extraction of thermodynamic parameters of protein unfolding using parallelized differential scanning fluorimetry 2017 thaiesha a. wright学术.pdf

该【extraction of thermodynamic parameters of protein unfolding using parallelized differential scanning fluorimetry 2017 thaiesha a. wright学术 】是由【探春文档】上传分享，文档一共【19】页，该文档可以免费在线阅读，需要了解更多关于【extraction of thermodynamic parameters of protein unfolding using parallelized differential scanning fluorimetry 2017 thaiesha a. wright学术 】的内容，可以使用淘豆网的站内搜索功能，选择自己适合的文档，以下文字是截取该文章内的部分文字，如需要获得完整电子版，请下载此文档到您的设备，方便您编辑和打印。:..,,RichardChristopherPage,.,eptedManuscript?DOI:.6b02894?PublicationDate(Web):09Jan2017Downloadedfrom,2017epted“epted”manuscriptshavebeenpeer-,“epted”munitytoexpeditetheeptance.“epted”manuscriptspaniedbyanHTMLabstract.“epted”manuscriptshavebeenfullypeerreviewed,(DOI?).“epted”,the“epted”,itwillberemovedfromthe“JustAccepted”,“epted”.,Washington,?,,orworksmonwealthrealmCrowngovernmentinthecourseoftheirduties.:..,,*,andDominikKonkolewicz*222324DepartmentofChemistryandBiochemistry,MiamiUniversity,Oxford,OH45056,United252627States282930AUTHORINFORMATION313233CorrespondingAuthor343536*(******@).*DominikKonkolewicz(******@).373839404142434445464748495051525354555657585960ACSParagonPlusEnvironment1:..TheJournalofPhysicalChemistryLettersPage2of18123ABSTRACT4567Thermodynamicpropertiesofproteinunfoldinghavebeenextensivelystudied;however,,wepresentafacile,simple,andparallelizeddifferentialscanning1213fluorimetry(DSF)-state,reversibleproteinunfolding1617mechanismandprovidesthecapacitytoquicklyanalyzethebiophysicalmechanismsof1819changesinproteinstabilityandtomorethoroughlycharacterizetheeffectofmutations,2021additives,inhibitors,,carbonicanhydrase,chymotrypsin,horseradish2425peroxidase,paredtosimilarbiophysicalanalysesbycircular2627dichroism,DSFallowsfordeterminationofthermodynamicparametersofunfolding2829whileprovidinggreaterthan24-,denaturation,freeenergy,enthalpy,entropy,:..Page3of18TheJournalofPhysicalChemistryLetters123Althoughthemeltingtemperature(T)ofaproteinisoftenusedasaproxyforstability,the4m56Gibbsfreeenergyofunfolding(?uG),determining?Gallowsforextractionoftheenthalpyofmu91011unfolding(?uH)andentropyofunfolding(?uS).Numeroustechniqueshavebeenutilizedfor122-85-1013estimating?uG,includingchemicaldenaturation,differentialscanningcalorimetry(DSC),1415differentialscanningfluorimetry(DSF)10,hydrogen-deuteriumexchangemassspectrometry1617111218(HDX-MS),,thebulkofthesetechniquesentail1920significantpreparationtime,requirehighconcentrationsofprotein,,weaddressthisissuebypresentingaparallelizedDSFapproachthat232425enablesasetofthermodynamicparametersofproteinunfoldingtobeextractedforproteins2627assumingareversible,two-:2829generalapplicabilitytomanyproteins,thesmallquantityofproteinrequired,,providingtheabilitytoassessproteinstabilitywithrespecttovariousadditives,353637inhibitors,%404113-,theGibbsfreeenergyofthefoldedandunfoldedstatesare424344equivalent,and?-19,DSF20-24,circulardichroism(CD)24-27,or474828-,conclusivedeterminationproteinstabilitycannotbeobtainedfrom49503351simpledeterminationofthemidpointofthefolded-to-unfoldedtransition(Tm).Forexample,5253cytochromecexhibitsaTmof80°C,whichwouldintuitivelysuggestitmorestablethan5455lysozymewhichexhibitsaTof74oC34;however,understandardconditionsthe?Glysozyme56mu57585960ACSParagonPlusEnvironment3:..TheJournalofPhysicalChemistryLettersPage4of18123(-1)-1higherthanthe?Gforcytochromec(-1),?uG,?uH,and?uSfromtheslopeofthefolded-to--statetransitionfromthe1415native,-3818Althoughseveralproteinsinvolvemultipleintermediatestates,itwillbeshownthrough1920benchmarkingexperimentsthatthetwo-stateapproximationissufficienttorecapitulate?uG2122valuesreportedintheliterature,-statefolded/unfoldeddenaturation,,plexdenaturation2829pathwaysinvolvingintermediates,,wewill3334showthatthisDSF-basedmethodiscapableofextracting?,wehighlightthe3839potentialofourmethod,,,whichcanbeconvertedtotheGibbsfree5657585960ACSParagonPlusEnvironment4:..Page5of18TheJournalofPhysicalChemistryLetters123energyofunfolding(?G)neartheprotein’smeltingpoint,()in91011Prism(GraphPad,Inc.)withleastsquaresminimization,whereFministheminimalfluorescence1213value,Tisthetemperature,Tmistheprotein’smeltingtemperature,andmisaparameterto1415characterizethebreadthofthetransition.
1617=
+(1)18192021AlternativeestimatesofTmfromtheDSFdatacanbeobtainedasthepointofinflectiononthe2223increasingportionoftheDSFcurve,orthemid-,thefractionoftheproteinthatremainsfolded,26m2728Pf,iscalculatedateachtemperatureas:2930= 1? (2)313233Equation2assumesatwo-,,whichdecreasestheobservedfluorescence,
,fluorescenceintensityatthemeltingtemperature(Tm)andthedefinitionthatatTm50%484950theproteinisfoldedtofindFmaxas515253
=
?
+
 (3)5455GivenPfateachtemperature,Pu,thefractionofunfoldedprotein,iscalculatedas5657585960ACSParagonPlusEnvironment5:..TheJournalofPhysicalChemistryLettersPage6of18123!=1? (4)456Theequilibriumconstantofunfolding,Ku,maythenbecalculatedutilizingtherelationship78#$"!= (5)9#%1011TheGibbsfreeenergyofunfolding,?uG,-1-1constant(),Tistheabsolutetemperaturevalueateachfluorescenceintensity,?!'=?()*+"!(6)1920Calculated?uGvaluesareplottedagainsttemperaturevaluescorrespondingto10-50%?G,?S,and?,-statefolded/unfoldedsystems,,,?uG°,asthevalueof?uG4041extrapolatedbackto298Kusingthelineofbestfitfromtheplotof?-,whereaistheslopeoftheline,Tistemperature,andbisthey-?!'=, )+-(7)4748Once?G°isdetermined,?So,theentropyofproteinunfoldingisdeterminedas:uu4950?$/51?!.= (8)()5253Theenthalpyofproteinunfolding,?Ho,isthencalculatedusingtherelationship:u545556?!2 = )?!.(9)57585960ACSParagonPlusEnvironment6:..Page7of18TheJournalofPhysicalChemistryLetters123Itisimportanttonotethatintheaboveanalysisthechangeinheatcapacityuponfoldingisnot45426explicitlydetermined,,,evenwiththeassumptionsinplace,,22monlyused242526proteins:carbonicanhydrase,chymotrypsin,lysozyme,,chymotrypsin,lysozyme,-,yieldingsmalldifferencesinTm,°Corsmaller,:..(A),α-chymotrypsinfrombovinepancreas(B),heneggwhitelysozyme(C),and3637horseradishperoxidasetypeI(D)m,?uG°,?uS°,and?uH°.A3839Boltzmannfit(dottedline)enableddeterminationofTandspreadsheetcalculationswereusedm404142toestimate?uG°,?uS°,and?uH°