E ff ects of Dissolved Gases on the Amyloid Fibril Morphology - Chiang et al. - 2021 - Unknown

《E ff ects of Dissolved Gases on the Amyloid Fibril Morphology - Chiang et al. - 2021 - Unknown》由会员上传分享，免费在线阅读，更多相关内容在学术论文-天天文库。

pubs.acs.org/LangmuirArticleEffectsofDissolvedGasesontheAmyloidFibrilMorphologyYa-LingChiang,Yu-JenChang,Yun-RuChen,*andIng-ShouhHwang*CiteThis:Langmuir2021,37,516−523ReadOnlineACCESSMetrics&MoreArticleRecommendations*sıSupportingInformationABSTRACT:Theonsetorprogressionofnumerousneurodegenerativediseasesoccursduetoaggregationofproteinsthatultimatelyformfibrils.Theassemblyandmorphologyoffibrilsaresusceptibletoenvironmentalfactors.Inthiswork,weusedatomicforcemicroscopy(AFM)toinvestigatetheeffectsofdissolvednitrogenandoxygenmoleculesonthemorphologyoffibrilsformedbyahydrophobicamyloidpeptideimplicatedinamyotrophiclateralsclerosis,15repeatsofglycine−alanine,onahighlyorientedpyrolyticgraphitesubstrate.Westartedwithpreformedfibrilsolutionsthatwerethendilutedwithbuffersofdifferentgasconditions,resultingintheaggregationofthefibrilsintodifferentmorphologiesthatwererevealedbyAFMafteradsorptiononthesubstrate.Straightfibrilswereobservedinbothdegassedandambientbuffers,butastrongerlateralassociationwasseenindegassedbuffers.SmallerandsofterfibrilswereobservedinO2-supersaturatedbuffers,andplaque-likefibrilaggregatesofconsiderablylargesizewereevidentinN2-supersaturatedbuffers.Inovernightincubationexperiments,weobservedchangesinboththemorphologyandheightofthefibrilaggregates,andtheirevolutionvariedwithdifferentgasconditions.Thesefindingsindicatethatthegastypeandconcentrationaffecttheaggregationofamyloidfibrilsandmayfacilitatethedevelopmentofbiomaterialapplicationsandtreatmentsforamyloid-relateddiseases.■INTRODUCTIONbetweengasesandmacromolecules,particularlytheeffectofdissolvedgasesonaggregationofamyloidfibrils.AmyloidsareatypeofproteinaggregationformingfibrilswithInthiswork,weusedatomicforcemicroscopy(AFM)tocross-βsheetstructuresassociatedwithmanydebilitatingneurodegenerativediseasessuchasAlzheimer’sdisease(AD),investigatetherolesofdissolvedN2andO2inaggregationoffibrilsofdipeptiderepeats(DPRs),whichwerediscoveredinParkinson’sdisease,Huntington’sdisease,andamyotrophic1−3patientswithfrontotemporallobardementiaandALScarryinglateralsclerosis(ALS).Apossiblecauseoftheseneuro-24theC9ORF72hexanucleotideexpansion(C9FTD/ALS).degenerativediseasesistheaccumulationofspecificmisfolded4,5Thisabnormalhexanucleotideexpansionresultsintheproteinsorpeptides.InadditiontotheirroleinhumantranslationoffiveDPRs,amongwhichpolyglycine−alaninediseases,amyloidfibrilshavealsodrawninterestfortheir(GA)isthemosthydrophobicandthemostpronetoamyloidpossibleapplicationsinfieldssuchasnanotechnology,drug246−8formation.Inthisstudy,weemployedapolypeptideDownloadedviaUNIVOFCONNECTICUTonMay16,2021at10:17:17(UTC).delivery,andmolecularbiomaterials.Therefore,furthercomposedof15repeatsofGA(i.e.,apeptidepolymerwithexplorationisrequiredtounderstandthefundamentalnature30aminoacids).Ourearlierstudybasedontransmissionofamyloidfibrils.TheassemblyandmorphologyoffibrilsareSeehttps://pubs.acs.org/sharingguidelinesforoptionsonhowtolegitimatelysharepublishedarticles.electronmicroscopy,dynamiclightscattering,atomicforcesusceptibletoalterationbyenvironmentalfactorsincludingsalt9−17microscopy(AFM),andFouriertransforminfraredspectros-concentration,pH,substrate,andpeptidesequence.copyindicatesthat(GA)15peptidesrapidlyformamyloidUnderambientconditions,aqueoussolutionsalwayscontainfibrilscontainingacross-βstructureafterincubationinacertainconcentrationofdissolvedairgases.Itisofinterestto24ambientphosphatebuffer.Inthepresentstudy,wediluteddeterminewhetherthesegasesaffecttheassemblyandfibrilsolutionswithdegassed,ambient(nogascontrol),O2-morphologyofamyloidfibrils.Becauseofthelowsaturationsupersaturated,andN2-supersaturatedbuffers,respectively,concentrationofairgases(molecularratio∼10ppmforN2andinvestigatedtheeffectofdissolvedgasesontheand∼5ppmforO2),thisquestionislargelyignored.However,morphologyofpreformedfibrilsonahighlyorientedpyrolyticmanystudieshaveindicatedthatdissolvedgaseshaveastrong18−22graphite(HOPG)substrate.WeadoptedHOPGherebecauseenrichmenttendencynearhydrophobicsurfaces.Hence,dissolvedN2andO2moleculesmayaffecttheassemblyandmorphologyofbiologicalorbiorelatedmolecules,asmanyReceived:November5,2020suchmoleculeshavehydrophobicdomains.Recently,ithasPublished:December22,2020beenreportedthatambientairgases(specificallyN2andO2)arepresentinsidetypicalmembranelipidbilayersandthatgas23accumulationenhancestherigidityoflipidvesicles.There-fore,anurgentneedhasarisentoinvestigatetheinteraction©2021AmericanChemicalSocietyhttps://dx.doi.org/10.1021/acs.langmuir.0c03215516Langmuir2021,37,516−523

1Langmuirpubs.acs.org/LangmuirArticleFigure1.Formationofdifferent(GA)15fibrilaggregatestructuresonHOPGafterdilutioninambient,degassed,O2-,andN2-supersaturatedbuffersonthe1stday.Thefinal(GA)15concentrationwas2μM.(a−d)AFMheightimagestakeninambient,degassed,O2-,andN2-supersaturatedbuffers,respectively.Thetimefordataacquisitionisindicatedatthetopofeachheightimage.(e−h)CorrespondingAFMadhesionimagesof(a−d),respectively.Thefibrilsin(e,f)exhibitcertainpreferentialorientations,asindicatedbywhitearrows.Yellowarrowsin(c,g)indicateagas-containingstructure.(i−l)Higher-resolutionheightimagestakeninambient,degassed,O2-,andN2-supersaturatedbuffers,respectively.(m−p)Heightprofilesalongthewhitedashedlinesin(i−l),respectively.(q)Averageheightofthe(GA)15peptideinbufferwithdifferentgasconcentrations.Scalebaris100nm.AFMimagingwasconductedwithin2hafterdepositionofasolution.ThelevelofstatisticalsignificanceisexpressedasaPvalue.*P<0.05;***P<0.005.517https://dx.doi.org/10.1021/acs.langmuir.0c03215Langmuir2021,37,516−523

2Langmuirpubs.acs.org/LangmuirArticleitisahydrophobicsubstrate;generally,hydrophobicmoleculesfibrilsolutionwasdilutedinphosphatebufferwithdifferentgasandstructurestendtoadsorbonhydrophobicsubstratesbutconditions,thesolutionwasimmediatelydepositedonanotonhydrophilicones.freshlycleavedHOPGsubstrate,anddifferentsurfacemorphologiesofthepeptidefibrilstructuresformedonthe■substratewereobserved(Figure1).FordilutionwithambientEXPERIMENTALSECTIONbuffer(oxygenconcentration6.5mg/L),AFMimagesshowedPeptideSynthesisandPreparation.(GA)15isa30residueindividualstraightpeptidefibrilsontheHOPGsurface(Figurepeptidecomposedof15repeatsofglycine−alaninedipeptides;boththeseaminoacidsarehydrophobic.(GA)wasproducedbysolid-1a,e,i);alateralassociationbetweenneighboringfibrilswas15phasepeptidesynthesisattheGenomicsResearchCenter,Academiaobservedoccasionally.Fordilutionwithdegassedbuffer(O2Sinica,Taiwan.The(GA)15peptidewasdissolvedat4mg/mLinconcentration1.6mg/L),thefibrilsexhibitedstrongerlateralhexafluoroisopropanol(HFIP,Sigma)toeliminatepreaggregatesandassociationsandformedrelativelymoretwo-dimensional(2D)wasincubatedatroomtemperaturefor2h.HFIPwasevaporatedinapatchesofvariousshapes(Figure1b,f,j)comparedwithvacuum,andthepeptidewasfurtherdissolvedindichloroaceticacidambientbuffer.Notably,forboththesecases,thefibrils(DCA,Sigma).(GA)15inDCAwasthenaddedto100mMNa2HPO4exhibitedthreepreferentialorientationsreflectingthethreefoldbufferandthesolutionwasadjustedtohaveapHof7.4.ThefinalsymmetryoftheHOPGsubstrate(Figure1e,f).FordilutionDCAconcentrationwas0.1%.Thepeptidesuspension(concentrationof100μM)wasstoredforseveralhoursatroomtemperature,withO2-supersaturatedbuffer(O2concentration>50mg/L),resultinginfibrilformation.Thefibrilsolutionwasmixedbyavortexweobservedfibrilaggregatesthatweresmallerandmoremachineandthengentlydilutedto2μMwitheitherdegassedbuffer,circularthanintheaforementionedtwocases(Figure1c,g,k).ambientbuffer,orgas-supersaturatedbufferbeforebeingappliedtoaThefibrilswerenotstraightandappearedtobecurvedorfreshlycleavedHOPGsubstrateforAFMimaging.entwined,suggestingthattheywerenotasstiffasinthePreparationofBuffersofDifferentGasConditions.aforementionedcases.FordilutionwithN2-supersaturatedPreparationofanambientbuffersolutionstartedwith100mMbuffer(O2concentration1.7mg/L),largefibrilaggregatesofNa2HPO4buffer,andthesolutionwasadjustedtopH7.4andirregularshapewereobserved(Figure1d,h,l).NopreferentialsupplementedwithasmallamountofDCA(concentration0.1%.).Forpreparationofdegassedbuffer,severaltubesofambientbuffer(2orientationswereobservedforthefibrilsinO2-andN2-mLeach)wereplacedinadesiccatorandpumpedto∼0.1atmwithsupersaturatedbuffers.Themeasuredheightsofpeptidefibrilsanoil-freevacuumpump(Rocker410,Rocker).Thebufferwasrevealedmainlysingle-layerfibrilsinambientanddegassedstoredinthedesiccatorforonenightbeforeuse.Forpreparationofbuffers(Figure1m,n).However,thefibrilsappearedtostackinthegas-supersaturatedbuffer,severaltubesofambientbuffer(5−10theaggregatesformedintheO2andN2-supersaturatedbuffersmLeach)wereplacedinapressuretank(TNKB1-3;Misumi)andbecauseofthelargeraggregateheights(Figure1o,p).Thethenpumpedtoapproximately0.1atmfor30minwithanoil-freeaverageheightoffibrilswas0.53±0.06nm(n=32),0.59±vacuumpump(Rocker410,Rocker).Thetankwasthenpressurized0.10nm(n=31),0.77±0.15nm(n=31),and1.02±0.11to3.7atmwithhigh-purity(99.999%)N2orO2.Anairfilternm(n=31)forambient,degassed,O-supersaturated,andN-(MSAF8A-0.01,Misumi)withaporesizeof0.01μmwasconnected22supersaturatedbuffers,respectively(Figure1q).Thelargebetweenthehigh-pressuregascylinderandthestainless-steelpressuretank.ThebufferwasstoredinthepressurizedtankforseveraldaysheightvariationinthecaseofO2-supersaturatedbuffer(Figurebeforeuse.Onetubeofthesolutionsplacedinthesamedesiccatoror1o,q)suggestedvariablesingletomultilayerstackingofthepressuretankwaschosenforthemeasurementofO2concentrationfibrils.usinganoxygenmeter(PCD650,Eutech).AllprocedureswereThemajorfeaturesofthefibrilmorphologyonHOPGdidconductedatroomtemperature.notchangeovertimeinthefirst2−3hafterdepositionoffibrilAFM.AFMmeasurementswereperformedonaBrukerAXSsolutions,eventhoughadsorptionofmorefibrilswasobservedMultimodeNanoScopeVequippedwithacommercialfluidcelltipintheveryearlystageofdeposition(FigureS2).Wedidnotholder.Thesubstrateforsampleadsorptionwasasquarepieceofobservecharacteristicsofself-assemblyofindividualpeptidesHOPG(lateralsizeof12mm×12mm,ZYH;StructureProbe,Inc.).orproteinsonthesubstratesurface,suchascontinuousgrowthThepeptide-containingsolutionwasinjectedontoaHOPGsubstrateintheliquidcell.Thesurfacewasthenimagedwiththepeakforceofthefibrillengthorincreasesintheheightorlateralwidthoftapping(PFT)modeunderaliquidenvironment.ForthePFTfibrils.25,26mode,thesamplewasoscillatedinaverticaldirectionwithanMorphologicalEvolutionof(GA)15FibrilsAfteramplitudeof35−100nmandatafrequencyof2kHz(SupportingOvernightIncubationinOxygen-SupersaturatedBuf-Information,FigureS1).Theverticalpiezomovementresultedinfer.ForthefibrilsampledilutedwithO2-supersaturatedcyclesofapproachingandretractingtraces,inwhichthetipmadephosphatebuffer,weobservedchangesinthemorphologyafterintermittentcontactwiththesamplesurface;inthisway,aforce−overnightincubation(Figure2).Figure2ashowsanAFMdistancecurvewasacquiredforeachcycle.Topographyinformationimageacquiredonthe1stday,withfibrilaggregatesdistributedwasobtainedfromtheheightcorrectionperformedbythefeedbacklooptokeepaconstant“peak”offorce.Severalproperties,suchasontheHOPGsurfacewithpreferentialadsorptionnearthestiffnessandadhesion,couldthenbeinferredfromtheforce−distancesubstratestepedges.Theheightofthefibrilaggregateswascurve.BacksideAu-coatedSicantilevers(Nanosensors,FM-AuD)0.7−0.8nm(Figure2b).Thissamplehadthesamewitharesonancefrequencyof22−32kHzinthebuffersolutionandaexperimentalconditionsasthatforFigure1c.Figure2c,dspringconstantof2−4N/mwereused.Thenominaltipradiuswasshowsthecorrespondingstiffnessandadhesionmaps,approximately10nm.PriortotheAFMmeasurements,theAFMrespectively,acquiredalongwithFigure2a.InadditiontoprobewascleanedwiththeUVlight.InAFMimaging,thescanratethefibrils,manyround-shapedstructuresexhibitedlowerwasapproximately1Hzandthepeakforcewassetat125−550pN.stiffness(darkcontrastinstiffness,Figure2c)andstrongerAllAFMimagingwasperformedatroomtemperature.adhesion(brightcontrastinadhesion,Figure2d)relativeto■theHOPGsubstrate,andthelateralsizewastypicallyseveralRESULTSANDDISCUSSIONhundrednanometers.Surprisingly,thenearlyround-shapeEffectsofDissolvedAirGasesontheFibrilstructureswerenotvisibleandappearedasbareHOPGMorphologyof(GA)15onthe1stDay.Aftera(GA)15surfacesintheheightimage(Figure2a).Theseareprobably518https://dx.doi.org/10.1021/acs.langmuir.0c03215Langmuir2021,37,516−523

3Langmuirpubs.acs.org/LangmuirArticlestructuresbecamesmallerafterovernightincubation.Someoftheroundstructurescouldbeobservedascap-shapednanostructuresintheheightimage,asshowninFigure2e.Theshapeoftheseroundstructuresandtheirevolutionresemblethenucleationprocessofsurfacenanobubbles27reportedinapreviousAFMstudy.ThatstudyindicatesthatgasadsorptiononanHOPGsurfaceinitiallyformsa2Dthinwettinglayerwithanalmostcircularshape,whichmaybelatertransformedintoacap-shapednanostructure(asurfacenanobubble)afterfurthergasadsorption.Afterthetrans-18formation,theheightincreasesandthelateralsizedecreases.OurobservationsshowninFigure2matchwellwiththoseofthepreviousstudy,sowecanconcludethatO2surfacenanobubblesalsoformedinFigure2.Thereasonthatmanygas-containingstructurescouldnotbeseenintheheightimages(Figure2a)isbecausethetipwashydrophobic,probablycausedbyadsorptionofthehydrophobic(GA)15moleculesontothetip.Thisisalsoevidencedbytherelativelylargeadhesionmeasuredonthesegas-containingstructures(Figure2d,h).Arelativelylargesnap-inmightoccurwhena28hydrophobictipcontactsgas-containingstructures,whichcancausethetiptopenetrateacertaindepthintothefluid-likegas-containingstructures.Ifthethicknessofthegas-containingstructuresislessthanthepenetrationdepth,thestructuresdonotappearintheheightimagebecausethetippenetrates29throughthestructuresandreachestheHOPGsubstrate.However,thestiffnessandadhesionmapsstillreflectthepresenceofthefluid-likegas-containingstructures.Nano-bubblesthickerthanthepenetrationdepthwillshowupintheheightimages,buttheapparentheightissmallerthanthetrue29,30heightbythepenetrationdepth.Thestiffnessandadhesionmapsshownherecanonlybeusedforqualitativeinterpretationandfordistinguishingbetweendifferentstructures.Mostofthestructuresstudiedhereweretoothin(<2nm)forextractionofquantitative31stiffness.Inaddition,extractionofquantitativestiffnessvaluesusingPFTrequirescarefulcalibrationofmany32parametersandmeetingsomefrequencyrequirements.ForFigure2.Evolutionof(GA)15fibrilsonHOPGinoxygen-theadhesionmap,thequantitativevaluesdependonthesupersaturatedbufferwithovernightincubation.Theconcentrationchemistryofthetip,whichvariesovertimeduetoadsorptionof(GA)15was2μM.(a)Heightimageacquiredat52minafterofhydrophobicmoleculesorfibrils.Themorphologyoffibrilsdepositionofthedilutedbuffer.(b)Heightprofilealongthewhitelinewithinthedashedboxin(a).(c,d)CorrespondingstiffnessandinFigure2ahasasomewhatdifferentappearancefromthatinadhesionmapsacquiredalongwith(a),respectively.(e)HeightFigure1c.Thisismainlybecausemostofthefibrilsappearedimageacquiredonthe2ndday.(f)HeightprofilealongthewhitelineneartheHOPGstepedgesinFigure2a,owingtotherelativelywithinthedashedboxin(e).(g,h)Correspondingstiffnessandhighdensityofstepedgesandthepresenceofmanygas-adhesionmapsacquiredalongwith(e),respectively.Whiteboxescontainingstructures.Thegas-containingstructureswereoutlinethesameareaintheseimages.Scalebar:100nm.mobileontheterraces(flatregions)intheinitialstageof27theirformation,andmightpushthefibrilstoadsorbneartwo-dimensional(2D)oxygen-containingstructuresatthestepedges;hence,thefibrilsdidnotappearcircularasdidHOPG−waterinterface,asexplainedlater.Notably,afterthoseonflatterraces(Figure1c,g,k).Therewerealsofibrilsovernightincubation,many(GA)15aggregatesdisappearednearstepedgesinFigure1c,g,k;althoughtheirappearancewasfromthesurface,withonlyafewfragmentsremaining(Figurealsoaffected,theheightdistributionandthenonstraight2e).Theheightoftheremainingfibrilsdecreasedtofeaturesweresimilartothoseofthefibrilsonflatterraces.approximately0.5nm(Figure2f).AnenlargedviewoftheMorphologicalEvolutionof(GA)15FibrilsAfteroutlinedregioninFigure2a,eisshowninFigureS3.WeOvernightIncubationinNitrogen-SupersaturatedBuf-observeddisappearanceof(GA)15aggregatesintwooutoffer.AsimilarexperimentwasconductedinN2-supersaturatedthreeindependentexperimentsusingO2-supersaturatedbuffer.buffer,andadifferentovernightevolutionofthefibrilFigure2g,hshowsthecorrespondingstiffnessandadhesionaggregateswasobserved(Figure3).Figure3a−cshowsheight,mapsacquiredalongwithFigure2e.Therewereroundstiffness,andadhesionmaps,respectively,acquiredonthe1ststructuresoflowstiffnessandhighadhesion.Thepositionsofday.Theheightimage(Figure3a)showsplaque-likefibriltheseroundstructurescorrespondwelltothoseofthenearlyaggregateswithaheightofapproximately1nm,similartothatround-shapedstructuresseeninFigure2c,d(oneisindicatedshowninFigure1d.Thestiffnessmap(Figure3b)showsawithayellowarrow),butthelateralsizeoftheroundlargedarkarea(indicatedwithagreenarrow),whichisalsoa519https://dx.doi.org/10.1021/acs.langmuir.0c03215Langmuir2021,37,516−523

4Langmuirpubs.acs.org/LangmuirArticlesimilarlargeplaque-likefibrilaggregatesonthe1stdayandsimilarevolutionafterovernightincubation(FigureS4).Theobservationsindicatethatthepresenceof2Dgas-containingstructuresandsurfacenanobubblesontheHOPGsurfacedidnotaffectthemorphology.Theimagesseenonthe1stdayshowedsimilarmorphologyoffibrilaggregates(FigureS4)andnosignofassemblyfrompeptidesonthesubstratesurface,indicatingthattheplaque-likefibrilaggregatesmighthavealreadyformedintheN2-supersaturatedsolutionpriortotheirdepositiononthesubstrate.MorphologicalEvolutionof(GA)15FibrilsAfterOvernightIncubationinAmbientBuffer.Forthesolutiondilutedwithambientphosphatebuffer,weobservedthedevelopmentoftwistedstrandsonanHOPGsurfaceafterovernightincubation(FigureS5).Theseresultsindicatedifferentovernightevolutionbehaviorsinthefibrilmorphol-ogyinbuffertreatedwithdifferentO2andN2concentrations.Weencounteredseveraltechnicalproblemsinlong-timeAFMimagingoftheevolutionof(GA)15fibrilsonHOPG.Atypicalproblemistipwear,whichcanbeminimizedthroughcarefulparametersetting,suchasasmallersetforceandaslowerscanspeed.Themajorcomplicationisadsorptionofhydrophobicmoleculesorfibrilsonthetip,renderingitmorehydrophobicandstrengtheninghydrophobicinteractionsbetweenthetipandthehydrophobicsurface.ThismaycauseseriousproblemsinAFMimaging,suchasstrongimagingforceorinstability,dependingonthehydrophobicityofthetip.Inaddition,adsorptionoflargehydrophobicstructuresonthetipcausesseriousdegradationinthespatialresolutionoftheacquiredimages,whichisacommonproblemweoftenencounteredduringourlong-timeimaging.Tominimizeadsorptionof(GA)15moleculesorfibrilsonthetip,wecleanedtheAFMprobewithaUVlightbeforehandtomakethetipmorehydrophilic.Thetipcouldusuallyobtainhigh-qualityimagesatthebeginningbutbecameincreasinglyhydrophobicandtheimageresolutiondegradedovertime.Anotherproblemthatoccurredwas,whenimaginginagas-supersaturatedbuffer,bubbleswouldoccasionallyformanddisrupttheimagingprocess.FurtherDiscussion.InthisAFMstudy,westartedwithFigure3.Evolutionof(GA)15fibrilsonHOPGinN2-supersaturatedpreformedfibrilsolutionspreparedwiththesamemethod,andbufferwithovernightincubation.Theconcentrationof(GA)15was2thendilutedthesolutionswithbuffersofdifferentgasμM.(a)Heightimageacquiredat31minafterdepositionoftheconditions.ThesefibrilaggregatesadsorbedonanHOPGbuffer.ThewhitearrowindicatesareferencepointforcomparisonsubstrateandwereobservedbyAFM.Differentfibrilamongimages.(b,c)Correspondingstiffnessandadhesionmapsacquiredalongwith(a).(d)Heightprofilealongthedashedwhitemorphologieswereobservedunderdifferentgasconditions.linein(a).(e)Topographicimageacquiredonthe2ndday.(f,g)Foreachexperiment,thefibrilmorphologyremainedroughlyCorrespondingstiffnessandadhesionmapsacquiredalongwith(e).thesameafterdepositiononthe1stday(FiguresS2andS4),(h)Heightprofilealongthedashedwhitelinein(e).Greenandblueandnocontinuousgrowthinlength,width,andheightofthearrowsindicatethepositionsofgas-containingstructures.Scalebar:fibrilswasobserved.Basedontheseobservations,we100nm.concludedthatthedifferentfibrilmorphologiesresultedfromdifferentaggregationinthebuffersolutionsunderdifferentgasfluid-likegas-containingstructureprecedingtheformationofaconditions,ratherthanbypeptideassemblyonthesubstrate.surfacenanobubble.ThisstructureexhibitedabrightcontrastThesubstratemayhaveonlyminoreffectsonfibrilintheadhesionmap(Figure3c).Afterovernightincubation,morphology,suchaspreferentialalignmentofthefibrilsthelateraldimensionofthe(GA)15plaque-likestructurealongthesubstratecrystaldirectionsinthedegassedandincreased(Figure3e),andtheheightofthefibrilsdecreasedtoambientbuffers(Figure1a,e,b,f)aswellaspreferential0.4−0.7nm(Figure3h).Thecorrespondingstiffness(Figuredepositionoffibrilaggregatesnearstepedgesinthepresence3f)andadhesion(Figure3g)mapsshowhighcontrastfortheofmanygas-containingstructures(Figure2a).Forthecasesgas-containingstructures(someareindicatedwithblueusinggas-supersaturatedbuffers(Figures1cand2fortheO2arrows).case;Figures3andS4fortheN2case),thegas-containingInanothersimilarexperimentwithAFMimaginginaregionstructuresonthesubstratesurface,includingsurfacenano-withnopresenceofgas-containingstructures,weobservedbubbles,didnotshowmucheffectonthefibrilmorphologies.520https://dx.doi.org/10.1021/acs.langmuir.0c03215Langmuir2021,37,516−523

5Langmuirpubs.acs.org/LangmuirArticleWeobservedacleardifferenceinthemorphologyof(GA)15Comparedwiththecaseofambientbuffers(nogascontrol),fibrilsbetweendegassedandambientsolutions,indicatingthatstrongerlateralassociationofstraightfibrilswasseenindissolvedairgasesinfluencethelateralassociationoffibrils.degassedbuffer,softer(moreflexible)andsmallerfibrilsorPreviousstudieshaveshownstrongenrichmentofdissolvedairfibrilaggregateswereseenintheO2-supersaturatedbuffer,and18−22gasesinthewaternexttoahydrophobicflatsurface.We,plaque-likefibrilaggregatesofconsiderablylargesizeweretherefore,conjecturethatdissolvedairgasesmightalsobeevidentintheN2-supersaturatedbuffer.Afterovernightenrichedinthewaternearthehydrophobicfibrilsandstabilizeincubation,weobserveddifferentlateralassociationsofpeptidethemandthatstrongerlateralassociationinthedegassedfibrilsunderdifferentgasconditions:theamountofsolutionisduetostrongerhydrophobicattractionsbetweenrecognizablepeptideaggregatesdecreasedinO2-supersatu-neighboringfibrilsintheabsenceofsuchgasenrichment.ratedbuffer;plaque-likestructuresinN2-supersaturatedbufferFurtherstudiesarerequiredtoconfirmthisproposal.becamethinnerwithalargerlateralsize;andfibrilsinambientWefurtherobservedthatenhancedconcentrationofO2andbufferdevelopedintosheetsandribbonstangledontheN2affectedthefibrilmorphologyandevolutionoffibrilHOPGsurface.ThecausesofthesesurprisingfindingswillassociationonanHOPGsurface.Wangetal.previouslyrequirefurthertheoreticalandexperimentalinvestigations.reportedthataphosphate-bufferedsalinesolutionpressurizedSuchinvestigationswillprovideafundamentalunderstandingwithairaffectedthestructureandmorphologyofassembledoftheconnectionbetweenamyloidsandgasconditionsthat33p11peptidefibrilscomparedwithanambientsolution.Bothmightfacilitatethedevelopmentofbiomaterialapplicationsstudiesconsistentlyindicatethatdissolvedgasesinanaqueousandtreatmentsforamyloid-relateddiseases.solutionaffectthemorphologyoffibrilsformedfromanamyloidpeptide.Inparticular,werevealedthedistincteffects■ofthegastypeontheaggregationoffibrilsinthesolution.InASSOCIATEDCONTENTthepresentstudy,weobservedmuchsmallerandsofterfibrils*sıSupportingInformationinO2-supersaturatedbuffers,andmuchlargerfibrilaggregatesTheSupportingInformationisavailablefreeofchargeatinN2-supersaturatedbuffers,comparedwiththecaseofhttps://pubs.acs.org/doi/10.1021/acs.langmuir.0c03215.ambientbuffer.TheobservationsindicatethatN2andO2Thepeakforcetappingmode;thetemporalevolutionofinteractdifferentlywiththefibrils,butthemechanismbehindthemorphologyoffibrils;andtheevolutionof(GA)15thesedifferencesremainstobeexplored.AsmisfoldedpeptidesfibrilsonHOPGinN2-supersaturatedbufferandoramyloidproteinshavebeenimplicatedinseveralneuro-ambientbuffer(PDF)degenerativediseases,itisimportanttocomprehensivelyinvestigatetheeffectsandrolesofdifferentgasspeciesinvariouspeptideaggregationsthroughexperimentaland■theoreticalapproaches.SuchstudieswillprovidefundamentalAUTHORINFORMATIONknowledgeconcerningneurodegenerativediseasesandincreaseCorrespondingAuthorsourunderstandingoftheeffectsofdifferentgasspeciesontheYun-RuChen−GenomicsResearchCenter,AcademiaSinica,34earth’sbiota.Taipei115,Taiwan;TaiwanInternationalGraduatePolymorphismofamyloidfibrilshasbeenproposedinPrograminInterdisciplinaryNeuroscience,NationalTaiwan3536,37variousamyloidproteinssuchastau,amyloidβ,andUniversityandAcademiaSinica,Taipei115,Taiwan;38transthyretin.Strain-specificpolymorphismofamyloidsmayorcid.org/0000-0002-6596-6338;Email:yrchen@alsocontributetothediversityofprogressionandsusceptibilitygate.sinica.edu.tw39−41toamyloidogenesis.DeterminationoftheaggregationIng-ShouhHwang−InstituteofPhysics,AcademiaSinica,stateandmorphologyofamyloidogenicproteins,suchasTaipei115,Taiwan;orcid.org/0000-0002-7670-1212;amorphousaggregatesandfibrils,involvesfactorspresentEmail:ishwang@phys.sinica.edu.tw42,43duringincubation.Here,wedemonstratedforthefirsttimethetransformationofamyloidfibrilsinresponsetoAuthorsenvironmentalgasconditions;ithasbeenreportedthatYa-LingChiang−InstituteofPhysics,AcademiaSinica,amyloidaggregationisacceleratedatthegas−liquidinter-Taipei115,Taiwan44,45face.Inourstudy,amyloidfibrilsreacteddifferentlywithYu-JenChang−GenomicsResearchCenter,AcademiaSinica,eitherO2orN2pressurizedinthesolution.HyperbaricO2Taipei115,Taiwan;TaiwanInternationalGraduate46,47therapyhasbeenadministeredtopatientswithAD.PrograminInterdisciplinaryNeuroscience,NationalTaiwanAdditionally,ischemicstrokeresultinginlackofoxygenintheUniversityandAcademiaSinica,Taipei115,Taiwanbloodisrelatedtoamyloiddepositioninthebloodvesselsand48Completecontactinformationisavailableat:withinthebrainparenchyma.Alongwithourfindings,thesehttps://pubs.acs.org/10.1021/acs.langmuir.0c03215studiessupporttheimportanceofunderstandingtheeffectsofdissolvedgasesonamyloidsandshouldfacilitatepotentialNotesclinicaldevelopment.■Theauthorsdeclarenocompetingfinancialinterest.CONCLUSIONS■Inthiswork,atomicforcemicroscopywasusedtoinvestigateACKNOWLEDGMENTStheeffectsofdissolvedN2andO2inthefibrilmorphologyofTheauthorsthankAcademiaSinica(AS-CFII-108-201and(GA)15onHOPGsubstrates.OurobservationsindicatethatAS-TP-109-LM-08)andtheMinistryofScienceandfibrilsaggregateintodifferentmorphologiesinbuffersofTechnology(MOST106-2112-M-001-025-MY3,MOSTdifferentgasconditions.Themorphologieswererevealedby109-2112-M-001-048-MY3,andMOST105-2314-B-001AFMafteradsorptionofthefibrilaggregatesonthesubstrate.-008-MY3)ofTaiwanforsupportingthisstudy.521https://dx.doi.org/10.1021/acs.langmuir.0c03215Langmuir2021,37,516−523

6Langmuirpubs.acs.org/LangmuirArticle■(19)Lu,Y.-H.;Yang,C.-W.;Hwang,I.-S.MolecularlayerofgaslikeREFERENCESdomainsatahydrophobic−waterinterfaceobservedbyfrequency-(1)Knowles,T.P.;Vendruscolo,M.;Dobson,C.M.Theamyloidmodulationatomicforcemicroscopy.Langmuir2012,28,12691−stateanditsassociationwithproteinmisfoldingdiseases.Nat.Rev.12695.Mol.CellBiol.2014,15,384.(20)Ko,H.-C.;Hsu,W.-H.;Yang,C.-W.;Fang,C.-K.;Lu,Y.-H.;(2)Goedert,M.Alzheimer’sandParkinson’sdiseases:TheprionHwang,I.-S.High-resolutioncharacterizationofpreferentialgasconceptinrelationtoassembledAβ,tau,andα-synuclein.Scienceadsorptionatthegraphene−waterinterface.Langmuir2016,32,2015,349,No.1255555.11164−11171.(3)Skovronsky,D.M.;Lee,V.M.-Y.;Trojanowski,J.Q.(21)Yang,C.-W.;Lu,Y.-H.;Hwang,S.CondensationofdissolvedNeurodegenerativediseases:newconceptsofpathogenesisandtheirgasmoleculesatahydrophobic/waterinterface.Chin.J.Phys.2013,therapeuticimplications.Annu.Rev.Pathol.Mech.Dis.2006,1,151−51,174−186.170.(22)Dammer,S.M.;Lohse,D.Gasenrichmentatliquid-wall(4)Morgado,I.;Fandrich,M.AssemblyofAlzheimer̈’sAβpeptideinterfaces.Phys.Rev.Lett.2006,96,No.206101.intonanostructuredamyloidfibrils.Curr.Opin.ColloidInterfaceSci.(23)Lee,C.W.;Chiang,Y.L.;Liu,J.T.;Chen,Y.X.;Lee,C.H.;2011,16,508−514.Chen,Y.L.;Hwang,I.S.EmergingRolesofAirGasesinLipid(5)Renton,A.E.;Majounie,E.;Waite,A.;Simon-Sánchez,J.;́Bilayers.Small2018,14,No.1802133.Rollinson,S.;Gibbs,J.R.;Schymick,J.C.;Laaksovirta,H.;Van(24)Chang,Y.-J.;Jeng,U.-S.;Chiang,Y.-L.;Hwang,S.;Chen,Y.-R.Swieten,J.C.;Myllykangas,L.;etal.AhexanucleotiderepeatTheglycine-alaninedipeptiderepeatfromC9orf72hexanucleotideexpansioninC9ORF72isthecauseofchromosome9p21-linkedexpansionsformstoxicamyloidspossessingcell-to-celltransmissionALS-FTD.Neuron2011,72,257−268.properties.J.Biol.Chem.2016,291,4903−4911.(6)Rajagopal,K.;Schneider,J.P.Self-assemblingpeptidesand(25)Rico,F.;Su,C.;Scheuring,S.Mechanicalmappingofsingleproteinsfornanotechnologicalapplications.Curr.Opin.Struct.Biol.membraneproteinsatsubmolecularresolution.NanoLett.2011,11,2004,14,480−486.3983−3986.(7)Du,X.;Zhou,J.;Shi,J.;Xu,B.Supramolecularhydrogelators(26)Shi,J.;Hu,Y.;Hu,S.;Ma,J.;Su,C.MethodandApparatusofandhydrogels:fromsoftmattertomolecularbiomaterials.Chem.Rev.UsingPeakForceTappingModetoMeasurePhysicalPropertiesofa2015,115,13165−13307.Sample.U.S.PatentUS20120131702A12008.(8)Maji,S.K.;Schubert,D.;Rivier,C.;Lee,S.;Rivier,J.E.;Riek,R.(27)Fang,C.-K.;Ko,H.-C.;Yang,C.-W.;Lu,Y.-H.;Hwang,S.Amyloidasadepotfortheformulationoflong-actingdrugs.PLoSNucleationprocessesofnanobubblesatasolid/waterinterface.Sci.Biol.2008,6,No.e17.Rep.2016,6,No.24651.(9)Chiang,Y.-L.;Chang,Y.-C.;Chiang,I.-C.;Mak,H.-M.;Hwang,(28)Song,Y.;Zhao,B.;Zhang,L.;Lü,J.;Wang,S.;Dong,Y.;Hu,J.S.;Shih,Y.-L.AtomicforcemicroscopycharacterizationofproteinTheOriginofthe“Snap-In”intheForceCurvebetweenAFMProbefibrilsformedbytheamyloidogenicregionofthebacterialproteinminEonmicaandasupportedlipidbilayer.PLoSOne2015,10,andtheWater/GasInterfaceofNanobubbles.ChemPhysChem2014,No.e0142506.15,492−499.(10)Khan,J.M.;Khan,M.S.;Ali,M.S.;Al-Shabib,N.A.;Khan,R.(29)Lu,Y.-H.;Yang,C.-W.;Fang,C.-K.;Ko,H.-C.;Hwang,S.H.Cetyltrimethylammoniumbromide(CTAB)promoteamyloidInterface-inducedorderingofgasmoleculesconfinedinasmallspace.fibrilformationincarbohydratebindingprotein(concanavalinA)atSci.Rep.2015,4,No.7189.physiologicalpH.RSCAdv.2016,6,38100−38111.(30)Yang,C.-W.;Lu,Y.-H.;Hwang,S.Imagingsurfacenanobubbles(11)Castillo,V.;Graña-Montes,R.;Sabate,R.;Ventura,S.atgraphite−waterinterfaceswithdifferentatomicforcemicroscopyPredictionoftheaggregationpropensityofproteinsfromtheprimarymodes.J.Phys.:Condens.Matter2013,25,No.184010.sequence:aggregationpropertiesofproteomes.Biotechnol.J.2011,6,(31)Yang,C.-W.;Investigatio,C.-H.C.;Ding,R.-F.;Liao,H.-S.;674−685.Hwang,S.Multiparametriccharacterizationofheterogeneoussoft(12)Kowalewski,T.;Holtzman,D.M.Insituatomicforcematerialsusingcontactpointdetection-basedatomicforcemicros-microscopystudyofAlzheimer’sβ-amyloidpeptideondifferentcopy.Appl.Surf.Sci.2020,522,No.146423.substrates:Newinsightsintomechanismofβ-sheetformation.Proc.(32)Amo,C.A.;Garcia,R.Fundamentalhigh-speedlimitsinsingle-Natl.Acad.Sci.U.S.A.1999,96,3688−3693.molecule,single-cell,andnanoscaleforcespectroscopies.ACSNano(13)Hoyer,W.;Cherny,D.;Subramaniam,V.;Jovin,T.M.Rapid2016,10,7117−7124.self-assemblyofα-synucleinobservedbyinsituatomicforce(33)Wang,Y.;Shen,Z.;Guo,Z.;Hu,J.;Zhang,Y.Effectsofmicroscopy.J.Mol.Biol.2004,340,127−139.nanobubblesonpeptideself-assembly.Nanoscale2018,10,20007−(14)Zhang,F.;Du,H.N.;Zhang,Z.X.;Ji,L.N.;Li,H.T.;Tang,L.;20012.Wang,H.B.;Fan,C.H.;Xu,H.J.;Zhang,Y.;Hu,J.;Hu,H.Y.;He,J.(34)Friedman,M.;Wilkins,S.,Jr.;Rothfeld,A.;Bromberg,P.EffectH.Epitaxialgrowthofpeptidenanofilamentsoninorganicsurfaces:ofventilationandperfusionimbalanceoninertgasrebreathingEffectsofinterfacialhydrophobicity/hydrophilicity.Angew.Chem.,Int.variables.J.Appl.Physiol.1984,56,364−369.Ed.2006,45,3611−3613.(35)Zhang,W.;Falcon,B.;Murzin,A.G.;Fan,J.;Crowther,R.A.;(15)Dai,B.;Kang,S.-g.;Huynh,T.;Lei,H.;Castelli,M.;Hu,J.;Goedert,M.;Scheres,S.H.W.Heparin-inducedtaufilamentsareZhang,Y.;Zhou,R.SaltsdrivecontrollablemultilayereduprightpolymorphicanddifferfromthoseinAlzheimer’sandPick’sdiseases.assemblyofamyloid-likepeptidesatmica/waterinterface.Proc.Natl.eLife2019,8,No.e43584.Acad.Sci.U.S.A.2013,110,8543−8548.(36)Fitzpatrick,A.W.P.;Debelouchina,G.T.;Bayro,M.J.;Clare,(16)Dong,M.;Hovgaard,M.B.;Mamdouh,W.;Xu,S.;Otzen,D.D.K.;Caporini,M.A.;Bajaj,V.S.;Jaroniec,C.P.;Wang,L.;E.;Besenbacher,F.AFM-basedforcespectroscopymeasurementsofLadizhansky,V.;Müller,S.A.;MacPhee,C.E.;Waudby,C.A.;Mott,matureamyloidfibrilsofthepeptideglucagon.Nanotechnology2008,H.R.;DeSimone,A.;Knowles,T.P.J.;Saibil,H.R.;Vendruscolo,19,No.384013.M.;Orlova,E.V.;Griffin,R.G.;Dobson,C.M.Atomicstructureand(17)Wang,J.;Zhu,Z.;Bortolini,C.;Hoffmann,S.;Amari,A.;hierarchicalassemblyofacross-βamyloidfibril.Proc.Natl.Acad.Sci.Zhang,H.;Liu,L.;Dong,M.DimensionalityofcarbonnanomaterialU.S.A.2013,110,5468−5473.impactingonthemodulationofamyloidpeptideassembly.Nano-(37)Colletier,J.-P.;Laganowsky,A.;Landau,M.;Zhao,M.;Soriaga,technology2016,27,No.304001.A.B.;Goldschmidt,L.;Flot,D.;Cascio,D.;Sawaya,M.R.;Eisenberg,(18)Yang,C.-W.;Miyazawa,K.;Fukuma,T.;Miyata,K.;Hwang,S.D.Molecularbasisforamyloid-βpolymorphism.Proc.Natl.Acad.Sci.DirectcomparisonbetweensubnanometerhydrationstructuresonU.S.A.2011,108,16938.hydrophilicandhydrophobicsurfacesviathree-dimensionalscanning(38)Ihse,E.;Ybo,A.;Suhr,O.B.;Lindqvist,P.;Backman,C.;forcemicroscopy.Phys.Chem.Chem.Phys.2018,20,23522−23527.Westermark,P.Amyloidfibrilcompositionisrelatedtothephenotype522https://dx.doi.org/10.1021/acs.langmuir.0c03215Langmuir2021,37,516−523

7Langmuirpubs.acs.org/LangmuirArticleofhereditarytransthyretinV30Mamyloidosis.J.Pathol.2008,216,253−261.(39)Sanders,D.W.;Kaufman,S.K.;DeVos,S.L.;Sharma,A.M.;Mirbaha,H.;Li,A.;Barker,S.J.;Foley,A.C.;Thorpe,J.R.;Serpell,L.C.;Miller,T.M.;Grinberg,L.T.;Seeley,W.W.;Diamond,M.I.DistinctTauPrionStrainsPropagateinCellsandMiceandDefineDifferentTauopathies.Neuron2014,82,1271−1288.(40)Morales,R.;Abid,K.;Soto,C.Theprionstrainphenomenon:Molecularbasisandunprecedentedfeatures.Biochim.Biophys.Acta2007,1772,681−691.(41)Toyama,B.H.;Kelly,M.J.S.;Gross,J.D.;Weissman,J.S.Thestructuralbasisofyeastprionstrainvariants.Nature2007,449,233−237.(42)Vetri,V.;Canale,C.;Relini,A.;Librizzi,F.;Militello,V.;Gliozzi,A.;Leone,M.AmyloidfibrilsformationandamorphousaggregationinconcanavalinA.Biophys.Chem.2007,125,184−190.(43)Ni,C.-L.;Shi,H.-P.;Yu,H.-M.;Chang,Y.-C.;Chen,Y.-R.Foldingstabilityofamyloid-β40monomerisanimportantdeterminantofthenucleationkineticsinfibrillization.FASEBJ.2011,25,1390−1401.(44)Jean,L.;Lee,C.F.;Vaux,D.J.Enrichmentofamyloidogenesisatanair-waterinterface.Biophys.J.2012,102,1154−1162.(45)Jiang,D.;Dinh,K.L.;Ruthenburg,T.C.;Zhang,Y.;Su,L.;Land,D.P.;Zhou,F.Akineticmodelforbeta-amyloidadsorptionattheair/solutioninterfaceanditsimplicationtothebeta-amyloidaggregationprocess.J.Phys.Chem.B2009,113,3160−3168.(46)Shapira,R.;Efrati,S.;Ashery,U.HyperbaricoxygentherapyasanewtreatmentapproachforAlzheimer’sdisease.NeuralRegener.Res.2018,13,817.(47)Harch,P.G.;Fogarty,E.F.HyperbaricoxygentherapyforAlzheimer’sdementiawithpositronemissiontomographyimaging:acasereport.Med.GasRes.2018,8,181−184.(48)Goulay,R.;Romo,L.M.;Hol,E.M.;Dijkhuizen,R.M.FromStroketoDementia:aComprehensiveReviewExposingTightInteractionsBetweenStrokeandAmyloid-betaFormation.Transl.StrokeRes.2020,11,601−614.523https://dx.doi.org/10.1021/acs.langmuir.0c03215Langmuir2021,37,516−523