LinköpingUniversity|DepartmentofPhysics,ChemistryandBiology

Bachelor’sthesis,16hp|ChemicalBiology:Physics,ChemistryandBiology

Springterm2018|LITH-IFM-G-EX—18/3513--SE

MethodDevelopmentforThermal

StabilityAnalysisbyCircular

Dichroism

ApplicationtotheAbp1pSH3domainfromyeast

LindaSjöstrand

Examinator,PatrikLundström

DatumDate2018-06-05

Avdelning,institutionDivision,DepartmentDepartmentofPhysics,ChemistryandBiologyLinköpingUniversity

URLförelektroniskversion

ISBNISRN: LITH-IFM-G-EX--18/3513--SE_________________________________________________________________Serietitelochserienummer ISSNTitleofseries,numbering ______________________________

SpråkLanguage Svenska/Swedish Engelska/English

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RapporttypReportcategory Licentiatavhandling Examensarbete C-uppsats D-uppsats Övrigrapport

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TitelTitleMethodDevelopmentforThermalStabilityAnalysisbyCircularDichroismApplicationtotheAbp1pSH3domainfromyeastFörfattareAuthorLindaSjöstrand

NyckelordKeywordAbp1p,SH3,thermalstability,chemicalstability,circulardichroismspectroscopy,fluorescencespectroscopy,differentialscanningcalorimetry,nuclearmagneticresonancespectroscopy.

SammanfattningAbstract

Thermalstabilityisanimportantandinterestingphysicalpropertyofproteins.Acommonmethodtostudyitbyiscirculardichroism(CD)spectroscopy.TheaimofthisstudywastotestmethodstoimprovethermalstabilityanalysisbyCDspectroscopy.ExperimentswereperformedusingtheAbp1pSH3domainfromyeastasamodelprotein.Thermaldenaturationwasmonitoredatmultiplewavelengths.Itwasconcludedthatfordatasetsofreasonablequalitythechoiceofwavelengthdoesnotaffecttheresults.AnapproachtoestimatestabilityofthermophilicproteinswastestedwherethermalstabilitywasmeasuredatdifferentconcentrationsofthedenaturantGuHCl.ThethermochemicaldatawasusedtoestimatethestabilityinabsenceofGuHClbyextrapolation.TheresultswerecomparedtothoseobtainedfromCDspectroscopyanddifferentialscanningcalorimetry.ItwasfoundthatastabilizingeffectfromlowconcentrationsofGuHClcomplicatedtheextrapolation.Itislikelythatthismethodismoresuccessfulifthereisnostabilizingeffect.TheeffectofΔCpinstabilityparametercalculationswasinvestigatedwithanexperimentallyandtheoreticallydeterminedΔCp.Thiswasfurtherinvestigatedwithsyntheticdatasets.TheΔCpusedincalculationshadnonotableeffect,aslongastherewasnocolddenaturation.AlthoughΔCpisnotnecessaryincalculations,itisaninterestingparameteritself.ΔCpcanbecalculatedfromthethermochemicaldatausedforextrapolation.TheresultsinthisstudydemonstraterobustnessinthermalstabilityanalysisbyCDspectroscopyandapotentialfordevelopment.

AbstractThermalstabilityisanimportantandinterestingphysicalpropertyofproteins.Acommonmethodtostudyitbyiscirculardichroism(CD)spectroscopy.TheaimofthisstudywastotestmethodstoimprovethermalstabilityanalysisbyCDspectroscopy.ExperimentswereperformedusingtheAbp1pSH3domainfromyeastasamodelprotein.Thermaldenaturationwasmonitoredatmultiplewavelengths.Itwasconcludedthatfordatasetsofreasonablequalitythechoiceofwavelengthdoesnotaffecttheresults.AnapproachtoestimatestabilityofthermophilicproteinswastestedwherethermalstabilitywasmeasuredatdifferentconcentrationsofthedenaturantGuHCl.ThethermochemicaldatawasusedtoestimatethestabilityinabsenceofGuHClbyextrapolation.TheresultswerecomparedtothoseobtainedfromCDspectroscopyanddifferentialscanningcalorimetry.ItwasfoundthatastabilizingeffectfromlowconcentrationsofGuHClcomplicatedtheextrapolation.Itislikelythatthismethodismoresuccessfulifthereisnostabilizingeffect.TheeffectofΔCpinstabilityparametercalculationswasinvestigatedwithanexperimentallyandtheoreticallydeterminedΔCp.Thiswasfurtherinvestigatedwithsyntheticdatasets.TheΔCpusedincalculationshadnonotableeffect,aslongastherewasnocolddenaturation.AlthoughΔCpisnotnecessaryincalculations,itisaninterestingparameteritself.ΔCpcanbecalculatedfromthethermochemicaldatausedforextrapolation.TheresultsinthisstudydemonstraterobustnessinthermalstabilityanalysisbyCDspectroscopyandapotentialfordevelopment.

AcronymsandAbbreviationsAbp1p SaccharomycescerevisiaeActin-BindingProtein1CD CirculardichroismCm MidpointofchemicaldenaturationDof DegreesoffreedomDSC DifferentialScanningCalorimetryGuHCl GuanidinehydrochlorideHis6-tag HexahistidinetagHSQC HeteronuclearsinglequantumcoherenceIMAC ImmobilizedmetalaffinitychromatographyNaPi SodiumphosphateatarbitrarypHNi NickelNMR NuclearmagneticresonanceOD600 Opticaldensityat600nmSH3 Srchomology3domainTEV TobaccoetchvirusTm MidpointofthermaldenaturationΔCp DifferenceinheatcapacitybetweenthenativeanddenaturedstateΔH Differenceinenthalpybetweenthenativeanddenaturedstateδ Chemicalshiftinunitsofppmχ2 Chi-square,targetfunctioninfittingmodel

TableofContent

1.Introduction.................................................................................................................................12.TheoreticalBackground..........................................................................................................22.1ProteinStability..................................................................................................................................22.1.1FactorsGoverningProteinStability......................................................................................................22.1.2ThermalStabilityAnalysisbySpectroscopy.....................................................................................22.1.3ChemicalStabilityAnalysisbySpectroscopy....................................................................................4

2.2TheModelProtein:Abp1pSH3.....................................................................................................52.3ProteinPreparation..........................................................................................................................62.3.1ExpressionSystem........................................................................................................................................62.3.2ProteinPurification......................................................................................................................................6

2.4CircularDichroismSpectroscopy.................................................................................................72.5DifferentialScanningCalorimetry...............................................................................................82.6FluorescenceSpectroscopy............................................................................................................82.7NuclearMagneticResonanceSpectroscopy..............................................................................9

3.MaterialsandMethods...........................................................................................................113.1ProteinPreparation........................................................................................................................113.1.1Transformation............................................................................................................................................113.1.2Expression......................................................................................................................................................113.1.3Harvest............................................................................................................................................................123.1.4ImmobilizedMetalAffinityChromatography.................................................................................123.1.5RefoldingandProteolyticCleavage....................................................................................................123.1.6ReverseImmobilizedMetalAffinityChromatography...............................................................123.1.7SampleConcentration...............................................................................................................................13

3.2SDS-PAGEAnalysis...........................................................................................................................133.3CircularDichroismSpectroscopy...............................................................................................133.4DifferentialScanningCalorimetry.............................................................................................143.5FluorescenceSpectroscopy..........................................................................................................143.6NuclearMagneticResonanceSpectroscopy............................................................................143.7GenerationofSyntheticData.......................................................................................................15

4.ResultsandDiscussion...........................................................................................................154.1ProteinPreparation........................................................................................................................154.2FarUVCDSpectra............................................................................................................................174.3ThermalDenaturationMonitoredatMultipleWavelengths............................................184.4ReversibilityofDenaturation......................................................................................................214.5StabilityAnalysisbyDifferentialScanningCalorimetry.....................................................224.6StabilityAnalysisbyNuclearMagneticResonance..............................................................234.7ChemicalStabilityAnalysis...........................................................................................................244.8ThermalStabilityinPresenceofGuHCl....................................................................................254.9DeterminationofΔCp......................................................................................................................284.10EffectofΔCpinCalculationofStabilityParameters...........................................................29

5.ConcludingRemarks...............................................................................................................316.FutureProspects.......................................................................................................................327.Acknowledgement...................................................................................................................328.References..................................................................................................................................339.Appendix.....................................................................................................................................36

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1.IntroductionProteinsareessentialtoallbiologicalsystems.Theytransportandstoremolecules,catalysereactions,transmitsignals,providestructure,generatemovementandcontrolgrowth.Proteinsunderpineveryreactioninbiologicalsystems.1Thefunctionofaproteinisdependentonitsstructure.Whenaproteindenaturesandlosesitsnativestructureitgenerallylooseitsfunctiontoo.2Thereforestudyingproteinsabilitytomaintaintheirstructure,theproteinstability,isofgreatinterest.Stabilityanalysiscangiveinsightintothecauseofcancer.3Itisalsousedtodevelopmorestableandefficienttherapeuticagents.4Whenanalysingproteinstabilitythedifferencebetweenstabilityparametersforthenativeanddenaturedstatearedetermined.Thestabilityofaproteincanbedefinedastheabilitytowithstandchangesintemperature,presenceofadenaturingagentorpHchanges.Thedenaturationcanbemonitoreddirectlybycalorimetryorindirectlybyspectroscopicprobes2.Acommonmethodtomonitorthermaldenaturationiscirculardichroism(CD)spectroscopy.Itisquick,doesnotrequirelargeamountsofproteinandthereisfreesoftwareavailableonlinetohelpinterprettheresults.5,6InthisstudythesoftwareCDpalwillbeusedtoanalyseresults.6ThermaldenaturationisusuallymonitoredbyCDspectroscopyatonesinglewavelength.Thiswavelengthischosenbasedonthesecondarystructurecontentofthestudiedproteinandatwhichwavelengththatsecondarystructureabsorbs.Thereisnoconventionforwhatwavelengthstochose.Forexamplesomerecommend216nmandothers218nmformonitoringdenaturationofβ-strands.5,7Thisstudywillinvestigatewhetherthechoiceofwavelengthaffectstheresult.Tobeabletodeterminethestabilityparameterstheproteinhastobefullydenaturedinthemeasurement.ThisisaproblemwhenanalysingthermophilicproteinsbyCDspectroscopy,asthetemperaturecontrollermaybedamagedattemperaturesabove90°C.7Thisstudywilltestanewapproachtosolvethisproblem.Thestabilityisstudiedinpresenceofdifferentconcentrationsofadenaturant,inthiscaseguanidinehydrochloride(GuHCl).Thestabilityparametersarethenplottedagainsttheconcentration.Thisthermochemicaldataisusedtopredictthestabilityinabsenceofdenaturantbyextrapolation.ThedefaultsettinginCDpalistosetthedifferenceinheatcapacitybetweenthenativeanddenaturedstate(ΔCp)tozerowhencalculatingthestabilityparameters.Thisassumptionmeansthattheenthalpyandentropyareindependentoftemperature,whichgenerallyisnottrueforproteins.ThisisassumedbecauseΔCpoftenisnotknownandbecausethatnoteventhecorrectΔCpvaluewouldgivetrueparameters.6InthisstudyitwillbeinvestigatedwhetherthestabilityparameterschangessignificantlywhenthetrueΔCpvalueisusedinthecalculationsinsteadofzero.

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TheaimofthisstudyistotestmethodstoimprovethermalstabilityanalysisbyCDspectroscopy.ExperimentswillbeperformedusingtheSrchomology3(SH3)domainfromSaccharomycescerevisiaeActin-BindingProtein1(Abp1p)asamodelprotein.TheresultwillbeanalysedinthesoftwareCDpal.AparallelgoalistoproducedatatotestthesoftwareCDpal2.0(Möller,etal.unpublished).

2.TheoreticalBackground

2.1ProteinStabilityInthissectionthefactorsgoverningproteinstabilityandtheparametersusedtoquantifystabilityispresented.Detailsinproteinstabilitymeasurementsandanalysisarediscussedforboththermalandchemicaldenaturation.Furthermore,thechemicaldenaturantusedinthisstudyisdescribed.

2.1.1FactorsGoverningProteinStabilityProteinsaremarginallystable.Thestabilizationisabalancingactofopposingforces.Whiletheforcesinvolvedinproteinfoldingandunfoldingarewellknown,theirrelativecontributionsarestilldebated.Whenanunfoldedpeptidechainisplacedinsolution,watermoleculeswillarrangethemselvestoformahydrationlayeraroundthehydrophobicpartsofthepeptide.Thishydrationprocessisdrivenbytheincreaseinenthalpy,althoughitislargelyunfavouredbythelossofentropy.Proteinfoldingisalsounfavouredbyentropyloss.However,proteinfoldingminimizestheexposedhydrophobicsurface,leadingtolesshydration.Thusthefoldedstateismorefavouredthantheunfoldedstate,simplybecauseitminimizesthelargelyunfavourablehydration.Thisphenomenoniscalledthehydrophobiceffect.8Thehydrophobiceffectisamajorcontributortoproteinfolding.Otherforcesaffectingproteinstabilityarehydrogenbonds,vanderWaalsinteractions,electrostaticforcesandhydrophobicinteractions.9

2.1.2ThermalStabilityAnalysisbySpectroscopyThethermalstabilityofaproteinistheabilitytowithstandheat.Whenanalysingthermalstabilitythesampleisheatedinsteps.Ateachtemperaturethesampleisallowedtoequilibrateforacertaintimebeforethemeasurementisperformed.InthisstudyCDspectroscopy,whichisdescribedinsection2.4,isusedtomonitorthermaldenaturation.TocalculatethestabilityparametersthedataisfittedusingthesoftwareCDpal.Theformulausedtofitthedataisderivedbelow.Theequilibriumbetweenthenativeanddenaturedstateforatwo-statefoldingprocessiswrittenas

! ⇌ ! (1)

whereNdenotesthenativestateandDthedenaturedstate.TheequilibriumconstantKisdefinedas

! = [!][!] =

!!!! (2)

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where[D]istheconcentrationofproteininthedenaturedstateand[N]theconcentrationofproteininthenativestate.XDandXNdenotesthemolefractionsinthedenaturedandnativestate,respectively.TheconformationalstabilityofaproteinisdefinedinthermodynamicsbythedifferenceinGibbsfreeenergybetweenthenativeanddenaturedstate.ThedifferenceinGibbsfreeenergyatstandardconditions(ΔG°)isdefinedas

∆!° = ∆!°− !∆!° (3)

whereΔH°istheenthalpychangeofunfoldingatstandardconditions,TisthetemperatureinkelvinandΔS°istheentropychangeofunfoldingatstandardconditions.Gibbsfreeenergycanalsobeexpressedintermsoftheequilibriumconstantas

∆!° = −!" ln! (4)

whereRisthegasconstant.9

Themidpointofthermaldenaturation,Tm,isdefinedasthetemperaturewheretheconcentrationsofdenaturedandnativeproteinarethesame.9AtthispointK=1andΔG°=0accordingtoequations2and4respectively.Equation3canthenberewrittenas

∆!° = ∆!°!!. (5)

Assumingthatthedifferenceinheatcapacityiszero,meaningthattheenthalpyandentropyaretemperatureindependent,combiningequation3withequation4givestheformula

! = exp !∆!°!∆!°!" . (6)

Combiningequation5and6givestheformula

! = exp !!!− !

!∆!°! . (7)

Themeasuredsignalateachtemperatureistheweightedsumofthesignalforthenativeanddenaturedstate.Assumingthatthereisalinearrelationshipbetweenthesignalforthestateandthetemperature,thesignalcanbedescribedas

! = !!!! + !!!! . (8)

Sdenotesthemeasuredsignal,SNthesignalforthenativestateandSDthesignalforthedenaturedstate.ThemolefractionsXNandXDcanbeexpressedasfunctionsoftheequilibriumconstantusingequation2,givingtheequation

! = !! !!!! + !! !

!!! . (9)

SubstitutingKusingequation7givesthefinalformula

! = !!!"# !

!!!!!∆!°!

!!!"# !!!!

!!∆!°!

+ !! !

!!!"# !!!!

!!∆!°!

. (10)

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Thesignalasafunctionofthetemperaturedescribesthethermaldenaturationprofile.ThesoftwareCDpalisusedtofindtheoptimalfitofthedatatoequation10byadjustingtheparametersTm,ΔHandthebaselinesignalsSNandSD.WhenthebaselinesareslopingSNandSDareexchangedtolinearfunctionsofthetemperature.6The stability parameters of interest in this study are TmandΔH. Thekeyassumptioninthisapproachisthatthedenaturationisreversible(equation1).Formanyproteinsthisisnottrue.Therefore,itshouldbeverifiedthattheinitialsignalcanberecoveredbyloweringthetemperaturetotheinitialvalueafterdenaturation.Itisalsoassumedthateachmeasurementisperformedatthermalequilibrium.Theequilibrationtimeof60secondsoftenusedislikelyfarfromadequate,thustheassumptionisviolated.DespitethisthedataisoftenwellfittedbyCDpalandsimilarsoftware.Itishoweverimportanttonotoverinterprettheresultsandtobecarefulwhencomparingdatafordifferentsystemsobtainedbydifferentmethods.6

2.1.3ChemicalStabilityAnalysisbySpectroscopyChemicalstabilityistheabilitytowithstandperturbationbydenaturantsorpHchanges.Toanalysechemicalstability,sampleswithdifferentconcentrationsofperturbingagentaremixedandallowedtoequilibrate.Thenmeasurementsareperformed.Inthisstudyfluorescence,describedinsection2.6,isusedtomonitorthedenaturation.Astheproteinunfoldthefluorescenceintensitydecrease.ThedataisfittedinCDpaltoequation9,withtheequilibriumconstantexpressedas

! = exp − !!" !! − ! . (11)

Cmisthemidpointconcentrationofchemicaldenaturation,theconcentrationofdenaturantatwhich[D]=[N].CistheconcentrationatwhichthesignalismeasuredandmistherateofchangeinΔG.6Theperturbingagentusedinthisstudyisguanidinehydrochloride(GuHCl).GuHClisasmallflatmoleculewiththechemicalformulaCN3H6Cl.ThestructureisshowninFigure1.Itinteractswiththehydrophobicregionsoftheproteinwithitsflatunpolarside,whileexposingthechargedandpolaraminogroupstothesolvent.Thisinteractionlowersthehydrophobiceffect,facilitatesthesolvationoftheproteinanddecreasesitsstability.10

Figure1.Structureoftheguanidiniumion.ThefigurewasretrievedfromWikimediaCommons.

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2.2TheModelProtein:Abp1pSH3SaccharomycescerevisiaeActin-BindingProtein1(Abp1p)isamemberofthehighlyconservedfamilyofactin-bindingproteins.Itbindstoactinfilamentsandisinvolvedinendocytosisandactinorganization.TheSrchomology3domain(SH3)intheC-terminusmediatesinteractionbetweenAbp1pandotherproteins.11TheSH3domainisahighlyconserveddomainfoundinalleukaryoticspecies.Itrecruitssubstratestoenzymes,mediateinteractionbetweenproteins,coupleintracellularpathways,regulateenzymeactivityandtakepartinproteinlocalization.Itisasmalldomainofapproximately60residuesfoldedintotwoorthogonalβ-sheetswithfiveantiparallelβ-stands.Thestrandsareseparatedbythreeunstructuredloops.Thereisonelongloop,calledtheRT-Srcloop,andtwoshorterloops.Threeresiduesareina310-helixconformation.12–14TheproteinstructureisshowninFigure2.

Figure2.Three-dimensionalstructureoftheAbp1pSH3domaininthreedifferentviews.ThefiguresweregeneratedusingthePDBfile1JO815andtheprogramPyMol(SchrödingerLLC).

TheSH3domaintypicallybindspeptidescontainingprolinerichregionswithPxxPmotifs,wherexcanbeanyaminoacid.TheinteractionbetweenastandardSH3domainandligandcanbesplitintotwoparts.ThebindingpocketisahydrophobiccleftthatbindsthePxxPmotif.Itisflankedbytwoloopsconstitutingtheso-calledspecificitypocketwhereelectrostaticinteractionsregulatethebindingspecificityandligandorientation.TheAbp1pSH3domainhasanunusualbindingspecificity.ItdoesnotseemtobindthetypicalSH3ligands.ThisispossiblyexplainedbythefactthatithasanegativelychargedresidueintheusuallyhydrophobicbindingpocketthatcoulddecreasethePxxPaffinity.TheAbp1pSH3domaininsteadtargetstheconsensussequence+xxxPxxPx+PxxL,wheretheplussigndenotesapositivelychargedresidueandLdenoteslysine.AnexampleofaligandisthekinaseArk1p.15

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2.3ProteinPreparation

2.3.1ExpressionSystemThemostcommonsystemusedtoexpressrecombinantgenesisthepETexpressionsystem.Thissystemallowsproteinexpressionwithhighspecificityandefficiency.InthepETvectorthetargetgeneisundercontrolofthestrongbacteriophageT7promoterandthelacoperator.16TotranscribeageneundercontrolofaT7promoteraspecificpolymeraseisrequired,namelytheT7RNApolymerase.Thevectoristhereforetransformedintoabacterialstrainwhichhasthegeneforthispolymerase,usuallyEscherichiacoliBL21(DE3).Thisstrainischosenbecauseitisabletogrowfastinminimalmediaandathighcelldensity.Italsohaslowproteaseabundanceallowingefficientproteinproduction.Anotherfeatureisthatitisgeneticallymodifiedtopreventitfrombeinginfectious,thisfacilitateslaboratorywork.17UnderT7repressiveconditionsboththepolymerasegeneandthetargetgenearesilencedandthecellsgrowatnormalrate.Theexpressionisinducedbyadditionoftheinducerisopropyl-β-d-thiogalactoside(IPTG),whichbindstothelacoperon.TheT7RNApolymeraseisefficientandout-competesthehostpolymerase,afterafewhoursthetargetproteinconstitutesthemajorityoftheproteinsinthecell.Afterinductionthecellgrowthslowsorstops.Thecellsarethereforegrowntoahighdensitypriortoinduction.16

2.3.2ProteinPurificationTofacilitatetheproteinpurificationthetargetproteinisoftenexpressedasafusionprotein,wheretheintroducedpeptidehashighaffinityforacertainresin.Acommonmethod,usedinthisexperiment,istoattachatagcontaining6histidineresidues(His6-tag).Thehistidinesidechainhasahighaffinityfornickel(Ni)ions.Thisisusedinimmobilizedmetalionaffinity(IMAC)wherethesampleisloadedonacolumncontainingaresinwithimmobilizedNi2+ions.TheHis6-taggedproteinbindstotheresinwhereasotherproteinsarewashedout.TheHis6-taggedproteincanbeelutedwithabuffercontainingimidazole,sinceimidazolecompeteswiththeHis6-taginbindingtotheresin.16E.coliisknowntoproducenativeproteinswithaffinityforNi,eitherduetometalbindingsitesorhistidines.TheseimpuritiesarenotremovedbyIMAC.18InthisexperimentthelysisandIMACareperformedunderdenaturingconditions.Therearetworeasonsforthis.WhenaproteinisdenaturedtheHis6-tagisfullyexposedfacilitatingthebindinginIMAC.Moreover,therefoldingcomposesanadditionalpurificationstepasthelargerandmorecomplexE.coliproteinsunabletoreform,formaprecipitatethatcanberemovedbycentrifugation.16TheHis6-tagiscleavedoffwithaTobaccoetchvirus(TEV)proteaseafterrefolding.ThecleavedoffHis6-tag,theHis-taggedTEVproteaseandremainingnativeE.coliimpuritiesabletobindtotheNicolumnisremovedefficientlybyreverseIMAC.Theimpuritiesbindtothecolumnwhilethetargetproteincomesoutwiththeflowthrough.16

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2.4CircularDichroismSpectroscopyCircularpolarizedlightiscomposedoftwocomponentsofequalmagnitude,onerotatinginaleft-handed(L)mannerandtheotherinaright-handed(R)manner.Anopticallyactive(chiral)sampleabsorbstheLandRcomponentstodifferentextent.ThisdifferenceinabsorptionismeasuredinCD.TheCDinstrumentmeasuresthedifferenceinabsorbance(ΔA=AR-AL)andusuallyreportsthesignalintermsoftheellipticity(θ)indegrees.TherelationshipbetweenΔAandellipticityis

! = 32.98×∆!.Thesignalcanbepositiveornegative,dependingonwhichcomponentisabsorbedmore.19InproteinstheopticallyactivepeptidebondscangiverisetoCDsignal.Aspectrumisobtainedwhenellipticityismeasuredasafunctionofwavelength.ACDsignalcanonlyarisewhenthelightisbeingabsorbed.Asdifferentstructuresabsorbatdifferentwavelengthsthesignalinaspectrumcanbeassignedtodistinctfeatures.α-heliceshavecharacteristicminimaat222and208nm,β-sheetsat216nmandunstructuredrandomcoilat198nm.AsecondarystructurereferencespectrumisshowninFigure3.Thisinformationcanbeusedtoestimatethesecondarystructurecontentofaprotein5.Thedifferenceinstructureandsignalbetweenthenativeanddenaturedstatecanbeusedtoanalyseproteinstability.6Thesignalismeasuredatdifferenttemperaturesandfittedasdescribedinsection2.1.2.

Figure3.CDreferencespectraforpuresecondarystructures:α-helix(black),β-sheet(red),randomcoil(green).20ThefigurewasgeneratedusingGrace(GraceDevelopmentTeam).

ProteinsamplesforCDspectroscopyshouldbefreefromaggregatesandparticlesthatcouldscatterlightanddecreasethesignaltonoiseratio.Itisimportanttochooseabufferthatdoesnotabsorbinthewavelengthrangebeingmeasured.Forexamplechlorideions,ethylenediaminetetraaceticacid(EDTA)andGuHClabsorbsbelow210nm.Dithiothreitol(DTT)absorbsbelow220nm.19

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2.5DifferentialScanningCalorimetryDifferentialScanningCalorimetry(DSC)isadirectmethodtomeasureproteinstabilitywheretheheatcapacityismeasuredasafunctionoftemperature.Heatcapacityistheenergyrequiredtoraisethetemperature1Kunderconstantpressure.21Theheatcapacityofasubstancedependsonhowmanywaystheheatenergycanbedistributed,forexamplebyvibrations,bondstretching,bendingandbreaking.Liquidwaterhashighheatcapacitysincetheheatenergyisusedtobreakhydrogenbondsratherthanraisethetemperature.22TheDSCinstrumenthastwowells.Foraprotein/bufferscanthesampleisloadedinonewellandbufferintheother.Thedifferenceinheatcapacitybetweenthetwowellsistheheatcapacitycontributionoftheproteinalone.Toaccountforanydifferencesinshapeorvolumebetweenthecellsabuffer/bufferscanismeasuredandsubtractedfromtheprotein/bufferscan.21TheDSCprofilehasonebaselineforthenativestateandoneforthedenaturedstate.Generallythedenaturedstatehashigherheatcapacitythanthenative,sincethedenaturedstatecaninteractwiththesolventinmoreways.Duringtheunfolding,energyisrequiredtobreakthebondsandtheheatcapacityishigh.ThisresultsinapeakfromwhichTmcanbedetermined,basedontheposition.Ifthewellvolumeandproteinconcentrationisaccuratelyknown,ΔHandΔCpcanbecalculated.ΔHiscalculatedfromtheareaunderthecurve.21ΔCpisgivenbytheheatcapacityshiftbetweenthebaselines.23

2.6FluorescenceSpectroscopyMoleculescanabsorblighttoreachanexcitedstate.Somemoleculesemitlightwhenreturningtogroundstate.Thesemoleculesarecalledfluorophores.Onenaturallyoccurringfluorophoreisthearomaticaminoacidtryptophan.Inthissectionthebasicprinciplesoffluorescenceanditsapplicationtoproteinstabilityisdescribed.Ingeneralfluorophoresareintheirgroundelectronicstateandlowestvibrationalenergylevel.Absorbanceoflightwithappropriatewavelengthwillexcitethemoleculeintoanuppervibrationallevelofthefirstelectronicexcitedstate.Theexcessenergyislost,usuallyasheat,untilitreachesthelowestvibrationallevel.Themoleculecanthenreturntogroundstatebyspontaneouslyemittinglight.Theintensityandwavelengthofthisemittedlightisthesignalmeasuredinthefluorometer.ThisprocessisvisualizedinFigure4.Wavelengthsofemittedlightareusuallylongerthanthoseofabsorbedlight.

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Figure4.AbsorptionofaphotonbringsthemoleculefromS0,thegroundelectronicstate,toS1,anexcitedstate(purplearrow).Energyislost,briningthemoleculetoalowervibrationalenergylevel(redarrows).Lightisspontaneouslyemitted(greenarrow)asthemoleculereturnstothegroundstate.ThefigurewasretrievedfromWikimediaCommons.

Whentheexcitedfluorophorecollideswithaquencheremissionisprevented.Watermoleculesandionsareexamplesofquenchers.Themoreexposedafluorophoreistothesolventandquenchers,thelowerintensitytheemittedlightwillhave.Whenaproteindenatures,thetryptophansbecomemoreexposedandfluorescenceintensitydecrease.Thismethodtomonitorchemicaldenaturationisusedinthisstudy.Anothermethodistostudytheemissionwavelengthshift.Theemissionwavelengthdependsonthepolarityoftheenvironment.Astheproteindenaturestheenvironmentbecomesmorepolarandtheemissionwavelengthincrease.24

2.7NuclearMagneticResonanceSpectroscopyNuclearmagneticresonance(NMR)spectroscopyisaversatiletool.Itcanbeusedtostudybindingproperties,stability,structureanddynamicsofproteins.ThissectionwilldescribethebasicprinciplesofNMR,thesamplerequirementsandtheapplicationofNMRinthisstudy.AllatomicnucleihaveapropertycalledspinthatischaracterizedbythespinquantumnumberI.Iiseither0oramultipleof1/2.Thespinofnucleicanbeorientedin2I+1numberofways.FornucleiwithI=1/2thisgivestwoorientations.Inabsenceofanexternalmagneticfieldthetwospinorientationsareofequalenergyandthenetmagnetizationiszero.Inanexternalmagneticfieldtheorientationparalleltothefieldhaslowerenergythantheorientationantiparalleltothefield.Thusthespinenergylevelssplit.Thisleadstoanunevendistributionbetweentheorientationsandanetmagnetizationofthenucleiinthesamedirectionasthemagneticfield.

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Thedifferenceinenergybetweenthesetwostatescorrespondstotheenergyofradiofrequencywaves.Thuspulsesofradiofrequencywavescanbeusedtomanipulatethenucleimagnetizationmoment.Thelengthofthepulseiscalculatedtotipthemagnetizationby90or180degrees.ThenetmagnetizationofthenucleiwillrotatewithacertainfrequencycalledtheLarmorfrequency(ν)andinduceavoltageinacoil.ThisvoltageistheobservedNMRsignal,whichiscalledfreeinductiondecay(FID).AFIDisvoltageasafunctionoftime,thisistransformedintovoltageasafunctionoffrequencyviaFouriertransformation.25Thelocalmagneticenvironmentofanucleusisaffectedbyanumberoffactors,givingnucleidifferentLarmorfrequencies.TheobtainedLarmorfrequencyforeachnucleusalsodependsontheinstrumentation,makingitdifficulttocomparespectraobtainedfromdifferentspectrometers.Thereforechemicalshift(δ)iscalculated,wheretheLarmorfrequencyiscomparedtoareferencenucleusdefinedashavingthechemicalshift0ppm.ThechemicalshiftisrelatedtotheLarmorfrequencyas

! = !!!!"#!!"#

×10! !!"

whereppmistheunitandνrefistheLarmorfrequencyofthesameisotopeinareferencecompound.ForproteinNMR,thereferencecompoundfor1His2,2-dimethyl-2-silapentane-5-sulfonicacid(DSS).Protonsofwaterhaveachemicalshiftat~4.7ppmat20°C.25,26AsampleintendedtobeanalysedbyNMRisusuallylabelledwithmagneticisotopessuchas13Cand15N,theyhaveI=1/2.Labellingisachievedbygrowingthecellsinadefinedculturemediumwithoutaminoacids,supplyingonlythedesiredisotopes.Thisforcesthecellstoproduceaminoacidsfortheproteinsfromtheatomspresent,leadingtoallproteincontainingthedesiredisotopes. Heteronuclearsinglequantumcoherence(HSQC)spectroscopyisanNMRexperimentgivinga2Dspectrumwith1Hsignalsononeaxisand15Nsignalsontheother.Everyaminoacidinaproteingivesapeak,exceptforproline.Thepeaksinthespectrumcanbeassignedtotheaminoacidsinaproteinbyrunningassignmentexperiments.TheHSQCspectrumisauniquefingerprintforeachproteinandcanbeusedtocontrolthataproteinisproperlyfolded.27InthisstudyaHSQCspectrumwasusedforjustthat.AdditionallythechangeintheHSQCspectrumuponheatingthesamplewasstudied.

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3.MaterialsandMethods

3.1ProteinPreparationTheSH3domainwasexpressedinaconstructwithaHis6-tag,TEVproteascleavingsiteandthioredoxindomain.Thesequenceoftheconstructispresentedinappendix.ApETvectorencodingampicillinresistancewasusedtoexpresstheconstruct.Inthissectionallthestepsintheproteinpreparationaredescribed.

3.1.1TransformationThreealiquotsof50µlelectro-competentE.coliBL21(DE3)cells(Novagen)werethawedonice.Transformationwasperformedintwoaliquots.Thethirduntransformedaliquotwasusedasacontroltoensurethatthecellsdidnotcontainanyplasmidbeforehand.0.5µlrecombinantplasmidwasaddedtotwoofthealiquots.Thebacteria-plasmidmixturewastransferredtosterileelectroporationcuvettes.TheywereelectroporatedusingtheEc1programonaMicroPulser(Bio-Rad).Itwascontrolledthatthetimeconstantswereintherange4-5.5.1mlLBmediawasaddedtotheelectroporationcuvettesandtothecontrol.Thecellswereallowedtorecoverfor45minutesinroomtemperature.50-100µlofallsampleswereplatedonLB-agarplatescontaining100µg/Lampicillin.Theplateswereincubatedinvertedwithoutshakinginroomtemperaturefor6hoursfollowedby37°Covernight.Itwasverifiedthatthecontrolplatehadnocolonies.

3.1.2ExpressionAlawnoftransformedbacteriafromoneplatewasdissolvedin2mlLBmediaandtransferredto50mlLBmediaina250mlbaffledflask.Itwasincubatedwithshakingat37°C.Thegrowthwasmonitoredbymeasuringtheopticaldensityat600nm(OD600)untilitreached1.1.Theculturewastransferredtofalcontubesandcentrifugedat3500rpmforapproximately8minutes.ThesupernatantwasdecantedandthebacteriawereresuspendedintheremainingLBmedia.100mlstarterM9culturemediumwaspreparedina500mlbaffledflaskwith1mMMgSO4,0.1mMCaCl2,0.5g/L15NlabelledNH4Cl(Sigma-Aldrich),4g/Lglucose,100µg/Lampicillin,1mg/Lofthiaminehydrochloride(SigmaAldrich)and1mg/Lbiotin(SigmaAldrich).TheresuspendedcellswereaddedtothestarterflaskyieldinganOD600of0.29.Theflaskwasincubatedwithshakingat37°CuntilOD600reached0.85.1LmainM9culturemediumwiththesamecompositionasthestarterculturemediumwaspreparedina5Lbaffledflask.Thestarterculturewasaddedtothemainflaskanditwasincubatedwithshakingat37°CuntilOD600reached0.67.Asampleof0.5mlwastakenoutforSDS-PAGEanalysis.ThesamplewasplacedinanEppendorftube,thecellswerecentrifugedat10000rpmfor1minuteandthesupernatantwasdiscarded.Thecellswerestoredat-20°Cuntilanalysis.Toinducetheproteinexpressioninthemainflask,IPTGwasaddedwiththefinalconcentration0.5mM,followedbyincubationwithshakingovernightat16°C.OD600afterincubationwas1.43.A0.5mlsamplewasremovedforSDS-PAGEthesamewayaspreviouslydescribed.

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3.1.3HarvestTheinducedculturewastransferredtocentrifugebottlesandthecellswerecentrifugedat6000rpmfor10minutesat4°C.Thesupernatantwasdecantedandthecellswereresuspendedin30mlof0.1Msodiumphosphate(NaPi),pH8.0,6MGuHCland2mMimidazole.Thesolutionwassonicated(Bransondigital,model450)at25%amplitudefor6×20secwith40secintervals.Thiswasfollowedbycentrifugationat13500rpmfor30minutesat4°C.Thepelletwasdiscardedandthesupernatantwasfilteredusinga0.45µmfilter.

3.1.4ImmobilizedMetalAffinityChromatographyA5mlprepackedNicolumn(GeneralElectric)wasusedforIMAC.Thecolumnwaswashedwith30mldistilledwaterandequilibratedwith30mlof0.1MNaPi,pH8.0,6MGuHCland2mMimidazole(bufferA).Thefilteredsamplewasappliedwiththerate0.5ml/min.Thecolumnwaswashedwith30mlofbufferA.Thesamplewaselutedwith6MGuHCl,0.2Maceticacid(bufferF)andtheflowthroughwascollectedinfivefractionsof5ml.ThewashandelutionwasfollowedbyproteinvisualizationwithBradforddye.50µlBradforddyewasmixedwith10µlofthesample.Samplescontainingproteingaveabluecolourduetoprotein-dyecomplexwhereasunbounddyeshowsabrowncolour.TheconcentrationintheelutedfractionswasdeterminedusingaP330Nanophotometer(IMPLEN).Theabsorbanceat280nmwasmeasuredandtheconcentrationwascalculatedwiththeextinctioncoefficient36565M-1cm-1andthemolecularweight24kDa(appendix).Allfractionswerepooledanddilutedtotheproteinconcentration0.5mg/mlwithbufferF.

3.1.5RefoldingandProteolyticCleavageThesamplewasdialyzedovernightagainst4Lof10mMTris,pH8.0,1MNaCland2mMDTT.Anadditionaldialysiswasperformedovernightagainst4Lof10mMTris,pH8.0,250mMNaCl,2mMDTTandonetabletofproteaseinhibitorcOmplete,EDTAfree(RocheDiagnostics).AstheGuHClconcentrationdecreasedandtheexpressedconstructrefolded,morecomplexproteinimpuritiesunabletorefoldformedaprecipitate.Theprecipitatewasremovedbycentrifugationat10000rpmfor10minutesat4°C.Thesupernatantwassterilefilteredusinga0.45µmfilter.Asampleof100µlwasremovedforSDS-PAGEanalysisbefore3mgHis6-taggedTEVprotease(madein-house)wasadded.Themixturewasincubatedfor24hoursatroomtemperatureandstoredat4°C.Anadditional100µlofthesamplewasremovedforSDS-PAGEanalysis.

3.1.6ReverseImmobilizedMetalAffinityChromatographyTheproteinsolutioncontainedDTTthatwouldinterferewiththeIMACandfurtheranalysis.ToremovetheDTTandchangebufferthesamplewasdialyzedovernightagainst55mMNaPi,pH8.0,110mMNaCl.TheSH3domainwasseparatedfromtheHis6-tagandtheTEVproteasewithreverseIMAC.ThereversedIMACwasperformedusingthesameequipmentaspreviouslydescribed.Thecolumnwasequilibratedwith55mMNaPi,pH8.0,110mMNaCl(bufferB).Thesamplewasappliedwiththeflowrate1ml/minandtheflowthoughcontainingtheSH3wascollectedinaflask.EDTAandNaN3wereaddedtotheflowthoughgivingthefinalconcentrations2.2mMand220µMrespectively.Thecolumnwaswashedwith

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bufferBandelutedwith44mMNaPi,pH8.0,88mMNaCland400mMimidazole.WashandelutionwasfollowedbyproteinvisualizationwithBradforddye.

3.1.7SampleConcentrationThesamplewasconcentratedtothefinalvolume7mlusingtheAmiconultrastirredcell(Merck)witha3.5kDacutoffmembranedisc.pHwasadjustedto7.0andtheproteinconcentrationwasmeasuredasdescribedinsection3.1.4,usingtheextinctioncoefficient20970cm-1M-1andthemolecularweight6990Da(appendix).Theconcentrationwas2.9mg/ml,or420µM.Theproteinwasstoredat4°Cin55mMNaPi,pH7.0,110mMNaCl,2.2mMEDTAand220µMNaN3.Thisproteinstocksolutionwasusedforallexperiments.

3.2SDS-PAGEAnalysisOneSDS-PAGEgelwasruntoensurethatthetransformationandcleavingwassuccessful,anothertoensurethepurityafterpurification.Mini-PROTEANTGX(Bio-Rad)precastgels4-20%with10-wellcombswereused.ThesampleswerepreparedbyadditionofloadingdyecontainingDTTandheatingat95°C.Thecellsampleswereheatedfor10minutesandtheproteinsamplesfor1minute.Thegelswereloadedwith5µlPageRulerprestainedproteinladder(ThermoFisherScientific)and20µlofcellsamples.10and20µloftheproteinsampleswereloadedinseparatewells.Thegelswererunat280Vfor25minutes.Thegelwasplacedindistilledwaterandheatedclosetotheboilingpoint.Thewaterwasthenpouredof.ThiswashingprocedurewasrepeatedintotalfourtimesbeforethegelwascoveredwithSimplyBlueSafeStainandheatedonceagain.Thegelwasstainedfor30minutesatroomtemperaturewithgentleshaking.Thestainwaspouredoffandthegelwasplacedinwater.

3.3CircularDichroismSpectroscopyAllcirculardichroism(CD)measurementswereperformedonaChirascanspectrometer(AppliedPhotophysics)usinga1mmcuvette.Thermaldenaturationswererecordedintheholdertemperaturerange20-94°Cwithstepsof2°C,60secsettingtimeand10repeats.Theaverageofthe10repeatswascalculatedforeachtemperature.Aprobemeasuringthetemperatureinthesamplewasused,theholdertemperaturewasoverwrittenwiththeprobetemperature.DataanalysiswasperformedinCDpalwhereerrorsareestimatedbytheJackknifemethod28andCDpal2.0whereerrorsareestimatedwiththeJackknifedelete-dmethod.Inordertofindwavelengthssuitableformonitoringdenaturation,farUVspectrawererecordedattheholdertemperatures20and94°C.Asampleof42µMprotein,55mMNaPi,pH7.0,11mMNaCl,22µMEDTAand2.2µMNaN3wasused.Thespectrawererecordedinthewavelengthrange205-260nmwithtenaveragedrepeats.Thethermaldenaturationwasthenmonitoredatwavelengthsfrom210to240nm,with2nmsteps.Asamplewiththesameconcentrationsasforthespectrawasused.Theobtaineddenaturationprofileswerestudiedtoevaluatethequalityofthedata.Thewavelength216nmwaschosenformonitoringtheunfoldinginfurtherexperiments.

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Toincreasethesignalsamplesof63µMprotein,55mMNaPi,pH7.0,16.5mMNaCl,33µMEDTAand3.3µMNaN3wereusedforfurtherexperiments.Reversibilityofthedenaturationwascontrolledbymonitoringboththeunfoldingandrefoldingat216nm.AGuHClstocksolutionwaspreparedwith55mMNaPi,pH7.0and6MGuHCl(ultrapure,AppliChem).TheconcentrationofGuHClinthestocksolutionwasdeterminedbyrefractometry29.TheGuHClconcentrationerroroflessthan0.5%wasneglected.SampleswithGuHClconcentrationsintherange0-1.4Mwerepreparedandequilibratedovernightat4°Candthermalmeltswererecorded.

3.4DifferentialScanningCalorimetry2mloftheproteinstocksolutionwasdialyzedagainst2×0.5Lof55mMNaPi,pH7.0,16.5mMNaCl,33µMEDTAand3.3µMNaN3.ThecalculationofΔHandΔCpfromDSCdataisconcentrationdependent,asdescribedinsection2.5.Tomoreaccuratelydeterminetheproteinconcentration,25µlofthesamplewasmixedwith75µlof6MGuHClbuffertodenaturetheproteinandfullyexposethetryptophans.Theconcentrationwasthenmeasuredasdescribedinsection3.1.4,usingtheextinctioncoefficient20970cm-1M-1andthemolecularweight6990Da(appendix).Asamplewiththeconcentration1.2105mg/mlwaspreparedfortheexperiment.TheexperimentwasperformedonNano-DifferentialScanningCalorimetryIIImodelCSC6300(CalorimetrySciencesCorporation)inthetemperaturerange25to95°Cwith0.1°Cstepsunder3atmpressure.Onescanwasperformedforthedialysisbufferandoneforthesamplewithdialysisbufferasreference.Thebufferscanwassubtractedfromthesamplescan.ThedatawasanalysedusingthesoftwareCPcalcaccompanyingthemachine.Linearandpolynomialbaselineswerefittedmanually.

3.5FluorescenceSpectroscopySampleswith5µMprotein,55mMNaPi,pH7.0,1.3mMNaCl,26µMEDTA,2.6µMNaN3and0-6MGuHClwerepreparedandequilibratedovernightatroomtemperature.TheGuHClstocksolutiondescribedinsection3.3wasusedwhenpreparingthesamples.FluorescencemeasurementswereperformedonaFluoromax4HoribaSpectrophotometer(JobinYvon)witha4mmcuvette.Thetemperaturewassetto24°C.Theexcitationwavelength295nmandtheemissionrange400-600nmwereused.Bothslitsweresetto2nm.BackgroundwasrecordedforthephosphatebufferandfortheGuHClstocksolution.Thebackgroundwascalculatedandsubtractedfromeachmeasurement.ThedatawasanalysedinCDpalandtheerrorwasestimatedusingtheJackknifemethod.

3.6NuclearMagneticResonanceSpectroscopyAllNMRexperimentswereperformedusinga600MHzmagnet(VARIANInova)withacryogenicallycooledprobehead.ThedatawasprocessedusingthesoftwareNMRpipe30andvisualizedinSparky(GoddardandKneller,UniversityofCalifornia,SanFrancisco).A500µlsampleof378µMprotein,50mMNaPi,pH7.0,100mMNaCl,2mMEDTA,200µMNaN3and10%D2OwasplacedinasealedNMRtube.AsensitivityenhancedHSQCwasrecordedat25°Ctoconfirmtheintegrityoftheprotein.Theobtainedspectrumwascomparedtoanassignedspectrum31.ThechangesinthespectrumuponheatingwasstudiedbymeasuringsensitivityenhancedHSQCatthesamesampleat20-

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50°Cin5degreesteps.Thetemperatureofthesamplewasmeasuredwithaprobe.Thetruetemperaturedifferedupto2degreesfromthesettemperature.ToconfirmtheintegrityoftheproteinafterrecordingthefarUVCDspectrumaHSQCwasrecordedfortheanalysedsample.Thesamplecontained38µMprotein,50mMNaPi,pH7.0,10mMNaCl,200µMEDTAand20µMNaN3and10%D2O.Thespectrumwascomparedtotheonepreviouslyobtained.

3.7GenerationofSyntheticDataSyntheticdatawasgeneratedinCDpaltoinvestigatewhethertheΔCpvalueusedinfittinghasanynotableeffectonthecalculatedstabilityparameters.AlldatasetsweregeneratedwithTmfixedto49°CandΔHfixedto230kJ/molwhileΔCpwasfixedforvaluesvariedfrom0to15000kJ·mol-1·K-1.Thiswasrepeatedtwicewiththestandarddeviationsofnoisesetto0.01and0.03respectively.ThedatasetswerethenautofittedinCDpal.Theerrorwasestimatedwiththejackknifemethod.

4.ResultsandDiscussion

4.1ProteinPreparationThetransformationcontrolplateshowedzerocoloniesindicatingthattransformationwassuccessful.TheproteinexpressionwasanalysedbySDS-PAGEofcellsamplesbeforeandafterinduction,seeFigure5A.Thesampletakenafterinductionshowsadistinctband,correspondingtoapproximatelythesize25kDa,whichisnotvisibleinthesampletakenbeforeinduction.Thisindicatesthattheexpressionofthe24kDaconstructwassuccessful.ThetargetproteinwasexpressedasafusionproteinthatwascleavedtoreleasetheSH3domain.ThecleavingoftheconstructwasanalysedbySDS-PAGE.Thecleavedsampleshowsfourbands:onebandcorrespondingtotheTEVprotease,twocorrespondingtotheexpectedproductsofcleavingandoneunknownbandindicatinganimpurity,seeFigure5A.AnadditionalSDS-PAGEgelwasrunafterthepurificationtoensureproteinpuritybeforethesamplewasconcentrated,seeFigure5B.Thereisonevisiblebandonthegel.ThebandcorrespondstothemolecularweightoftheSH3domain.Thisindicatesthatthepurificationwassuccessful.Thebandsareweakduetolowproteinconcentration.

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Figure5.PicturesofSDS-PAGEgelswithPageruler10-180kDaladders(ThermoFisherScientific).A)Geltoanalyseexpressionandcleaving.(1)Beforeinduction,(2)afterinduction.Adistinctbandappearsafterinductioncorrespondingtothemolecularweight25kDa.(3+4)beforecleaving,(5+6)TEVprotease,(7+8)aftercleaving.Thegelindicatesasuccessfulcleavageandpresenceofanimpurity.B)Gelfromanalysingtheproteinpurityafterpurification.Theproteinsamplewasloadedinthreewells.Onesinglebandisvisible,markedbyanarrow,correspondingtoamolecularweightoflessthan10kDa.Thisindicatesthattheproteinispure.

Thesamplewasconcentratedto7ml,givingtheconcentration420µMandatotalyieldof20mg.AnHSQCexperimentwasruntoconfirmtheidentityandcorrectfoldingoftheprotein.Theobtainedspectrum,showninFigure6,matcheswithapreviouslyassignedspectrumoftheAbp1pSH3domain31.ThereareafewadditionalpeaksinthespectrumthatdonotbelongtotheSH3domain.

Figure6.HSQCspectrumforthepurifiedproteinsample,visualizedinSparky.TheaminoacidpeaksfortheAbp1pSH3domainarelabelledwiththeirassignment31.Redunlabelledpeaksbelongtosidechains.PeaksmarkedinbluedonotbelongtotheSH3domain,buttoanimpurity.

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Thefewextrapeaksindicatethatasmallpeptideimpurityremainsinthesample.Theextrapeakshavelowdispersionin1Hchemicalshiftindicatingthatthepeptideisunstructured.32Toquantifythefractionofimpurity,theaveragepeakintensitieswerecompared.TheaverageintensityoftheSH3peaksis1240000withthestandarddeviation288000.Theaverageintensityofthepeptidepeaksis182000withthestandarddeviation179000.Theaverageintensityoftheextrapeaksisapproximately15%oftheaverageSH3peakintensity.Duetolowerorderparameters,unstructuredproteinshavehigherintensitythanstructuredproteins.33Theconcentrationofunstructuredpeptideimpuritymustthusbelowerthan15%oftheSH3concentration.Basedonthis,thesamplewasconsideredpureenoughfortheintendeduse.

4.2FarUVCDSpectraToidentifythedifferencesinellipticitybetweenthenativeanddenaturedstate,farUVspectrawererecordedattheholdertemperatures20and94°C,seeFigure7.Theseholdertemperaturescorrespondsapproximatelytothesampletemperatures22and75°C.Theproteinisexpectedtobeinitsnativeformatthelowtemperatureandinitsdenaturedformatthehightemperature.

Figure7.FarUVspectraforAbp1pSH3innativeform(blue)anddenaturedform(red).

SincetheAbp1pSH3domaininitsnativestateconsistsmainlyofβ-strandsonewouldexpecttoseethecharacteristicnegativepeakat216nmseeninFigure3.Insteadthesignalat216nmisclosetozeroandthereisanegativepeakaround230nm.Theunexpectedspectrumgaverisetosuspicionsthattheproteinfoldwasnotintact.AnHSQCspectrumwasrecordedforthesamesample(datanotshown).NonotabledifferencewasdetectedfromthepreviousHSQCspectrum(Figure6).Thisindicatesthatthespectrumindeedisrecordedfortheproteininitsnativeform.FurthermorethespectraissimilartothosefoundforAbp1pSH3inlitterature14,34.Itistheorisedthatastructureintheproteinabsorbandgiverisetoapositivesignalthatcancelsoutthenegativepeakat216nm.Thisabsorbingstructurecouldbetheunstructuredloops(randomcoil)orperhapsthreeexposedtryptophansonthesurfaceoftheprotein.ThefarUVspectrumforthedenaturedstatehasastrongnegativesignal.Thespectrumisonlymeasuredto205nm,butitislikelythatthespectrumhastheminimumat198nmcharacteristicforrandomcoil(Figure3).Asignificantdifferenceinsignalbetweenthenativeanddenaturedstateisvisiblefrom240nmto205nm.

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4.3ThermalDenaturationMonitoredatMultipleWavelengthsTodeterminethethermalstabilityparametersandtoinvestigatewhetherchoiceofwavelengthaffectstheresults,thermaldenaturationwasmonitoredatsixteendifferentwavelengths.Wavelengthsintherange210-240nmwerechosenbecausetheyhaveasignificantdifferencebetweenthesignalforthenativeanddenaturedstate(Figure7).AlldatasetswerefittedindividuallyinCDpaltoevaluatethequalityofthedata.Somedatasetsarenoisyandhaveasmalldifferenceinsignalbetweenthenativeanddenaturedstate,seeFigure8.Thesealsohaveunreasonablestabilityparameterswithlargeerrors,seeTable1.Thesedatasetswerenotusedforfurtheranalysis.

Figure8.ThermaldenaturationprofilesfrommonitoringdenaturationofAbp1pSH3byCDspectroscopyatthewavelengths218,220226,228,230,232,234,236,238and240nm.Thesedatasetswereconsideredtobeoftoolowqualityforfurtheranalysis.ThefigurewasexportedfromCDpal.

Table1.Calculatedstabilityparametersforthedatasetsmeasuredatwavelengthsconsideredtobeoftoolowqualityforfurtheranalysis.CDpalwasnotabletofitanygraphstothedatasetsobtainedat230or236nm.

λ(nm) Tm(°C) ΔH(kJ/mol)218 40±10 140±90220 29.9±0.2 700±10000226 52±5 300±1000228 40±10 600±20000230 - -232 16±2 500±200234 52±1 3000±9000236 - -238 51.07±0.09 500±50000240 40±30 90±60000

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Otherdatasetshaveprofilestypicalforthermaldenaturationwithplateausandtransitionregions,seeFigure9.Thecalculatedstabilityparametershavereasonablevaluesanderrors,seeTable2.Thesedatasetswereusedforfurtheranalysis.

Figure9.ThermaldenaturationprofilesfrommonitoringdenaturationofAbp1pSH3byCDspectroscopyatthewavelengths210,212,214,216,222and224nm.Thesedatasetswereconsideredtobeofsufficientlyhighqualityforfurtheranalysis.ThefigurewasexportedfromCDpal.

Table2.Calculatedstabilityparametersforthedatasetsmeasuredatwavelengthsconsideredtobeofsufficientqualityforfurtheranalysis.

λ (nm) Tm(°C) ΔH(kJ/mol)210 50±1 290±50212 49±2 300±200214 48±2 170±50216 49±1 200±100222 49±3 180±80224 47±4 200±100

Therelativelyweaksignalchangeupondenaturationatwavelengthsabove224nmisinaccordancewiththefarUVCDspectrameasuredfornativeanddenaturedprotein(Figure7).Althoughthewavelengths218and220havealargesignalchange,asexpected,theyarenoisygivingunreasonablylargeerrorsinthecalculatedstabilityparameters.Thereisnoclearexplanationforwhythesedatasetsarenoisierthanothers.Itispossiblyduetochance.TheresultswereevaluatedinCDpal2.0toinvestigatewhethertheobtainedstabilityparametersvarywithwavelengthornot.Thestabilityparameters(TmandΔHseparately)wereplottedagainstwavelength,seeFigure10.Oneconstantandonelinearfunctionwerefittedtothedata,takingtheerrorsintoconsideration.

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Figure10.StabilityparametersTm(right)andΔH(left)plottedagainstwavelength.Linearfunctions(dashedline)andconstantfunctions(solidlines)arefittedtothedata.TheerrorforΔHat212nmof200kJ/molisoutsidethewindowrange.ThefigureswereproducedinGrace(GraceDevelopmentTeam).

Thegoodnessoffitwasevaluatedbycalculatingchi-square(χ2)anddegreesoffreedom(dof).Agoodfitisindicatedbyalowχ2.Alinearfunctionwillalwaysfitatleastasgoodasaconstantfunction,sincealinearfunctionwithaslopeofzeroisconstant.Anf-testwasperformedtoinvestigatedwhetherthefitofthelinearfunctionsaresignificantlybetterthanthoseoftheconstantfunctions.Thenullhypotheseswerethatthereisnosignificantdifferencebetweentheχ2-valuesandthealternativehypotheseswerethatthereisasignificantdifference.Thetestswereperformedwiththesignificancelevel0.05.ThetestsaresummarizedinTable3.Table3.Summaryofthetwof-testsperformedtodeterminewhetherthestabilityparametersvarywithwavelengthornot.χ2valueanddofaregivenforeachfunction,togetherwiththep-valueforeachtest.

Plottedparameter

Fittedcurve χ2 dof p-value

Tm Linear 0.3753 4 0.1308Constant 1.268 5

ΔH Linear 1.490 4 0.3688Constant 2.169 5

Thep-valuesfortheTmandΔHtestsare0.1308and0.3688respectively.Thesevaluesarebothlargerthanthesignificancelevelof0.05meaningthatthenullhypotheseswerenotrejected.Thelinearfunctionsdonotfitsignificantlybetterthantheconstantfunctions.ThismeansthatthestabilityparametersTmandΔHareindependentofwavelengthandthatdenaturationcanbemonitoredateitherwavelengthincludedinthetest. Awayofdeterminingsharedstabilityparametersfordatasetsisbyglobalfitting.Inglobalfittingitisassumedthatthedatasetsshareoneormoreparameters.InthiscasethesharedparameterswouldbeTmandΔH.Thedatasetsarethenfittedindividuallywiththesharedparametersandtheparametersareadjustediniterationstofindtheparametersgivingthebestfitforalldatasets.Thisanalysiswasperformedforthedata

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setsinTable2,usingCDpal2.0.TheresultingstabilityparameterswereTm=49±2°CandΔH=240±50kJ/mol.Globalfittingseemstoreducetheerrorcomparedtolocalfitting(Table2).

4.4ReversibilityofDenaturationOneoftheassumptionsindeterminingstabilityparametersisthattheunfoldingisreversible.Toverifythereversibility,denaturationandrefoldingwasmonitoredbyCDspectroscopyat216nm,whichisthewavelengthchosenformonitoringthermaldenaturationinfurtherexperiments.ThedatawasanalysedinCDpal,seeFigure11.

Figure11.Thermaldenaturation(filledcircles)andrefolding(emptysquares)profileforAbp1pSH3.Thedataisnotnormalized.ThefigurewasexportedfromCDpal.

TheobtainedstabilityparametersfortheunfoldingareTm=47.7±0.5°CandΔH=200±20kJ/mol.ThestabilityparametersforrefoldingareTm=49.5±0.8°CandΔH=160±20kJ/mol.95%ofthesignalisrecovereduponrefolding,indicatingthatasmallportionoftheproteinaggregatesathightemperature.Theslightdisplacementofthecurvesmightindicatethattheequilibriumtimeof60secondswasinsufficient7.Alongerequilibriumtimehasthedisadvantageoflongerrunningtimeandanincreasedriskforaggregationofthedenaturedprotein.35Itcouldbepossibletoalterthesettingtimeandbuffersystemtofindconditionsforcompletereversibility.Sincetherecoveryofsignalislargeandthedisplacementisslight,theunfoldingisconsideredmainlyreversible.Thisisassumedtobetrueforallexaminedconditionsofthisstudy.TheTmvalueobtainedfordenaturationis47.7±0.5°C.ThisisdifferentfrompreviouslyreportedTmvaluesfortheAbp1pSH3domain,whicharearound60°C.Inthosestudies,denaturationwasmonitoredbyCDspectroscopyat220nmandbyfluorescence.BufferswithpH3.5and8wereused.14,34InmystudythebufferhadpH7.ThedifferencesinmethodsandconditionscouldaccountforthedifferenceinTm.

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4.5StabilityAnalysisbyDifferentialScanningCalorimetryThestabilityparametersdeterminedforsampleswithoutdenaturantarelaterusedtoevaluatetheresultfromextrapolationofthermochemicaldata.Itisthereforeimportantthattheyareaccuratelydetermined.Toconfirmtheresults,anadditionalmethodwasusedtomonitordenaturationandcalculatestabilityparameters,namelyDSC.ThedenaturationprofileisshowninFigure12.

Figure12.DSCdenaturationprofile(solidline)andmanuallyfittedbaseline(dashedline).ThefigurewasproducedinGrace(GraceDevelopmentTeam).

TheobtainedstabilityparametersareTm=48.8°CandΔH=167kJ/mol.TheTmvalueisinagreementwiththevaluesdeterminedbyCDspectroscopy.TheΔHvalueisinthesameorderofmagnitudeasthosedeterminedbyCDspectroscopy,althoughslightlylower.SincethecalculationofΔHisconcentrationdependent,anincorrectconcentrationdeterminationwouldaffectthisresult.Itshouldalsobenotedthatthereisanexperimentalerrorintheobtainedstabilityparameters,althoughnotreported.Theerrorisnotreportedbecausethereisnosoftwareavailabletocalculateit.

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4.6StabilityAnalysisbyNuclearMagneticResonanceTofurtherinvestigatetheeffectofheatingtheprotein,HSQCspectrawererecordedatsixtemperaturesintherange24.4-48.1°C,seeFigure13.

Figure13.HSQCspectra,visualizedinSparky,measuredat24.4°C(purple),29.6°C(blue),33.2°C(green),38.6°C(yellow),43.4°C(orange)and48.1°C(red).Insetsshowenlargementofthepeaksforresiduenumber43and59.

Generallythepeaksmovetowardstherightofthespectrumwhenthetemperatureisraised,loweringthechemicalshiftoftheprotons.Thisisaknownandexpectedphenomenon.Asthetemperatureincreasetheaveragedistancebetweentheatomsincrease.Thisweakensthehydrogenbondsandlowersthehydrogenchemicalshifts.36Thesechangesinchemicalshiftuponheatingarethusnotnecessarilyrelatedtoproteindenaturation.32ThedeterminedvaluesofTmarecloseto48.1°C.ThismeansthatapproximatelyhalfoftheproteinisexpectedtobedenaturedintheHSQCrecordedat48.1°C.Knowingthatdenaturedproteinshavelowdispersionofchemicalshift,onecansaywithcertaintythattheproteinisnotcompletelydenaturedat48.1°C,sincethereisnonotabledifferenceinpeakdispersionbetweenthespectrafor24.4°Cand48.1°C.Therearetwowaysinwhichthedenaturationprocesscanbeobservedinaspectrum.Thepeakscaneithergraduallymovetowardsthedenaturedstateposition,orthepeakintensitiesgraduallydecreaseandnewpeaksappear.37ForAbp1pSH3thelatterseemstobethecase.TheinsetsinFigure13showenlargementsoftwopeaks.Itisvisiblethattheintensitydecreaseswhenthetemperatureisraised.Thisindicatesthattheproteinswitchbetweenthenativeanddenaturedstate.

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4.7ChemicalStabilityAnalysisChemicalstabilitywasstudiedtodetermineasuitableGuHClconcentrationrangeforthethermochemicalstabilityanalysis.Denaturationwasmonitoredbyfluorescence.ThedatawasfittedinCDpaltocalculatetheCmvalue,seeFigure14.

Figure14.ChemicaldenaturationprofileforAbp1pSH3withGuHCl.ThefigurewasexportedfromCDpal.

TheobtainedCmvalueis1.7±0.4M.Thecurvehasnoclearplateauforthenativestate.AmeasurementwasperformedwithoutGuHCl.WhennormalizedasinFigure14thispointhadthevalue1.23.Sinceitdeviatesmuchfromtheothersitwasremoved.Theremovalofthe0MpointdidnotaffecttheCmvaluenotably.IthasbeenobservedformultipleproteinsthatevenasmalladditionofGuHClchangestheintensitysignificantly,independentoftheunfolding(CeciliaAndrésen,personalcommunication).ItislikelythatmeasuringthefirstpointinasamplewithasmallGuHClconcentrationsuchas0.1Minsteadofzerowouldgiveaclearerplateau.

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4.8ThermalStabilityinPresenceofGuHClThethermalstabilityinabsenceofdenaturanthasbeendetermined.Next,anattempttopredictthestabilityinabsenceofGuHClfromstabilitymeasuredinpresenceofGuHClwasmade.TheGuHClconcentrationrangeof0.1-1.4MwaschosenwiththepreviouslydeterminedCmof1.7±0.4Minmind.ThethermaldenaturationwasmonitoredbyCDspectroscopy.TheresultingdenaturationprofilesareshowninFigure15.

Figure15.ThermaldenaturationprofilesforsampleswithdifferentconcentrationsofGuHCl:0.1M(magenta),0.2M(purple),0.4M(blue),0.6M(cyan),0.8M(green),1.0M(yellow),1.2M(orange),1.4M(red).ThefigurewasexportedfromCDpal.

Thisdatahaslessnoisethanthedatacollectedwhenmonitoringdenaturationatmultiplewavelengths(section4.3).Thiswasachievedbyraisingtheproteinconcentration.ThedatawasanalysedinCDpal,theresultingstabilityparametersaresummarizedinTable4.Table4.ThermalstabilityparametersforsampleswithvariousconcentrationsofGuHCl.

[GuHCl](M) Tm(°C) ΔH(kJ/mol)0.1 51.7±0.7 200±200.2 52.7±0.8 200±300.4 49±1 190±300.6 47.1±0.8 210±300.8 45±1 180±301.0 45±2 140±301.2 38±2 100±101.4 35±2 130±10

CDpal2.0wasusedtoplottheTmvaluesversustheconcentrationofdenaturant.ThesamewasdoneforΔH.Linearandquadraticfunctionswerefittedtoeachdataset,seeFigure16.

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Figure16.StabilityparametersTm(left)andΔH(right)plottedagainstwavelength.Linearfunctions(dashedlines)andquadraticfunctions(solidlines)arefittedtothedata.ThefigureswereproducedinGrace(GraceDevelopmentTeam).

Foreachplot,anf-testwasperformedtoanalysewhetherthequadraticfunctionsfitsignificantlybetterthanthelinearornot.Thenullhypothesesarethatthereisnosignificantdifferencebetweentheχ2-valuesandthealternativehypothesesarethatthereisasignificantdifference.Thetestswereperformedwiththesignificancelevel0.05.ThetestsaresummarizedinTable5.Table5.Summaryofthetwof-testsperformedtodeterminewhetherthestabilityparametersdependencyofGuHClconcentrationarebestdescribedbyalinearorquadraticfunction.χ2valueanddofaregivenforeachcurvetogetherwiththep-valueforeachtest.

Plottedparameter

Fittedcurve χ2 dof p-value

Tm Linear 360 6 0.0026Quadratic 52.8 5

ΔH Linear 178 6 0.0059Quadratic 39.3 5

Thep-valuesfortheTmandΔHtestsare0.0026and0.0059respectively.Thesevaluesarebothsmallerthanthesignificancelevelof0.05,thenullhypotheseswerethusrejected.ThismeansthatthequadraticfunctionsbetterdescribethestabilityparametersdependencyofGuHClconcentrationthanthelinearfunctions.Whenthequadraticfunctionsareextrapolatedto[GuHCl]=0Mthefollowingstabilityparametersareobtained:Tm=52.9±0.8°CandΔH=200±20kJ/mol.Theerroroftheextrapolationwasestimatedwiththejackknifemethod.Apparently,lowconcentrationsofGuHClhaveastabilizingeffectfortheAbp1pSH3domain.ThiseffecthasbeenobservedforbothSH3domainsandotherproteins.38–40IthasbeenshowninastudybyZarrine-Asfaretal.thatGuHClslowsdowntheunfoldingprocessofthehumanFynSH3byinteractingwiththeRT-Srcloopnearthepeptidebindingpocketinthenativestate.InthesamestudyitwasexaminedwhetherGuHClstabilizesotherSH3domainsaswell.NostabilizingeffectoftheAbp1pSH3domainwasobserved.38Thisiscontradictorytowhatwasshowninthisstudy.

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ThedenaturationwasmonitoredbyCDspectroscopyinbothstudiesbutatdifferentwavelengths.Therewerealsoslightdifferencesinthebuffersused.Thiscouldaffecttheresults.AmorestrikingdifferenceishoweverthatZarrine-Afsaretal.performedthemeasurementwithaHis-tagontheC-terminusoftheprotein.38His-tagsaresmallandoftenassumedtonotinterferewithstructureandfunctionformostproteins.AlthoughithasbeenshownthatHis-tagscanaffectthermalstabilityandenzymeactivity.41ItispossiblethattheHis-tagalterstheGuHClbindingpropertiesoftheAbp1pSH3domain.Theresultsofextrapolationwereevaluatedbycomparisontothestabilityparametersobtainedfromexperimentswherethestabilitywasstudiedinabsenceofdenaturant.Theseexperimentswere;denaturationmonitoredatmultiplewavelengthscombinedbyglobalfit(section4.3),denaturationmonitoredbyCDspectroscopyat216nm(section4.4)andDSC(section4.5).TheresultsaresummarizedinTable6.Table6.Summaryoftheobtainedstabilityparametersfromthedifferentexperiments.NoerrorwascalculatedintheDSCexperiment.

Experiment Tm(°C) ΔH(kJ/mol)CD,GuHClextrapolation 52.9±0.8 200±20CD,globalfit 49±2 240±50CD,216nm 47.7±0.5 200±100DSC 48.8 167

TheextrapolationofΔHseemssuccessful.Theextrapolatedvalueiswithintheerrorofthevaluesobtainedinotherexperiment(excludingDSCwhichhasanunknownerror).However,theerrorsarequitelargeforallΔHvalues.ItisthereforemoreinterestingtocomparetheTmvaluesthathavebeenmoreaccuratelydetermined.TheextrapolatedTmvalueisnotwithintherangeoftheresultsinotherexperiments.Thismeansthattheextrapolationwasunsuccessful.ItseemslikethefactthatGuHClbindstoandstabilizesthenativestatecomplicatestheextrapolation.Anotherfunctionisneededtofitthedataproperly.Thelinearandquadraticfunctionswouldlikelygiveextrapolationsclosertothetruthiftherewerenostabilizingeffect.

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4.9DeterminationofΔCpTobeabletoinvestigatetheeffectoftheΔCpvalueusedincalculationsofstabilityparameters,ΔCpforthemodelproteinwasdetermined.ΔCpcanbecalculatedfromexperimentswhereTmandΔHchangesasafunctionofperturbationsuchasadditionofdenaturantorchangedpH.WhenΔHisplottedagainstTmandalinearfunctionisfittedtothedata,theslopeofthefunctionequalsΔCp.7ThisanalysiswasperformedinCDpal2.0,usingtheresultsfromthermaldenaturationinpresenceofdifferentconcentrationsinGuHCl(section4.8).TheresultisshowninFigure17.

Figure17.ΔHplottedagainstTm.Errorbarsareshowninbothdirections.AlinearfunctionwiththeequationΔH=–68.971+5.1194×Tmisfittedtothedata.ThefigurewasproducedinGrace(GraceDevelopmentTeam).

Theslopeofthefittedlinearfunction,whichequalsΔCp,is5±1kJ·mol-1·K-1.TheerrorwasestimatedwiththeMonteCarlomethod.ΔCpcanintheorybecalculatedfromoneDSCexperimentasthedifferencebetweenthebaselinesforthenativeanddenaturestate.Inpractisethisisoftendifficult.Amorereliableresultisobtainedbyperformingmultiplemeasurementsandusingtheplottingapproachdescribedabove.23IntheDSCexperimentperformedinthisstudythebaselineforthedenaturedstateissloping(Figure12).ThismakesanestimationofΔCpdifficult.Theapproximateshiftbetweenthebaselinesis5kJ·mol-1·K-1.ThisisinaccordancewiththeΔCpvaluecalculatedfromtheCDdata.IfΔCphasnotbeenexperimentallydetermined,itcanbeestimatedfromthenumberofresidueswithformulas.Onesuggestedformulais

∆!! = 62! − 530

whereNisthenumberofresiduesandΔCphastheunitJ·mol-1·K-1.23FormulassuchasthiswillgenerallynotgiveanentirelyaccurateΔCpsinceitdoesnotdependonlyonthenumberofresiduesbutalsowhichsidechainsthereare,howtheyinteractwitheachotherandwiththesolvent.22UsingthisformulatoestimateΔCpforAbp1pSH3givesΔCp=3.3kJ·mol-1·K-1.Thisisinreasonableagreementwiththeexperimentallydeterminedvalue,althoughthereisanotabledifference.

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4.10EffectofΔCpinCalculationofStabilityParametersThedatafrommonitoringthermaldenaturationatmultiplewavelengths(section4.3)wasusedtoinvestigatewhethertheΔCpvalueusedinfittinghasanynotableeffectonthecalculatedstabilityparameters.ΔCpfortheproteinhasbeenestimatedexperimentallyto5kJ·mol-1·K-1andtheoreticallyto3.3kJ·mol-1·K-1(section4.9).GlobalfittingwithΔCpfixedto0,3.3and5kJ·mol-1·K-1respectivelywasperformedinCDpal2.0.TheresultsaresummarizedinTable6.Table7.SummaryofresultsfromglobalfittingwithdifferentfixedvaluesforΔCp.

ΔCp(kJ·mol-1·K-1) Tm(°C) ΔH(kJ/mol)0 49±2 240±503.3 49±2 240±605 49±1 230±50

Thereareslightdifferencesintheobtainedstabilityparameters,mainlyforΔH.Tofurtherinvestigatethis,syntheticdatasetsweregeneratedinCDpal.TheywereallgeneratedwithfixedTmandΔHvaluesof49°CandΔH=230kJ/molrespectively,whileΔCpwasvaried.Onesetofsimulationswasmadewithlowstandarddeviationofnoise,tomakeanydifferencesmorevisible,andonewithhigherstandarddeviation,tomorecloselyresemblerealmeasuredCDdata.ForallsyntheticdatasetsthetrueTm,ΔHandΔCpareknownfromthesettings.TheywereautofittedinCDpaltocalculatethevaluesofΔHandTmwhenΔCp=0kJ·mol-1·K-1.TheresultsarepresentedandcomparedtothetruevaluesinTable7.Table8.ResultsfromautofittingdatasetsgeneratedwithdifferentΔCpvalues.Parameterswitharangethatincludesthetruevaluearemarkedingreen.Parameterswitharangethatdoesnotincludethetruevaluearemarkedinred.

Standarddeviationofnoise

FixedΔCp(kJ·mol-1·K-1)

AutofittedTm(°C)

AutofittedΔH(kJ/mol)

0.01

0 48.9±0.2 225±63 49.3±0.2 230±105 49.5±0.2 229±910 49.9±0.2 230±1015 48.3±0.7 170±30

0.03

0 48.4±0.6 210±303 49.9±0.5 210±305 50.1±0.5 220±2010 49.1±0.5 230±3015 48.1±0.9 190±40

NotalltrueTmvalueswerewithintheobtainedrange.However,theywereallinthecorrectorderofmagnitude.ΔHwasaccuratelydeterminedfordatasetswithΔCpupto10kJ·mol-1·K-1.

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ForthedatasetswithΔCp=15kJ·mol-1·K-1theerrorsinthecalculatedstabilityparametersarelargeandΔHismoreinaccuratethanfortheotherdatasets.Inthisdatasetthesignalincreasefromlowtemperaturesgivingamaximumaround33°Cbeforedecreasinginthedenaturationtransitionregion,seeFigure18.Thisprofileindicatescolddenaturation.

Figure18.SyntheticallygenerateddatasetgeneratedwithTm=49°C.ΔH=230kJ/mol,ΔCp=15kJ·mol-1·K-1andthestandarddeviationofnoise0.01.Theprofileshowscolddenaturation.ThedataisautofittedandthefigurewasexportedfromCDpal.

Profilesshowingcolddenaturationcannotbeproperlyfittedwiththelinearnativebaselineusedinautofitting.Thisisthereasonbehindthelargeerrorsforthesedatasets.Whenthereiscolddenaturation,ΔCphastobeknowntofittheprofileproperly.Thisconclusionissupportedinliterature.42TheseresultsindicatethatitisvalidtoassumeΔCp=0kJ·mol-1·K-1unlessthereiscolddenaturation,inwhichcaseΔCphastobetakenintoaccount.Notethatduetorandomdistributionofthenoise,slightlydifferentresultsfromtheautofitwouldbeobtainedifthedatasetswereregenerated.

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5.ConcludingRemarksThermaldenaturationforAbp1pSH3wasmonitoredbyCDspectroscopyatmultiplewavelengths.Foralldatasetsofreasonablequalitythechoiceofwavelengthdidnotaffecttheresultingstabilityparameters.However,sincethisisnotnecessarilytrueforotherproteins,itshouldbeexperimentallyconfirmedineachcase.Monitoringatmultiplewavelengthstakeslongertimethanmonitoringatasinglewavelength.Ontheotherhand,itrequiresnoextraprotein.Thisexperimentcanbeausefulfirststeptodeterminewavelengthorwavelengthssuitableforfurthermeasurements.Anotherapplicationforthisapproachisdeterminingstabilityparameterswhenoneisshortofsample.Onesamplewithlowconcentrationcanbeusedtocollectmultipledatasetsthat,combinedbyglobalfit,givereasonablyaccuratestabilityparameters.Inglobalfittingthedatasetsareassumedtohavesharedstabilityparameters.Ifthemeasurementsareperformedunderdifferentconditionsitshouldnaturallybeconfirmed,forexamplebyanf-test,thatthestabilityparametersdonotdiffersignificantly.Ifanexperimenthasbeenrunmultipletimesunderthesameconditions,itisreasonabletoassumethatthestabilityparametersareshared.GlobalfittinginCDpal2.0isthenaconvenientandstatisticallycorrectwaytocombinetheresultstogiveonevalueforeachparameter.ThestabilityparametersTmandΔHinabsenceofGuHClwerepredictedinCDpal2.0byextrapolationofdatameasuredforsampleswithvariousconcentrationsofGuHCl.ThisextrapolationmethodwasunsuccessfulforAbp1pSH3duetothestabilisingeffectofGuHClatlowconcentrations.ThisextrapolationmethodismeantforthermophilicproteinsthatcannotbefullydenaturedintheCDinstrumentwithoutperturbation.Iflowconcentrationsofdenaturantdoesnotdestabilizetheproteinenoughtoenablefulldenaturation,anystabilizingeffectwhichdeceasethereliablyofextrapolationwouldnotbeobserved.Beforeconductingthisexperimentitshouldthereforebeconfirmedthatthedenaturantdoesnotbindtothenativestateoftheprotein,forexamplebyisothermaltitrationcalorimetry.Ifthedenaturantdoesbindtothenativestateoftheprotein,anotherdenaturantorperturbationbypHchangecanbetested.Anotherfactortotakeintoaccountwhenchoosingperturbingagentistoxicityandhandlingconsiderations.GuHClishighlyirritatingtoskinandeyeswhereasureaonlycauseslightirritation43.Ureasolutionsshouldbeusedwithin24hoursafterpreparation,whereasGuHClisstableatroomtemperaturefordays.44Measuringstabilityatdifferentconditionsisrelativelytimeconsuming.Eachmeasurementtakesapproximately1.5hours.Thismeansthatanexperimentwith8measurementstakes1.5workingdays.Eachmeasurementalsorequiresanewproteinsample.Inmyexperimentapproximately1mgproteinwasusedintotal.AnalternativemethodtodeterminestabilityofthermophilicproteinsisDSC.InDSCthemeasurementisperformedunderpressure,allowingmeasurementsathighertemperatures.RunningaDSCexperimenttakesapproximately4hours.Anadditionalscanisrunforthebuffer.MeasuringoneproteinsamplewithDSCthustakes8hoursandrequires0.6-1mgofprotein.Thetimeandproteinrequirementforthetwomethodsaresimilar.However,inourdepartment’sexperience,DSCinstrumentsarelessavailableandmorecomplicated

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tousethanCDmachines.AnotherdisadvantagewithDSCisthatanaccurateproteinconcentrationisneededforcalculationsofΔH.ThisstudyshowsthattheknowledgeofΔCpisnotnecessarytodetermineTmorΔHfortheprotein,aslongasnocolddenaturationtakesplace.UsingthecorrectΔCpmighthowevergivemoreaccuratestabilityparameters.Furthermore,ΔCpitselfisaninterestingparametertostudy.Itisrichininsightalthoughhardtounderstandinphysicalterms.Itgivesinformationaboutsolvationpropertiesanddescribesthetemperaturedependencyofentropyandenthalpy.IfonewishestoaccuratelydetermineΔCp,multiplemeasurementswithperturbationareneededforbothCDspectroscopyandDSC.InthiscaseCDspectroscopyisconsiderablylesstimeconsuming.ΔCpcanbecalculatedfromthesamedatasetsusedtoestimatestabilityinabsenceofperturbation.

6.FutureProspectsWhenmonitoringthermaldenaturationatmultiplewavelengths,twowavelengthshadalowsignaltonoiseratioalthoughtherewasasufficientdifferenceinsignalbetweenthenativeanddenaturedstate.Itwashypothesisedthatthiswasduetochance.ItwouldbeinterestingtorepeattheexperimenttoconfirmwhetherthesewavelengthscanbeusedtomonitordenaturationofAbp1pSH3ornot.ItwasconcludedthatchoiceofwavelengthdoesnotaffecttheresultinmonitoringthermaldenaturationforAbp1pSH3.Theresultsinthisstudygivenoindicationtowhetherthisistrueforotherproteinsornot.Thiswouldbeinterestingtostudy.Especiallyforamorecomplexproteinthatcontainsmoretypesofsecondarystructure.Perhapsmonitoringthedenaturationofbothα-helicesandβ-strandsatthesametimecouldgiveamorecompletepictureofthedenaturation.FurtheritwasfoundthatextrapolationwasnotasuccessfulmethodforestimatingthestabilityofAbp1pSH3inabsenceofGuHCl,duetothestabilizingeffectoflowconcentrationsofGuHCl.ItwouldbeinterestingtotrythisapproachforAbp1pSH3withanotherdenaturantorwithanothermodelprotein.Intheextrapolationexperimentitwouldbebeneficialtocollectdataformoreconcentrations,asthiswouldfacilitatethefittingandextrapolation.Iftheextrapolationissuccessfulwhenalldatapointsareused,itcouldbeinvestigatedhowmanydatapointsthatareactuallyneededbyremovingsomedatapointsandrepeatingtheanalysis.Theoptimaldistributionofthedatapointscouldalsobeinvestigated.Inaddition,whentherearemanydatapointsΔCpcanbemoreaccuratelydetermined.

7.AcknowledgementFirstandforemostIwishtothankmysupervisorPatrikLundströmfortheexcellentguidancethroughouttheproject.Thankyouforalwaysmakingtimeforme,fortheencouragementandforeverythingIhavelearnt.IalsowishtothankRickieMöllerforthehelpwithCDpal2.0.Lars-GöranMårtenssonandCeciliaAndrésenfortheappreciatedhelpinthelab.JohannaHultmanandFredrikBengtssonforthecompanyandallstimulatingdiscussions.

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9.AppendixTheTrx-His-TEV-Abp1pSH3constructThesequenceforthefullconstructis: MSDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDEIADEYQGKLTVAKLNIDQNPGTAPKYGIRGIPTLLLFKNGAVAATKVGALSKGQLKEFLDANLAGSGSGHMHHHHHHSSGLVPRGSGMKETAAAKFERQHMDSPDLGTENLYFQGAMAPWATAEYDYDAAEDNELTFVENDKIINIEFVDDDWWLGELEKDGSKGLFPSNYVSLGN. TheHis6-tagismarkedinyellow.TheTEVproteaserecognitionsiteismarkedingreen,theproteasecutsbetweenQandG.TheAbp1pSH3domainismarkedinbold.Theentireconstructhas231aminoacidsresidues,24kDa,ε=36565M-1cm-1.Abp1pSH3domainhas62aminoacidsresidues,6990Da,ε=20970M-1cm-1.ThemolecularweightsandextinctioncoefficientswerecalculatedfromthesequenceusingtheProtparamtoolfromExPaSy.