsurfaceofbareNPs.Moreimportantly,itpreventsdirectanyintrinsicfluorescencefromaromaticaminoacids,a785nm44contactbetweenproteinmoleculesandNPstherebyretaininglaserwaschosenasanexcitationsource.Beforecarryingout39,42thenativestructureoftheprotein.WefunctionalizedAgtheSERSmeasurements,wefirstrecordedthebulkRamanNPswiththreedifferenthalides,suchaspotassiumiodidespectrumofPrPbyusingdropdepositionRamanspectrosco-(KI),potassiumbromide(KBr),andpotassiumchloridepy.TheRamanshiftassignmentsrevealedthestructural(KCl).39,42elementsinPrP(amideI,1620−1700cm−1,andamideIII,TheUV−visabsorptionbandafterhalidemodification1220−1300cm−1)andseveralcharacteristicside-chain19,45−47exhibitedasmallred-shiftthatisexpectedduetohalidevibrations(Figure3A).TheamideIregionrepresents3189https://doi.org/10.1021/acs.jpclett.1c00240J.Phys.Chem.Lett.2021,12,3187−3194

3TheJournalofPhysicalChemistryLetterspubs.acs.org/JPCLLetterFigure3.(A)BulkRamanspectrumofPrP(23−231)indicatingmajorvibrationalfeatures(seeTableS2forinterpretationsofthepeaks).(B)DeconvolutionoftheamideIregion(1630−1720cm−1)bandforPrP(23−231).Theareaunderthecurvesrepresentsthepercentagecontributionofthesecondarystructuralelements:α-helix:magenta,randomcoil:cyan.(SeeTableS1forpercentagecontributionsestimatedfromdeconvolution.)Theblacklineistherawdataandtheredlineisthecumulativefit.Figure4.SolutionSERSspectraandbulkRamanspectra(black)ofPrP(23−231)(A),PrP(Y145Stop)(B),andPrP(90−231)(C).ThesolutionSERSspectrafordifferentproteinconcentrationsareshowninred(25μM),blue(5μM),magenta(500nM),andolive(250nM).SeeSupportingInformationfordetailsofdataacquisition,processing,andanalysis.primarilythecarbonyl(−CO)stretchwithsomecontribu-AftercarryingoutbulkRamanmeasurementsandassign-tionsfrom−C−Nstretchand−C−C−Ndeformationandisments,wenextsetouttoperformSERSstudiesonPrP(Figuresensitivetotheproteinsecondarystructures.Thedeconvolu-4A).Wewereabletoobtainhigh-qualitySERSspectraatlowtionoftheamideIregionofthespectrumallowedustomicromolardowntohundredsofnanomolarPrPconcen-estimatethecontentofdifferentsecondarystructures(∼27%trations.ThesespectracorroboratedthebulkRamanspectrain48α-helixand∼73%extended/coils;Figure3BandTableS1).termsofthepeakpositionandtherelativeintensityandwereThesecondarystructuralcontentsestimatedfromRamanfurtherestablishedbyplottingcorrelationspectra(FigureS7).spectroscopyalsocorroboratedtheresultsobtainedfromfar-TheelectrostatictetheringbetweenN-terminalIDRwiththeUVcirculardichroism(CD)spectroscopy(FigureS6).WenegativelychargedNPsenabledustodetectPrPatlowwereabletoassignseveralprominentside-chainmarkerssuchconcentrationsandthedetectionlimitwasapproximately250astryptophan(Trp),phenylalanine(Phe),andtyrosine(Tyr).nM.ManySERSsignalsat<250nMwerepresumablyduetoInbulkRamanmeasurements,wewereunabletoobserveafewerhotspotsatlowerproteinconcentrations.Theanalyticalwell-knowndisulfide(−S−S−)vibrationalsignaturethatenhancementfactorwasestimatedusingapreviouslydescribednormallyappearsintherangeof∼500−550cm−1.Wemethodandwasfoundtobeapproximatelyintheorderof10651presumethatthispeakwasmaskedunderabroadbandindicatinghighsensitivityofourmeasurements.TheSERSobservedat∼530cm−1correspondingtobackbonedeforma-spectrumwascarefullyanalyzedandtheobservedshiftsbothtionsthatarecommonlyobservedindisorderedpolypeptidesforbulkRamanandSERSweretabulated(Figure4AandowingtothepresenceofalargenumberofglycinesandTableS2).ThebackboneamidegroupmarkerssuchasamideI49,50alanines.andamideIIIregionsaswellasaromaticside-chainmarkers3190https://doi.org/10.1021/acs.jpclett.1c00240J.Phys.Chem.Lett.2021,12,3187−3194

4TheJournalofPhysicalChemistryLetterspubs.acs.org/JPCLLettersuchasPhe,Trp,andTyrwerecarefullyinspectedand(Y145Stop)(C-terminaltruncation)andPrP(90−231)(N-analyzed.TheSERSspectrumforPrP(23−231)wasterminaltruncation).ThesePrPconstructsretainedtheabilitydominatedbybandscorrespondingtoamideI,1658/1683topromoteagglomerationofsurface-modifiedsilvernano-cm−1;amideIII,1236/1257/1267cm−1;Phe,1005cm−1;Trp,particles(AgIMNPs)asevidentbyabsorptionandAFM1551and758cm−1andaFermidoubletat1344/1359cm−1;studies(FiguresS9andS10).ThisnanoparticleaggregationisTyr,643cm−1andaFermidoubletat829/854cm−1;andC−thoughttobedrivenbyelectrostaticsequestrationofHbending,1447cm−1.Besides,thepresenceofpositivelynegativelychargednanoparticlesbytheproteinconstructschargedresidues(Lys,Arg,andHis)facilitatedtheadsorptionsincePrP(Y145Stop)andPrP(90−231)alsocarryapositiveoftheunstructureddomaintothesurfaceofNPsresultinginacharge(+13.6and+4.3,respectively)underourexperimentalsignificantenhancementofthepeakat∼1581and1066cm−1condition.WefirstperformedthebulkRamanmeasurementscorrespondingtohistidineandC−Cstretching,respectively.andtheobtainedsecondarystructuralcontentsagreedwell34ThedeconvolutionoftheamideIandamideIIIregionofthewiththosereportedearlier(FigureS11;TableS1).WenextspectraandpercentageanalysisfortheSERSpeakofPrPrecordedtheSERSspectraforPrP(Y145Stop)thatmatchedrevealthepresenceofprimarilyα-helicesanddisordered/coilswiththebulkRamanspectralsignaturesasevidencedbytheasalsoshownpreviouslyusingNMRstudies(TableS1andcorrelationgraph(Figures4BandS12A).TheSERSspectrum34S2).TheuniquestructureofPrPrepresentsajuxtapositionforPrP(Y145Stop)wasdominatedbybandscorrespondingtoofdisorderedandordereddomains(Figure1C).TheamideIamideI,1662/1685cm−1;Phe,1004cm−1;Trp,1552and758deconvolutionindicatedthatthereisanenhancementfromcm−1anditsdoubletat1340/1360cm−1;Tyr,644cm−1andboththedisorderedN-terminalandorderedC-terminal;itsdoubletat825/853cm−1;andC−Hbending,1445cm−1however,uponcarefulinspectionandanalysis,weobserved(Figure4B).Besidesthis,therewasamajorenhancementofthattheSERSspectrumisdominatedbytheside-chainthepeakscorrespondingtohistidineandC−CstretchingatvibrationsfromN-terminalresiduesoftheprotein.Also,we1583and1063cm−1,respectively,similartothatobservedindidnotobserveanysignificantenhancementfordisulfidePrP(23−231).Thepeakat∼530cm−1correspondingtolinkagethatispresentintheC-terminalglobulardomain.Sincebackbonedeformationsofadisorderedpolypeptidechainistheelectromagneticenhancementdisplaysa1/r12distancealsoobserved.TheamideIregionoftheSERSspectrumfordependence,wespeculatethatthepositivelychargedPrP(Y145Stop)showed∼100%disordered/extended/ran-disorderedN-terminaltailattachestothenegativelychargeddom-coilconformation(TablesS1andS2).Thisisconsistentsurface-modifiednanoparticles,staysproximaltonanoparticles,withthefactthatC-terminaltruncationatresidue145yieldsandthereforeresultsinagreaterSERSenhancementasprimarilyadisorderedprotein.Ourresultsalsodemonstrated36comparedtotheglobulardomain.Additionally,thethattheintrinsicdisorderisretainedevenuponinteractionsdisorderedregioncanbeimaginedasafluctuatingdynamicwithnanoparticlesunderourSERSexperimentalcondition.structurewithalargehydrodynamicradius,whichenablesittoTheobservedsignalenhancementattheN-terminalsegmenthavealargesurfaceareaandgreatercaptureradiusforsimilartothatofthefull-lengthPrPindicatesanassociativeinteractionascomparedtoitsstructuralcounterpart.ThisledroleofhighnetpositivechargeandextendedsizeoftheustosurmisetheassociativeroleofthechargeandintrinsicallydisorderedN-terminusindominatingtheSERShydrodynamicradiiofintrinsicallydisorderedN-terminusinspectraofPrP.Theseresultsindicatedthatthepositively52dictatingtheSERSspectraofPrP.ThisexplanationisalsochargedN-terminaltailcanselectivelyilluminatethisconsistentwiththeobservationoflittleornoenhancementofdisorderedregioninourSERSmeasurements.OurnextthedisulfidebondpresentintheglobularC-terminaldomainquestionwaswhetherashorterN-terminaltailwouldallowusofPrP.todetecttheC-terminalglobulardomainofPrP.Inordertotestiftheobservedside-chainvibrationsintheTheSERSspectrumofPrP(90−231)thathasashorterN-SERSspectraindeedaroseduetotheintrinsicallydisorderedterminalsegmentwasdominatedbybandscorrespondingtoN-terminus,weadoptedtwostrategies.First,wecreatedaPrPamideI,1655/1681cm−1;Phe,1004cm−1;Trp,1551and750construct23−144withastopcodonmutationatresidue145cm−1;Tyr,639cm−1anditsdoubletat828/852cm−1;andC−[PrP(Y145Stop)]resultinginatruncatedproteinthatadoptsHbending,1448cm−1.ItshowedasimilaritywithitsbulkacompletelydisorderedstateasevidentbyitsCDsignatureRamanspectrumasalsoshowninthecorrelationgraph(FigureS8A).Wewouldalsoliketonoteherethatthis(Figure4C,S12B).TheanalysisoftheamideIbandrevealedamutationhasapathologicalsignificance,givingrisetotheGSS-higherfraction(∼45%)ofhelicalcontentsthatisexpectedfor53phenotype.IftheSERSenhancementinPrPindeedarisesanN-terminallytruncatedPrPandcorroboratestheprevious34fromanintrinsicallydisorderedregion,weshouldobserveresults(TablesS1andS2).InadditiontoprominentbandssimilarSERSspectraofPrP-Y145Stoptothatofthefull-lengthforamideI,amideIII,andotheraromaticside-chainmarkers,PrP(23−231)withsomeminordifferencesduetoaweakertherewerecertainstrikingdifferencesforPrP(90−231)enhancementoftheC-terminalglobulardomainofthefull-comparedtothefull-lengthprotein(Figure4).(i)Sincethelengthprotein.Second,weusedapartialN-terminaltruncationPrP(90−231)harborsasingleTrp,thesignalcorrespondingconstructPrP(90−231)havingashorterdisorderedN-toTrpwasmuchweakercomparedtothatobservedforseventerminaltailandanintactglobularC-terminaldomainthatisTrpresiduesinthefull-lengthPrP.(ii)Duetotheproximityofpredominantlyα-helicalinstructureasalsoindicatedbyitsCDtheC-terminaldomainwiththenanoparticlesurface,wespectrum(FigureS8B).WeexpectedthatthisproteinobservedasignificantenhancementforaminoacidspresentinconstructwouldprovideamuchstrongerRamanenhancementtheglobulardomainnamely,glutamate(Glu)andaspartateofresiduesbothfromdisorderedandfoldeddomainsduetoa(Asp)residues(1391cm−1)andmethionine(716,683,andmuchshorterlengthoftheN-terminaltail.800cm−1).(iii)Thepeakat∼530cm−1correspondingwithTotestourhypothesis,wenextsetouttoperformSERSbackbonedeformationswasmuchweakerforPrP(90−231)studieswiththesetwotruncatedPrPconstructsnamely,PrPduetoashorterdisorderedsegment.(iv)Asignificant3191https://doi.org/10.1021/acs.jpclett.1c00240J.Phys.Chem.Lett.2021,12,3187−3194

5TheJournalofPhysicalChemistryLetterspubs.acs.org/JPCLLetterFigure5.Schematicoftether-length-dependentSERSofPrPusingfunctionalizednanoparticles.enhancementinthedisulfide(−S−S−)vibrationalbandattetheringbetweenhighlypositivelychargedintrinsically∼500cm−1wasobserved.disordereddomainoftheproteinandsurface-modifiedThedisulfidelinkagebetweenCys179andCys214isnegativelychargednanoparticles(Figure5).ThissynergisticlocatedattheC-terminalglobulardomain(Figure1A)andwaseffectbetweentheSERSsubstrateandanalyteresultsinnotpreviouslyvisibleeitherbybulkRamanorbySERSofthesignificantlyenhancedandreproducibleRamansignalsinfull-lengthprotein.TheshorteningoftheN-terminaldomainaqueoussolutions.Bychangingthelengthofthepositivelybringsthisdisulfideandothernonaromaticaminoacidswithinthecriticalnear-fielddistancefromthenanoparticlesurfacechargedN-terminaldomain,wewereabletoselectivelyrequiredfortheplasmonicenhancement.Next,weanalyzedenhanceandcharacterizedifferentpartsoftheproteinusingtheconformationofthedisulfidebridgethatisahallmarkofSERS.ThehighsensitivityofSERSwillbeofprimethetertiarystructureofPrP.TheRamanpeakat∼500cm−1isimportanceinthedetection,quantification,andcharacter-suggestiveofagauche−gauche−gaucheconformationandizationofthePrPexpressionlevelsandmisfoldinginbiologicalreflectstheinternalrotationaboutC−SandC−Cbondsinfluids.Webelievethatourtether-length-dependentSERSCα−Cβ−S−S−Cβ−Cαconformations.Despitethepresenceofmethodologywillbeapplicableintheselectivesequestrationnonaromaticaminoacidssuchasaspartates,glutamates,andandcharacterizationofotherproteinscontainingpositivelymethionineaswellasadisulfidebondinfull-lengthPrP,wechargedintrinsicallydisorderedsegmentsinthemilieuofadidnotobservesuchhighenhancementssincetheSERSspectrumforfull-lengthPrPwasdominatedbyaromaticaminocomplexbiologicalmixture.Additionally,engineeringpos-acidsresidingattheN-terminaldomain(Figure4A).Thehighitivelychargeddisorderedtracksateitherterminusofaproteinscatteringcross-sectionduetothepolarizabilityofthewillserveasapotentstrategytoselectivelysequesteranddelocalizedelectronsysteminringstructuresofaromaticcharacterizetheproteinofinterestusingultrasensitiveand54,55aminoacidsrendersthemhighlyRaman-active.Inlabel-freevibrationalRamanspectroscopy.contrast,feweraromaticaminoacidresiduesalongwithashorterN-terminallengthprovidedamuchgreateropportunity■fortheaminoacidside-chainsoftheC-terminaldomaintobeASSOCIATEDCONTENTenhancedinourSERSexperimentsforPrP(90−231)(Figure*sıSupportingInformationS13).Additionally,tomakeaquantitativeassessmentoftheSupportingInformationcontains.TheSupportingInformationcontentofaromaticresidues(TrpandPhe),weplottedtheisavailablefreeofchargeathttps://pubs.acs.org/doi/10.1021/ratiooftherelativepeakintensities[(I758/(I758+I1005)]againstacs.jpclett.1c00240.39theratio[Trp/(Trp+Phe)](FigureS14).ThisplotshowedalinearcorrelationbetweentheintensityratioandtheMaterialsandMethodssectionandsupportingfigures,contentsofTrpandPheforallthreeconstructsofPrP.Theseincludingabsorbance,CD,SERS,andRamanspectra,ζ-resultsindicatethatourSERSstudiescanalsoallowustomakepotentialplots,SEMandAFMimages,correlationquantitativeassessmentsofaromaticaminoacidcontentsingraph,andaplotofrelativepeakintensityratios(Figurestheprotein.S1−14),andsupportingtables,includingthepercentageInsummary,wewereabletodemonstrateadirect,contributionofdifferentsecondarystructuralelementsandpeakassignmentsofsomeRamanandSERSspectraultrasensitive,label-free,in-solutionSERScharacterizationof(TablesS1andS2)(PDF)PrPunderthenativeconditionbyutilizingtheelectrostatic3192https://doi.org/10.1021/acs.jpclett.1c00240J.Phys.Chem.Lett.2021,12,3187−3194

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