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Transcript
Lecture5:8/29 CHAPTER5 TechniquesinProteinBiochemistry Chapter5Outline Theproteomeistheentiresetofproteinsexpressedandmodifiedbyacell underaparticularsetofbiochemicalconditions. Unlikethegenome,theproteomeisnotanunvaryingcharacteristicofthe cell. Proteinpurificationrequiresatest,orassay,thatdetermineswhethertheprotein ofinterestispresent. Anassayfortheenzymelactatedehydrogenaseisbasedonthefactthata productofthereaction,NADH,canbedetectedspectrophotometrically. Proteinpurificationsaremonitoredinpartbydeterminingthespecificactivityof theproteinbeingpurified. Inthecaseofanenzymepurification,specificactivityistheratioofenzymeactivity toproteinconcentration. Specificactivityshouldincreasewitheachstepofthepurificationprocedure. Differentialcentrifugation Cellsaredisruptedtoformahomogenate,whichisamixtureofallofthe componentsofthecell,butnointactcells. Thehomogenateisthencentrifugedatlowspeedtoyieldapelletconsistingofnuclei andasupernatant.Thissupernatantisthencentrifugedatahighercentrifugalforceto yieldanotherpelletandsupernatant.Thisprocess,calleddifferentialcentrifugation,is repeatedseveralmoretimestoyieldaseriesofpelletsenrichedinvariouscellular materialsandafinalsupernatantcalledthecytosol. Differentialcentrifugation Saltinginand Saltingout Saltingouttakesadvantageofthefactthatthesolubilityofproteinsvarieswith thesaltconcentration. Mostproteinsrequiresomesalttodissolveinwater,aprocesscalledsaltingin. Asthesaltconcentrationisincreased,differentproteinswillprecipitateat differentsaltconcentrations,aprocesscalledsaltingout. Competitionbetweenthesaltionsandproteinforwaterresultinsprotein precipitation. Thedependencyofproteinsolubilityonsaltconcentration Thesaltcanberemovedfromaproteinsolutionbydialysis. Theproteinsolutionisplacedinacellophanebagwithporestoosmallto allowtheproteintodiffuse,butbigenoughtoallowthesalttoequilibrate withthesolutionsurroundthedialysisbag. Dialysis Proteinmolecules(red)areretainedwithinthedialysisbag,whereas smallmolecules(blue)diffuseintothesurroundingmedium. Molecularexclusionchromatography(gelfiltrationchromatography) Molecularexclusionchromatography(gelfiltrationchromatography)allowsthe separationofproteinsonthebasisofsize. Aglasscolumnisfilledwithporousbeads.Whenaproteinsolutionispassedover thebeads,largeproteinscannotenterthebeadsandexitthecolumnfirst.Small proteinscanenterthebeadsandthushavealongerpathandexitthecolumnlast. Gel-filtrationchromatography Ionexchangechromatographyallowsseparationofproteinsonthebasisofcharge. Thebeadsinthecolumnaremadesoastohaveacharge. Whenamixtureofproteinsarepassedthroughthecolumn,proteinswiththesame chargeasonthecolumnwillexitthecolumnquickly. Proteinswiththeoppositechargewillbindtothebeads,andaresubsequently releasedbyincreasingthesaltconcentrationoradjustingthepHofthebufferthatis passedthroughthecolumn. Ion-exchangechromatography Thistechniqueseparatesproteinsmainly accordingtotheirnetcharge Affinitychromatography Affinitychromatographytakesadvantageofthefactthatsomeproteinshavea highaffinityforspecificchemicalsorchemicalgroups.Beadsaremadewiththe specificchemicalattached.Aproteinmixtureispassedthroughthecolumn.Only proteinwithaffinityfortheattachedgroupwillberetained.Theboundproteinis thenreleasedbypassingasolutionenrichedinthechemicaltowhichtheprotein isbound. Affinitychromatography High-pressureliquidchromatography(HPLC) Theresolvingpowerofanychromatographictechniqueisrelatedtothe numberofpotentialsitesofinteractionbetweentheproteinandthe columnbeads. Veryfinebeadsallowmoreinteractionsandthusgreaterresolvingpower, butflowratesthroughsuchcolumnsaretooslow. High-pressureliquidchromatography(HPLC)usesveryfinebeadsinmetal columnsandhigh-pressurepumpstomovetheliquidthroughthecolumn. Becauseoftheincreasednumberofinteractionsites,theresolvingpower ofHPLCisgreaterthannormalcolumns. High-pressureliquidchromatography(HPLC) Sodiumdodecylsulfate-polyacrylamidegelelectrophoresis(SDS-PAGE) Proteinswillmigrateinanelectricalfieldbecausetheyarecharged.Whenthemigration occursinagel,theprocessiscalledgelelectrophoresis. Sodiumdodecylsulfate-polyacrylamidegelelectrophoresis(SDS-PAGE)allowsaccurate determinationofmass.SDSdenaturesproteinsand,formostproteins,1moleculeof SDSbindsforeverytwoaminoacids.Thus,proteinshavethesamecharge-to-massratio andmigrateinthegelonthebasisofmassonly. Polyacrylamide-gelelectrophoresis Thestainingofproteinsafterelectrophoresis ProteinsseparatedbySDS-PAGEarevisualizedbystainingthegelwithdyes suchasCoomassie blue. Isoelectricfocusing Isoelectricfocusingallowsseparationofproteinsinagelonthebasisoftheirrelative amountsofacidicandbasicaminoacids. IfamixtureofproteinsisplacedinagelwithapHgradientandanelectricalfieldis applied,proteinswillmigrateuntiltheyreachtheirisoelectricpoint(pI),thepHat whichtheyhavenonetcharge. Theprincipleofisoelectricfocusing ApHgradientisestablishedinagelbeforethesamplehasbeenloaded.(A)Thesampleis loadedandvoltageisapplied.TheproteinswillmigratetotheirisoelectricpH,thelocationat whichtheyhavenonetcharge.(B)Theproteinsformbandsthatcanbeexcisedandusedfor furtherexperimentation. Two-dimensionalgelelectrophoresis(2Dgel) Intwo-dimensionalgelelectrophoresis,proteinsareseparatedinone directionbyisoelectricfocusing. ThisgelisthenattachedtoanSDS-PAGEgelandelectrophoresisis performedata90° angletothedirectionoftheisoelectricfocusing separation. Two-dimensionalgelelectrophoresis (A)Aproteinsampleisinitiallyfractionatedinonedirectionbyisoelectricfocusingas describedinFigure5.10.Theisoelectric-focusinggelisthenattachedtoanSDSpolyacrylamidegel,andelectrophoresisisperformedintheseconddirection,perpendicular totheoriginalseparation.ProteinswiththesamepI valuearenowseparatedonthebasisof mass. Two-dimensionalgelelectrophoresis Alterationsinproteinlevelsdetectedbytwo-dimensionalgelelectrophoresis. Theeffectivenessofapurificationschemeismeasuredbycalculatingthe specificactivityaftereachseparationtechnique. SDS-PAGEallowsavisualevaluationofthepurificationscheme. Electrophoreticanalysisofaproteinpurification Gradientcentrifugation Theestrogenreceptorbindsthesteroid hormoneestradioltightlyandwith greatspecificity. Theestrogenreceptorhasnoenzymatic activity,butcanbepurifiedby immunologicaltechniquesandtheuse ofgradientcentrifugation. Ultracentrifugationcanbeusedtoexamineproteins.Whensubjectedtoa centrifugalforce,therateofmovementoftheparticleisdefinedbythe sedimentationcoefficient,s. Wherem=mass, =thepartialspecificvolume(thereciprocaloftheparticle density),ρ =densityofthemedium,and=thefrictionalcoefficientofthe particle. 5.3ImmunologicalTechniquesAreUsedtoPurify andCharacterizeProteins SedimentationcoefficientsareusuallyexpressedasSvedbergunits(S)equalto10-13s. ThesmallertheSvalue(i.e.smallerthemolecularweight), theslowertheproteinmovesinacentrifugalfield. Adensitygradientisformedinacentrifugetube,andamixtureof proteinsinsolutionisplacedontopofthegradient. Toidentifytheestradiolreceptor,theproteinmixtureisfirstincubated withradioactiveestradiol,whichisreadilydetected.Onlytheestradiol receptorwillbindtothesteroid.Moreover,thesteroidaloneistoo smalltobeinfluencedbythecentrifugalforce. Afterthecentrifugationiscomplete,asmallholeismadeinthebottom ofthecentrifugetubeandportionsofthegradientarecollectedand testedforradioactivity. Zonalcentrifugation,a formadensitygradient Gradient-centrifugationanalysisoftheestradiol-receptorcomplex Anantibodyisaproteinsynthesizedinresponsetothepresenceofaforeign substancecalledanantigen. Theantibodyrecognizesaparticularstructuralfeatureontheantigencalledthe antigenicdeterminantorepitope. Antibodystructure Anyantibody-producingcellsynthesizesantibodiesthatrecognizeonlyoneepitope. Eachantibody-producingcellthussynthesizesamonoclonalantibody. Anyantigenmayhavemultipleepitopes.Theantibodiesproducedtotheantigenby differentcellsaresaidtobepolyclonal. Immortalcelllinesproducingmonoclonal antibodiescanbegeneratedbyfusing normalantibodyproducingcellswithcells fromatypeofcancercalledmultiple myeloma. Amonoclonalcelllineisisolatedby screeningfortheantibodyofinterest. Thepreparationofmonoclonalantibodies Amonoclonalantibodyfortheestrogenreceptorcanbeisolatedbysearchingforcell linesthatproduceanantibodythatbindstothereceptor. Ifanantibodyforthereceptorispresent,itwillbindtothereceptorandalterthe sedimentationconstantofthereceptor. Alterationofthesedimentationprofilewhenantibodybindstothereceptorprotein Oncethemonoclonalcelllineisisolated,theantibodycanbeusedtopurify theestrogenreceptor. Purificationbyimmunoprecipitation(IP) Purificationbyimmunoprecipitation Enzyme-linkedimmunosorbentAssay(ELISA) Antibodiesareusedasareagenttodeterminetheamountofaproteinorotherantigen present.Enzyme-linkedimmunosorbent Assay(ELISA)quantifiestheamountofprotein presentbecausetheantibodyislinkedtoanenzymewhosereactionyieldsareadily identifiedcoloredproduct. IndirectELISAandsandwichELISA(aclinicallyusedassay) Inwesternblottingorimmunoblotting,proteinsareseparatedinanSDSPAGEgel,transferredtoasheetofpolymer,andthenstainedwitha fluorescentantibody. Westernblotting ProteinsonanSDS-polyacrylamidegelaretransferredtoapolymersheetand stainedwithfluorescentantibody.Thefluorescentantibodyisexcitedbylightand thebandcorrespondingtotheproteintowhichtheantibodybindsisvisualizedwith anappropriatedetector. Determinationofaminoacidcomposition. Akeystepinunderstandingproteinfunctionistodeterminetheprimary structure,ortheaminoacidsequence,oftheprotein.Apreliminarystepisto determinetheaminoacidcompositionoftheprotein. Theproteinishydrolyzed,andtheconstituentaminoacidsareseparatedonan ion-exchangecolumn.Theaminoacidsarevisualizedbyreactionwith fluorescamine. Fluorescentderivativesofaminoacids. Fluorescamine reactswiththea-aminogroupofanaminoacid toformafluorescentderivative Determinationofaminoacidcomposition Edmandegradation TheaminoacidsequencecanbedeterminedbyEdmandegradation. Theproteinisexposedtophenylisothiocyanate (PTH),whichreacts withtheN-terminalaminoacidtoformaPTH-derivative. ThePTH-aminoacidcanbereleasedwithouthydrolyzingtheremainder oftheprotein,andthedegradationissubsequentlyrepeated. TheEdmandegradation BecausethereactionsoftheEdmandegradationprocedurearenot100% effective,itisnotpossibletosequencepolypeptideslongerthan50amino acids. Inordertosequencetheentireprotein,theproteinischemicallyor enzymaticallycleavedtoyieldpeptidesoffewerthan50aminoacids. Thepeptidesarethenorderedbyperformingadifferentcleavage procedureinordertogenerateoverlappeptides. MassSpectrometryCanBeUsedtoDetermineaProtein’sMass, Identity,andSequence Twomassspectrometrytechniquescanbeusedtodetermineaproteinsmass: matrix-assistedlaserdesorption(MALDI)andelectrosprayionization(ESI).In MALDI,proteinsareprecipitatedontoamatrixandalaserflashreleasesnegatively chargedions. Timeofflight(TOF)analysisisusedtomeasurehowrapidlytheionsmovetowarda detector. Onlypicomoles orfemtomoles ofaproteinarerequiredforMALDI-TOFanalysis. MALDI-TOFmassspectrometry (1)Theproteinsample,embeddedinanappropriatematrix,isionizedbytheapplication ofalaserbeam.(2)Anelectricfieldacceleratestheionsthroughtheflighttubetoward thedetector.(3)Thelightestionsarrivefirst.(4)Theionizinglaserpulsealsotriggersa clockthatmeasuresthetimeofflight(TOF)fortheions. MALDI-TOFmassspectrumofinsulinandb-lactoglobulin Massspectrometryallowsdeterminationofaprotein’sidentity.Forinstance,an unknownproteinvisibleinatwo-dimensionalgelcanberemovedfromthegel. TheproteinisthencleavedinsomefashionandsubjectedMALDI-TOF,revealinga seriesofpeptideswithknownmasses. Thesepeptidemassesarethencomparedtoproteinsinadatabasethatare “electronicallycleaved”byacomputerusingthesamecleavagetechniqueusedto generatetheproteinfragments. Aproteinssequencecanbedeterminedwiththeuseoftandemmassspectrometry. Peptidesequencingbytandemmassspectrometry 1. Primarystructuresfromdifferentproteinscanbecomparedto inferknowledgeaboutstructureandfunction. 2. Primarystructurecomparisonofsimilarproteinsfromdifferent speciesprovidesinformationaboutevolution. 3. Primarystructurecanbesearchedforinternalrepeatsthatmay yieldinformationonthehistoryoftheindividualprotein. Repeatingmotifsinaproteinchain 4. Primarystructurecanrevealthepresenceofaminoacidsequences thatregulateproteinfunctionandlocation. 5. Primarystructurecanprovideinsightintothemolecularbasisof disease. 6. Primarystructurecanbeusedasaguidetoexplorenucleicacid information.
