Interactions of an Anionic Antimicrobial Peptide with Zinc ( II ) Application to Bacterial Mimetic Membranes - Almarwani et al. - 2020 -

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pubs.acs.org/LangmuirArticleInteractionsofanAnionicAntimicrobialPeptidewithZinc(II):ApplicationtoBacterialMimeticMembranesBashiyarAlmarwani,NsokiPhambu,*YahiaZ.Hamada,andAndersonSunda-Meya*CiteThis:Langmuir2020,36,14554−14562ReadOnlineACCESSMetrics&MoreArticleRecommendations*sıSupportingInformationABSTRACT:Whilethemajorityofknownantimicrobialpeptidesarecationic,asmallnumberconsistofshortAsp-richsequencesthatareanionic.Theserequiremetalionstobecomebiologicallyactive.Here,wereportthestudyofthezinccomplexesofthepeptideGADDDDD(GAD5),anantimicrobialpeptide.Usingacombinationofdynamiclightscattering(DLS),ζ-potential,infrared,Raman,thermogravimetricanalysis(TGA),differentialscanningcalorimetry(DSC),andscanningelectronmicroscopy(SEM),wefindthataddingzincionstoGAD5forcesitintoacompactstructure.Higheramountsofzincionsfavoralargerstructure,possiblyadimer.SEMimagesshowthatzincionsreducethesizeofthefibrillarstructuresofGAD5.TGAcurvesshowthattheadditionofzincionsincreasesthethermalstabilityofthestructureofthepeptide.TGAandDSCindicatethattheassociationofGAD5withazwitterionicphospholipidinthepresenceofzincionsisthemoststable.Thestabilityofthatcomplexisduetothepresenceofasharpendothermicpeakinthe200−300°Crange,suggestingthepresenceofinterlamellarwaterthatisessentialtothestabilizationofthestructure.TheseresultsindicatethattheZn−GAD5complexprefersthebacteria-mimickingneutral(zwitterionic)membranes.Inthepresenceofnegativelychargedphospholipids,thecomplexremainsunorderedandunstable.Intermsofmechanismofaction,theZn−GAD5complexpromotesapossibleendocyticuptakewithrespecttoneutral(zwitterionic)membraneswhilepromotingmembranedisruptionbyformingporeswithrespecttonegativelychargedphospholipids.1.INTRODUCTIONtechniquessuchasdynamiclightscattering(DLS),ζ-potential,Mostantimicrobialpeptidesarecationic.1VeryfewstudiesRaman,thermogravimetricanalysis(TGA),differentialscan-havebeendevotedtoanionicantimicrobialpeptides.2−6ningcalorimetry(DSC),andscanningelectronmicroscopyAnionicantibacterialpeptides(AAPs)werefirstisolated(SEM)couldyieldmoredetailedinformationtounderstandfromepithelialcellsandpulmonarysecretions.3,6−8Thesethekillingmechanismofanionicantimicrobialpeptides.Tomoleculeshavebeenfoundtopreventbacterialgrowthintheunderstandthemembranedisruptionmechanismforzinc-presenceofdivalentmetalions.5,6Brogdenetal.3haveshownboundGAD5,theinteractionofthezinc−peptidecomplexDownloadedviaHELMHOLTZ-ZENTRUMHEREONonMay14,2021at15:08:47(UTC).thatthesesmallpeptides(lessthan1kDinsize)inhibitthewithmodelmembraneswasinvestigated.BacterialmembranesgrowthofbothGram-negativeandGram-positivebacteriainarehighlyanionicinnature,whereasouterleafletsofSeehttps://pubs.acs.org/sharingguidelinesforoptionsonhowtolegitimatelysharepublishedarticles.9−120.14MNaClwith2.5μMZnCl2.Antimicrobialactivityresideseukaryoticmembranestendtobeneutral.Therefore,inthecoreAsphexapeptidehomopolymericregion,andzwitterionicdipalmitoylphosphocholine(DPPC)andanionicgrowthinhibitionincreaseswiththenumberofAspresiduesinpalmitoyloleoylphosphoglycerol(POPG)lipidswerechosenas3,6thepeptide.Theyrequirezincformaximalactivityandformmodelsfor,respectively,eukaryoticandbacterialmembranes3acomplexwithit.Itwasspeculatedthatzincmayformatoelucidatetheeffectofelectrostaticinteractionsonthecationicsaltbridgethatallowsthepeptidetoovercomethenetactivityandspecificityofzinc−GAD5.DPPCisthemain3,6−8negativechargeonthemicrobialsurface.However,thecomponentofthemembranemodelofthepulmonarymechanismsunderlyingtheantimicrobialactionofthesesurfactant.10,13POPGhasbeenusedasapotentialmodelforanionicpeptidesremainunclearorhasnotbeendefinitivelyGram-positivebacteriasuchasStaphylococcusaureus.11,12,14,153,6−8elucidated.Aquestionthatbegstobeansweredis:whatisthenatureofthecomplexesformedbythepeptideandzincmetalionattheconcentrationof0.5mMGAD5in20μMReceived:August5,2020ZnClusedbyBrogdenetal.?3Thecalculatedzinc/peptideRevised:November11,20202molarratiois0.04,whichisnonstoichiometric.ThenatureofPublished:November23,2020thecomplexbetweenGAD5andzincionsintheirworkconditionsisfullycharacterizedhereusingthetraditionalspectrummethodssuchasinfrared.However,resultsfrom©2020AmericanChemicalSocietyhttps://dx.doi.org/10.1021/acs.langmuir.0c0230614554Langmuir2020,36,14554−14562

1Langmuirpubs.acs.org/LangmuirArticleFigure1.AminoacidsequenceoftheanionicantimicrobialpeptideGAD5atneutralpH.aTable1.AverageSizeandζ-PotentialDistributionsof10μMGAD5forDifferentConcentrationsofZnCl2Zn−GAD5molarhydrodynamicradius(peak#1)percenthydrodynamicradius(peak#2)percentζ-potentialsampleratio(nm)(%)(nm)(%)(mV)GAD5freepeptide164764124−13.4Zn−GAD50.0184100−12.3Zn−GAD50.0447100−12.8Zn−GAD50.0531100−12.9Zn−GAD50.0874100−11.1Zn−GAD50.192100−9.9Zn−GAD50.2111100−9.3Zn−GAD50.5105100−9.4Zn−GAD5116195195−1.2Zn−GAD521481000.7Zn−GAD551531001.4aAnerrorof4%wasobtainedfromthreeindependentmeasurements.2.EXPERIMENTALSECTIONappropriateamountof10μMGAD5inavolumetricflaskanddilutingtothemarkwithwateratpH6.7.Asuitabledilutionofthese2.1.Materials.DPPCandPOPGwerepurchasedfromAvantistocksolutionsdownto10μMallowedonetotitrateGAD5withPolarLipidInc.andusedwithoutfurtherpurification.Thepeptideincreasingamountsofzincions.Thecalculatedmolarratiosofzinc−GAD5(GADDDDD-OH)wasobtainedfromGenScript(>97%peptidesystemsare0.01,0.02,0.05,0.1,5,10,50,and100.purity)andusedasreceived.ZnCl2wasobtainedfromFisherandζ-Potentialstudiesforsurfacechargemeasurementswerecarriedusedasreceived.Ourworkconditionsaredifferentfromthose3outusingthesamemachineasthatusedforparticlesize.AtotalofreportedbyBrogdenetal.whousedmillimolarconcentrations,threemeasurementsof100runseachwerecarriedoutforallofthewhereasthisstudyusedmicromolarconcentrationsofthezincsaltsamples.Next,eachpreparationwastransferredtoadisposablethataremorerelevanttothebiologicalenvironments.Figure1cuvetteforsizeevaluationand,afterward,toaDTS1060cell(MalvernpresentstheaminoacidsequenceofthepeptideGAD5atneutralpH.22−24Instruments)forζ-potentialmeasurement.2.2.PreparationofCellMembraneMimeticVesicles.2.4.RamanSpectroscopy.RamanspectrawererecordedusingaPreparationofcellmembranemimeticvesicleshasbeenextensively13,15−19confocalmicroscopeRamanspectrometer(DXRRamanmicroscopedescribedinourpreviousreports.NeutralvesicleswerefromThermoScientific)equippedwithOMNICsoftware.ThepreparedusingpureDPPC,andnegativelychargedvesicleswereexcitationwavelengthwas532nm,andthespectralcoveragerangepreparedusingpurePOPG.Theappropriateamountofdriedlipidwas4000−400cm−1withaspectralresolutionof2cm−1.Theoutputwasweighedoutanddissolvedinchloroformandvortexedfor5min.powerwas8mW.TheRamanspectrumwasbaseline-corrected,andThesamplewasdriedunderastreamofnitrogengasfor6hand12,25−31undervacuumovernight.AthinlipidfilmwasformedonthewalloftheRamanintensitiesweremeasuredaspeakheight.thevial.ThethinlipidfilmwasthenhydratedwithwateratpH6.7to2.5.InfraredSpectroscopy.Infraredspectrawereobtainedusingatotallipidconcentrationof5mg/mL.SmallunilamellarvesiclesaFouriertransforminstrument(ThermoScientificiS10)equipped(SUVs)werepreparedbysonificationofthemilkylipidsuspensionwithasinglereflectionGeattenuatedtotalreflectanceaccessory.SolidusingaSonicDismembratorUltrasonicProcessor(modelFB-50sampleswereused.ThechangeinvibrationmodesofGAD5withandincludingastandard1/8″diametermicrotip,intitaniumalloy)fromwithoutzincionsand/orthelipidmixturewasmonitored.TheFisherScientificforaboutanhourinanicebathuntilthesolutionspectrawererecordedatroomtemperaturebetween4000and600cm−1ata4cm−1resolution,and64scanswereaccumulated.Routinebecametransparent.Forpeptidebindingexperiments,anappropriatevolumeof10μMGAD5wasmixedwitheitherDPPCorPOPGinsmoothingandnormalizationwereappliedtoalloftheinfrared28,32−34chloroformtomakeupthedifferentsolutionswithdifferentspectra.proportionsofmembranevesicles.17−19AvacuumapparatusFreeDeterminationofthesecondarystructure.ThedeterminationofZone6fromLABCONCOwasusedtoobtainthefreeze-driedthesecondarystructurefrominfraredspectrausingtheamideI17−19samplesusedforinfrared,Raman,TGA,DSC,andSEMtechniques.vibrationhasbeenextensivelydescribedinourpreviousreports.2.3.ParticleSizeandζ-PotentialMeasurements.ThesizeThedeconvolutionoftheamideIbandwasperformedwithOMNICanddistributionofaggregatesinsolutionweremeasuredbydynamicsoftware(ThermoFisherScientific,Waltham,MA)andanalyzedasalightscatteringusingaZetasizerNanoSeries(Nano-ZS)instrumentsumofGaussiancurves,withconsecutiveoptimizationofamplitudes,20−22bandpositions,half-widths,andGaussiancompositionofindividual(MalvernInstruments,Malvern,U.K.)anda633nmlaserdiode.Allmeasurementswereconductedatascatteringangleof90°.Allbands.Thetypesofsecondarystructureswereassignedbasedonthe35−37sampleswerefiltered,degassed,andscannedusinga1cmpathlengthdatafoundintheliterature.ThecharacteristicamideIpeakquartzcuvette.Thesampleswerelefttoequilibratefor3minat25describedintheliteraturecontainsdifferentsecondarystructures,includingstrongintermolecularβ-sheet(1622−1627cm−1),strong°C.ThedatawerefittedusingMalvernInstrumentDTSsoftware.intramolecularβ-sheet(1628−1638cm−1),weakβ-sheet(1690−TheDLStitrationofvesicleswithGAD5wasperformedasfollows:stocksolutionsofvesiclesofGAD5werepreparedbydissolvingan1703cm−1),randomcoils(1640−1644cm−1),α-helix(1645−165714555https://dx.doi.org/10.1021/acs.langmuir.0c02306Langmuir2020,36,14554−14562

2Langmuirpubs.acs.org/LangmuirArticleaTable2.PercentageofSecondaryStructuresin10μMGAD5andItsZn−GAD5ComplexSamplesGAD5(%)Zn−GAD5(%)Zn−GAD5−DPPC(%)Zn−GAD5−POPG(%)a-helix6901000β-sheet31000β-turn022034randomcoil078066aAnerrorof2%wasobtainedfromthreeindependentruns.Figure2.SEMimagesoflyophilizedsamplesofGAD5(A)andZn−GAD5complex(B).cm−1),3-helix(1658−1666cm−1),andturns(1668−1685cm−1).10throughtheentiremolarratiorange.TheZn−GAD5complexTheconformationrangesabovearebasedontheexperimentaldataadoptsacompactstructureatamolarratioof0.05.andassignmentscollectedfromvariousauthorsandevaluatedbyourWhilemeasuringtheeffectofzincionsonparticlesize,the17−19,28,32−34team.surfacechargeofthesolutionswasalsomeasuredusingtheζ-2.6.ThermogravimetricAnalysis(TGA)andDifferential38potentialtechnique.AsexpectedfortheanionicpeptideScanningCalorimetry(DSC).ThermalstabilityandphasetransitionsofcellvesiclesandtheircomplexeswererecordedbyGAD5,theζ-potentialis−13.4mVintheabsenceofzinc.TheDSCandTGAwithaLINSEISSTAPT1600instrument.Thisnegativeζ-potentialindicatesthepresenceofnegativelyinstrumentdeterminessimultaneouschanges(inasinglerun)ofmasschargedpeptideaggregates.Then,theζ-potentialofGAD5andcaloricreactionsofasample.Itperformstestsfromanultrahighincreaseswiththeincreaseinthezincconcentration,reachingvacuumof10−4mbarto5baroverpressure.Samplesweighing4−6apositivevalueof+1.4mVforaZn−GAD5molarratioof5.mgwereputinanaluminumpan,andanemptypanwasusedasaAtamolarratioof0.05,theζ-potentialwasnegativeasreference.Investigationswereperformedbetweenroomtemperatureexpectedatavalueof−12.9mV.AtthisneutralpH,GAD5isand700°Cwithaheatingrateof10°Cperminuteforpeptidesubjecttotautomerism,butattheconcentrationof5%zincrecruitingexperiments.Atthisheatingrateof10°Cperminute,thedegradationofthesampleswasreduced.TransitionparameterswerewhereGAD5remainslargelynegativelycharged,itcanbeobtainedwithincorporatedsoftware.ThealiquotsfromthesamereasonablyassumedthattheZn−GAD5−vesiclecomplexesarefreeze-driedsampleswereusedforRaman,infrared,SEM,TGA,andnotsignificantlyaffected.TheseDLSandζ-potentialdataagree17−1913,20,24,38−40DSC.withdatareportedintheliterature.2.7.ScanningElectronMicroscopy(SEM).TheSEMimagesTounravelthespecificfeaturesoftheZn−GAD5complexwereobtainedinaZeissMerlinFEG-SEMwithaGemini-IIcolumn.at5%,specificsamplesofGAD5werepreparedwithandAllsamplesweregoldsputter-coatedat30mAfor80s(K550Xwithout5%zincchlorideandanalyzedusingSEMandinfraredSputterCoater,QuorumTechnologiesLtd.,WestSussex,U.K.).An17−19techniques.Table2presentstheinfraredcurve-fittingresultsofacceleratingvoltageof5kVwasusedforallSEMobservations.GAD5anditscomplexes,whileFigure2presentstheSEMimagesofGAD5(Figure2A)andZn−GAD5complex(Figure3.RESULTSANDDISCUSSION2B).SEMimageofGAD5showselongatedfibrilsofabout103.1.EffectofZincIonsontheGAD5Structure.Toμmthicknessandplaques.SEMimageoftheZn−GAD5investigatetheeffectofzincionsonthestructureofpeptidecomplexshowsaggregatesincludingroundnodulesofabout6GAD5,theeffectofincreasingamountsofzinconthesizeandμm.FromTable2,thecorrespondingIRcurve-fittingdatasurfacechargeofthepeptideGAD5(Table1)wasmonitoredshowthatGAD5adoptsamixtureofhelixandsheetstructures,usingDLSandζ-potentialtechniques,respectively.InthewhiletheZn−GAD5complexadoptsamixtureofamorphousabsenceofzinc,DLSdataanalysisshowsthepresenceoftwoandturnstructurescorrespondingtothenodulesseenonSEMpopulations,onewhosehydrodynamicradiusdoesnotexceedimages.thevalueof41nm(24%)andtheotherbeingcharacterizedbyBasedonthat,itcanbeinferredthatthereasonwhythealargerradiusof164nm(76%).Then,thehydrodynamicconcentrationof0.5mMGAD5in20μMZnCl2waseffective3radiusofGAD5aggregatesdecreasedwiththeincreaseoftheinkillingbacteriaasstatedbyBrogdenetal.isthatitisonlyatzincconcentration,reachingaminimumvalueof31nmforathisconcentrationof5%zincchloridethatthepeptideGAD5Zn−GAD5molarratioof0.05.Beyondthemolarratioof0.05,adoptsaverycompactstructure,consistingofamixtureofturnthehydrodynamicradiusincreasedwiththeincreaseofthe(22%)andrandomcoil(78%)structuresbeforeinteractingzincconcentration.Aconclusionwasdrawnthatthewithbacteria.SincetheminimumhydrodynamicradiusofthecomplexationofzincaltersthesizeofGAD5aggregatesGAD5aggregateswasreachedusingaconcentrationof5%of14556https://dx.doi.org/10.1021/acs.langmuir.0c02306Langmuir2020,36,14554−14562

3Langmuirpubs.acs.org/LangmuirArticleTable3.AverageSizeDistributionofVesiclesMimickingEukaryotic(DPPC)MembranesforDifferentConcentrationsof10aμMZn−GAD5sample[Zn−GAD5]/[DPPC]molarratiohydrodynamicradius(peak#1)(nm)percent(%)Zn−GAD5freezinc:peptide31100[Zn−GAD5]/[DPPC]0.0183100[Zn−GAD5]/[DPPC]0.0246100[Zn−GAD5]/[DPPC]0.0371100[Zn−GAD5]/[DPPC]0.0572100[Zn−GAD5]/[DPPC]0.1153100[Zn−GAD5]/[DPPC]0.261100[Zn−GAD5]/[DPPC]0.3228100[Zn−GAD5]/[DPPC]1245100DPPC199100aAnerrorof4%wasobtainedfromthreeindependentmeasurements.Table4.AverageSizeDistributionofVesiclesMimickingBacterial(POPG)MembranesforDifferentConcentrationsof10aμMZn−GAD5[Zn−GAD5]/[POPG]molarhydrodynamicradius(peak#1)percenthydrodynamicradius(peak#2)percentsampleratio(nm)(%)(nm)(%)Zn−GAD5freeZn:GAD531100[Zn−GAD5]/[POPG]0.01166753825[Zn−GAD5]/[POPG]0.02135100[Zn−GAD5]/[POPG]0.03122882412[Zn−GAD5]/[POPG]0.05124802120[Zn−GAD5]/[POPG]0.1105100[Zn−GAD5]/[POPG]0.242100[Zn−GAD5]/[POPG]0.3180733627[Zn−GAD5]/[POPG]194100POPG8858267442aAnerrorof4%wasobtainedfromthreeindependentmeasurements.zincchloride,therestofourstudywasfocusedonthetobe88nmand42%ofPOPGparticlesarefoundtobeinteractionoftheGAD5peptidecontaining5%ofzincionsaround3μm.TheaggregationortheformationofnonlamellarwithDPPCandPOPGmodelmembranes.dispersionoflipidshasbeenfoundtoproducemultiplepeaks223.2.InvestigationoftheMembraneActivityandofthesizedistributionprofile.SelectivityofZn−GAD5Complex.AmechanismofInthepresenceofanincreasingamountofZn−GAD5,theinteractionbetweenZn−GAD5andthetwomodelmem-changesintheapparenthydrodynamicradiusofPOPGbraneswasproposedbasedonRaman,DSC,DLS,andζ-particlesaresignificantthroughtheentirerange(Table4).potentialdatausingdifferentlipid−peptidemolarratios.TheparticlesizeofPOPGvarieswithnocleartrend,showingStructuralstudieswereundertakenusingIR,TGA,andSEMsingleormultiplesizedistributionsatdifferentmolarratios.withaspecialemphasisonthephysicochemicalcharacteristicsTheassociationofZn−GAD5withPOPGreachedtheoftheZn−GAD5−vesiclecomplexesatlowpeptidesmallesthydrodynamicradiusof42nmatamolarratioofconcentration.Thepeptide−lipidmolarratioof1:100was0.2;atthismolarratio,thehydrodynamicradiusdecreasedusedinaccordancewithstandardreportsforlowpeptidefrom88to42nmforalloftheparticlesinsolution.13,17−19concentration.BasedonDLSdata,theinteractionbetweenZn−GAD53.2.1.CharacterizationofZn−GAD5−VesicleComplexesseemstobestrongerwithDPPCthanwithPOPG,butabyDLSandζ-Potential.ToinvestigatetheinteractionofthecomplementaryapproachusingstructuraldataisneededtoZn−GAD5complexwithDPPCandPOPG,theeffectofgiveaclearpictureoftheseinteractions.BecauselowpeptideincreasingamountsoftheZn−GAD5complexonthesizeofconcentrationwithrespecttotheamountoflipidismoreDPPCorPOPGatdifferentpeptide−lipidmolarratioswasrelevantinbiologicalsystems,therestofthisstudyislimitedtomonitoredusingDLStechnique(Tables3and4).DPPCandthezinc−peptide/lipidmolarratioof1:100.POPGsolutionswereusedascontrol.3.2.2.CharacterizationofZn−GAD5−VesicleComplexesIntheabsenceofZn−GAD5,thehydrodynamicradiusofbyRamanSpectroscopy.Ramantechniquewasusedtostudy12DPPCparticlesisfoundtobe199nm.InthepresenceofanthelocationofZn−GAD5withrespecttothelipidbilayer.increasingamountofZn−GAD5,thehydrodynamicradiusofZn−GAD5maybelocalizedinthehydrophobicregionwithDPPCincreasessignificantlythroughtheentirerange,withatheacylchains,atthesurfaceincontactwiththephosphatesinglesizedistributionprofile(Table3).Thissuggestsastrongheadgroups,orbetweenthesetworegions.Intheliterature,22the1000−1200cm−1regionhasbeenhelpfulininvestigatingassociationbetweenZn−GAD5andDPPC.IntheabsenceofZn−GAD5,thehydrodynamicradiusthedelocalizedC−Cstretchingvibrations,involvinglongprofileofPOPGparticlespresentsmultiplepeakscorrespond-portionsoftheacylchains,andthephosphategroup12,23,41,42ingtomultiplepopulations:58%ofPOPGparticlesarefoundmodes.TheRamanspectraoftheDPPCandPOPG14557https://dx.doi.org/10.1021/acs.langmuir.0c02306Langmuir2020,36,14554−14562

4Langmuirpubs.acs.org/LangmuirArticlevesicleswithandwithoutZn−GAD5wererecordedintheIncontrast,inthesameconditions,usingPOPG,itwasregioncitedaboveandanalyzed.OurreferenceRamanspectraobservedthattheratiosI1098/I1126andI1098/I1062increasedarethoseofDPPCandPOPGvesicleswithoutZn−GAD5.from0.588and0.465inthefree-peptidePOPGvesiclestoTheeffectofZn−GAD5ontheintensityandfrequencyofthe1.866and>1.0intheZn−GAD5−POPGvesicles,respectively.acylandphosphategroupbandsoftheDPPCandPOPGTheincreasesuggestsasignificantincreaseinthenumberofvesicleswassubsequentlymonitored.Figure3displayspeakgauchebonds,suggestingthatZn−GAD5disturbstheorderoftheacylchainsinthehydrophobiccore,whichimplythatZn−GAD5maybelocalizeddeepinsidethehydrophobiccoreofthePOPGvesicles.Infact,thefrequencyofthephosphategroupat1093cm−1shiftedfrom1093to1081cm−1,theshifttolowerfrequencysuggestingthatZn−GAD5isstronglyinteractingwiththephosphategroupatthesurfaceofthePOPGvesicles.ThisresultshowsthattheRamantechniquedetectstheinteractionasithasbeenshowntobeavery22,23,27sensitivetoolinthedetectionofphosphategroups.Inaddition,RamanspectrashowthattheCObandataround1735cm−1shiftedtoahigherwavenumber1745cm−1,suggestingthattheCOgroupsarenotinvolvedintheinteractionwithZn−GAD5.FromtheRamandata,onecaninferthatZn−GAD5islocatedbetweenthehydrophobiccoreandthemembranesurface.Insummary,ata1:100molarratio,theinteractionZn−GAD5withPOPGisdrivenbyelectro-staticinteractionswiththeheadgroupregionofthebilayerFigure3.NormalRamanspectraoflyophilizedsamplesofvesiclesmimickingeukaryotic(DPPC)membranes,bacterial(POPG)withnodisturbanceoftheacylchainregion.Thus,Zn−GAD5membranes,andtheircomplexeswithZn−GAD5inthephosphateactivityismediatedbyamechanismofactionwelldescribedby−1otherresearcherssuchasdosSantosCabreraetal.50Theyregion(1000−1200cm).showedthatthepeptideaccumulatesontheheadgroupregioncharacteristicofvibrationsofhydrocarbonchainsintheofthenormalmembrane,leadingtotheincreasedpermeability1,000−1,200cm−1region.TheC−Cstretchingvibrationmodeofthemimeticmembranetoformporesordefects.isassociatedwithtrans/gaucheconformationchangesintheOneadditionalfeatureisthatwhenZn−GAD5−DPPCisfattyacidchains.23,43−46ThethreecharacteristicpeaksatcomparedtoZn−GAD5−POPGinthe1500−1800cm−1amidaround1062,1098,and1126cm−1havebeenassignedintheregion,itwasobservedthatthepeaksonZn−GAD5−DPPCliterature.26−28The1062and1126cm−1peakscorrespondtoaresharp,suggestingahighlyorderedstructure,whilethetheout-of-phaseandin-phasetransconformationoftheC−CpeaksonZn−GAD5−POPGarebroad,suggestingalargeskeleton,andthe1098cm−1peakcorrespondsnotonlytothenetworkofhydrogenbondsand,thus,thepresenceofgaucheconformationoftheC−Cskeletonbutalsototheamorphousaggregates,asseenandconfirmedbyinfraredinsymmetricPO−stretchingmode.TheintensityratiosI/thenextsection.21098I1126andI1098/I1062reflecttheproportionoftrans/gauche3.2.3.CharacterizationofZn−GAD5−VesicleComplexesconformationinthechain,whichareoftenusedtocharacterizebyThermalAnalysis.TGAtechniquewasusedtoexaminethethelipidchaindisorder.26−28stabilityofthecomplexes,whiletheDSCtechniquewasusedThechangesinRamanintensityratiosI1098/I1126andI1098/toevaluatethemoisturecontentofthecomplexes.ThelevelofI1062oftheDPPCvesicleswerealsomonitored.Thestrikinghydrationhasbeenfoundtoaffectthethermalstabilityof51,5251findinginthisstudyisthatatamolarratioof5%,itwaspeptides.AccordingtoLiberatoetal.,watermoleculesobservedthattheratiosI1098/I1126andI1098/I1062decreasedmayformabridgebetweenhydrophobicmoleculesleadingtofrom0.852and0.764inthefree-peptideDPPCvesiclestoanextendednetwork.Also,whentheyareintercalatedintothe0.469and0.424intheZn−GAD5−DPPCvesicles,respec-matrixoftheassemblies,theywouldstimulatetheformationoftively,suggestingasignificantdecreaseinthenumberofgaucheH-bonds,whichwouldaccountfortheself-assemblyoflargerbonds.ThisdecreaseindicatesthatZn−GAD5causedthestructures.Inaddition,interlamellarwaterhasbeenfoundto53,54orderofthelipidacylchainandmembranefluidityoftheincreasethestabilityofsamplescontainingmetalions.ToDPPCvesicles,andthusZn−GAD5isnotlocatedinthesummarize,ourfocushasbeenontheregionbetween200and12450°C,whichcorrespondstothebreakingofthebonds.Thishydrophobiccoreregionofthemembrane.Inaddition,thefrequencyofthephosphategroupat1103cm−1peakslightlytemperaturerangeincludescomplexeventssuchasdecarbox-decreasedto1099cm−1confirmingthatZn−GAD5isylation,deamination,desulfuration,anddephosphorylation.interactingwiththephosphategroupofthelipidsandliesForDSCexperiments,theregionofinterestisbetween20023,43−46ontothemembranesurface.Inaddition,Ramanspectraand300°C,whichcorrespondstotheappearanceofshowthattheCObandataround1740cm−1hasalmostinterlamellarwater.53,54disappeared,suggestingthattheCOgroupsarealsoinvolvedFigure4displaystheDSCthermogramsofDPPCandintheinteractionwithZn−GAD5.FromtheRamandata,onePOPGandtheircomplexeswithZn−GAD5.DSCwasappliedcaninferthatZn−GAD5mayaccumulateovertimeatthewiththegoaloffurtherunderstandinghowtheZn−GAD5membranesurfaceformingbundlesorclustersofpeptidesatcomplexinteractswiththelipidvesiclesata1:100peptide−47−4917−19thesurfaceinagreementwiththeliterature.Infact,lipidmolarratio.Figure4showstheappearanceofa47Masudaetal.suggestthattheaccumulationofpeptidesonsharppeakataround248°ContheZn−GAD5−DPPCthesurfacemaypromotemembranebending.complex,suggestingthepresenceofinterlamellarwater14558https://dx.doi.org/10.1021/acs.langmuir.0c02306Langmuir2020,36,14554−14562

5Langmuirpubs.acs.org/LangmuirArticlechargedphospholipids,thecomplexremainsunorderedandunstable.3.2.4.StructuralAnalysis.InfraredspectroscopywasusedtodeterminethesecondarystructureoftheZn−GAD5complexwithinthemodelmembranes.ThesecondarystructureoftheZn−GAD5complexwithandwithoutDPPCandPOPGcellmembraneswasexaminedbycomparingFouriertransforminfraredspectroscopy(FTIR)spectraatamideI(1700−1600cm−1)andamideII(1600−1500cm−1)26,27,56regionsthatarecharacteristicofpeptides.ThegoalistoobservewhethertheconformationoftheZn−GAD5changesinthepresenceofDPPCandPOPGmodelmembranes.Figure6presentstheoriginalFTIRspectraofthesamplesintheFigure4.DSCthermogramsforlyophilizedsamplesofvesiclesmimickingeukaryotic(DPPC)membranes,bacterial(POPG)membranes,andtheircomplexeswithZn−GAD5.53,55necessaryforincreasedstability.ThispeakindicatesanincreaseinstabilityofDPPCaftertheadditionoftheZn−GAD5complex.ThereisnosuchpeakintheZn−GAD5−POPGcomplex.TheseDSCdataaresuggestingthattheZn−GAD5−DPPCcomplexisstrongerthantheZn−GAD5−POPGcomplexinagreementwithDLSandRamandataintheprevioussections.Togainmoreinsightsintothestabilityofthecomplexes,Figure5showstheTGAthermogramsofDPPCandPOPGFigure6.FTIRspectraoflyophilizedsamplesofGAD5anditscomplexesinthecarbonylandamideIandIIregions.regionscitedabove.TheFTIRspectrumofZn−GAD5showsabroaddissymmetricpeakwithtwomaximaintheamideIregion.TheamideIbandsintheseregionsaresobroadthatitisdifficulttodeterminethesecondarystructureofthepeptide.17−19AnFTIRcurve-fittingprocedurewasapplied.TheresultsofquantitativeanalysisofpeptideconformationrevealthatZn−GAD5consistsofamixtureofturn(22%)andrandomcoil(78%)structures(Table2).Knowingthatthecompositionofmodelmembranesisdifferent,thegoalwastomonitorifthesedifferencestranslatetoadifferentimpactonthepeptideinterface.InthepresenceofDPPCmodelmembranes,Zn−GAD5atFigure5.TGAthermogramsforlyophilizedsamplesofvesicleslowconcentrationcontainsamajorhelixstructure(100%).mimickingeukaryotic(DPPC)membranes,bacterial(POPG)The78%ofamorphousstructureofZn−GAD5areconvertedmembranes,andtheircomplexeswithZn−GAD5.intoamoreorderedstructurethatishelixstructureinthepresenceofDPPC,probablybecauseDPPCentersincontactandtheircomplexeswithZn−GAD5.At50%massloss,thewiththeZn−GAD5atthesurfaceofthemembrane,asshownZn−GAD5−DPPCcomplexisdecomposingathighertemper-byRamanspectra.InthepresenceofPOPGmodelaturethanpureDPPC,whileZn−GAD5−POPGisdecom-membranes,Zn−GAD5atlowpeptideconcentrationstillposingalmostatthesametemperaturethanitscorrespondingcontainsamajorrandomcoilstructure(66%).The22%ofpurePOPG,confirmingtheconclusionfromDSCdata.TheturnstructureisnotconvertedintoanamorphousstructureininvolvementoftheNH+groupintheinteractionofDPPCthepresenceofPOPG,probablybecausezincionsdonot4withZn−GAD5mayleadtoanincreaseinthetransitioninfluencethelipidbecauseZn−GAD5isembeddedintothe53,54temperature.MeasurementsfromtheTGAcurveindicatehydrophobiccoreofthelipidandthelipiddoesnotsensethethatthemasslossofGAD5−Zn−DPPCduetointerlamellarpresenceofzincions,contrarytoDPPC.Theseobservationswaterisabout10%ofitstotalmass,correspondingtoanagreewiththeRamandatapresentedinthesectionabove.enthalpychangeof244J/gdeducedfromtheDSCcurve.Overall,thestructuralmeasurementsprovidethefirstTheseresultsfromDSCandTGAindicatethattheZn−GAD5explanationofhowGAD5bindstolipidbilayersinthecomplexshowspreferencesforbacteria-mimickingneutralpresenceofzincions.Themechanismofinteractionbetween(zwitterionic)membranes.InthepresenceofnegativelythesemembranesandZn−GAD5atlowpeptideconcentration14559https://dx.doi.org/10.1021/acs.langmuir.0c02306Langmuir2020,36,14554−14562

6Langmuirpubs.acs.org/LangmuirArticleFigure7.SEMimagesoflyophilizedsamplesoftheZn−GAD5complex(A),ofvesiclesmimickingeukaryotic(DPPC)membranes(B),bacterial(POPG)membranes(C),andoflyophilizedsamplesoftheZn−GAD5complexwithvesiclesmimickingeukaryotic(DPPC)(D)andbacterial(POPG)membranes(E).involvesaconformationchange.IRcurve-fittingdatarevealthepresenceofDPPC,whiletheamorphousportionremainsthatZn−GAD5undergoesalipid-inducedconformationalsignificantinthepresenceofPOPG.ComparisonofpanelsB,transitionintoahelix-likestructureinthepresenceofDPPCCandD,EofFigure7revealsthatZn−GAD5hasadifferentmembrane,whereasZn−GAD5remainsmostlyamorphousineffectoneukaryoticandbacterialmembranes,asshownbythepresenceofPOPGmodelmembrane.Ramandata.SEMimagessuggestthatZn−GAD5disrupts47Masudaetal.reportedthatanamphiphilicpeptideR6W3eukaryoticandbacterialcellsmimickingmembranesby28−33stimulatedanendocyticuptakebyinducingmembraneformingsuperstructures.curvature.Theyobservedtheclusteringofthepeptideonthecellsurface,likeinourexperiments,andconcludedthatthe4.CONCLUSIONSmechanicalformationofconcavecurvaturebyexternallyaddedOurresultsprovidethefirstinsightsintotheeffectofzincionspeptidemayleadtotheendocyticevent,inagreementwithonthestructureoftheanionicpeptideGAD5.Revealingthatotherresearchers.ThiseventwasalsofollowedbyatransitionthepeptideGAD5adoptsaconformationdominatedbyaofthepeptideintoahelix-likestructureasseeninourinfraredcompactfoldedstructureinthepresenceofasmallamountofexperimentsandalterationofmembranemorphologyasseenzinc,measurementsshowthattheassociationoftheZn−inourSEMimages.ThissuggeststhattheZn−GAD5complexGAD5complexwithmodelmembranesisstrongtowardpromotesapossibleendocyticuptakewithrespecttoneutral47neutralphospholipids.Thecombinedinvestigationofthe(zwitterionic)membranesalthough,likeMasudaetal.,ourstructurebyinfraredandstabilitybyTGAandDSCledtotheunderstandingisthatthemechanismmaynotbeasingle,conclusionthattheaggregationofGAD5inthepresenceofcanonicalendocyticmechanism.zincistheresultofthestructuralchangeratherthanpurelya3.2.5.UltrastructuralAnalysis.Toobservehowthechangeinelectrostaticinteractions,andthethermalstabilityisdifferencesinmembranecompositionofbothmodelduetothepresenceofinterlamellarwater.SignificantmembranesareaffectedbytheZn−GAD5complexand17−19differenceswereobservedinthestrengthandthemodeoftranslateinmorphology,SEMtechniquewasused.TheactionofthepeptideasafunctionofchargeofthelipidSEMimagesshowninpanels(A−C)ofFigure7presenttheheadgroup.WhiletheinteractionisstrongwiththeimagesoftheZn−GAD5complex,vesiclesmimickingzwitterionicDPPCheadgroupandquaternaryammoniumeukaryotic(DPPC)andbacterial(POPG)membranes,group,theZn−GAD5interactionwiththeanionicPOPGisrespectively,whilethoseinpanelsDandEpresenttheimagesmarginalatourworkconditions.ThecorrelationsseeninouroftheZn−GAD5complexwithvesiclesmimickingeukaryoticstudiesbetweentheroleofmetalionsonthestructureof(DPPC)andbacterial(POPG)membranes.TheSEMimageantimicrobialpeptidesandtheirbiophysicalinteractionswithoftheZn−GAD5complex(panelA)presentsnodulesmodelmembranesillustratethesignificanceofsuchstudiesinembeddedinaggregates,whiletheSEMimagesofpurepredictinginteractionsofantimicrobialpeptideswithcells.DPPCandPOPG(panelB,C)presentlargeamorphousThisstudywouldsuggestthattheZn−GAD5complexaggregates.Figure7providesstrongevidencethataddingapromotesapossibleendocyticuptakewithrespecttoneutralzinc-boundpeptidetomodelmembranesinducessuper-(zwitterionic)membranes.structuresconsistingofsphericalballs.InthepresenceofDPPC(panelD),theballshavearoughsurfaceandarelarge■andisolated,whileinthepresenceofPOPG(panelE),theASSOCIATEDCONTENTballsareembeddedinlargeamorphousaggregates.Thesedata*sıSupportingInformationagreewithIRdatasuggestingthatalloftheamorphousportionTheSupportingInformationisavailablefreeofchargeat(78%)oftheZn−GAD5complexistransformedintohelixinhttps://pubs.acs.org/doi/10.1021/acs.langmuir.0c02306.14560https://dx.doi.org/10.1021/acs.langmuir.0c02306Langmuir2020,36,14554−14562

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