ProteinI教學(xué)講解課件

ProteinsIProteins

1830Mulder–whatremainedafterremovalofsugars,fats,salts,etc.1838Berzelius–“Proteins”fromtheGreek“of1strankorimportance”CentralDogma:DNARNAprotein(informationflow)DNA,RNAinformationisinthesequence-linearProteinfunctionisinthe3DstructureWhatareproteins?Proteinsarelinearpolymers(chains)ofaminoacids(morethan50aa)“Protein”O(jiān)neormorepolypeptidechains

Onepolypeptidechain-amonomericproteinMorethanone-multimericproteinHomomultimer-onekindofchainHeteromultimer-twoormoredifferentchainsHemoglobin,forexample,isaheterotetramerIthastwoalphachainsandtwobetachainsProteins-LargeandSmallInsulin-Achainof21residues,Bchainof30residues-totalmol.wt.of5,733Glutaminesynthetase-12subunitsof468residueseach-totalmol.wt.of600,000Connectinproteins-alpha-MW2.8million!betaconnectin-MWof2.1million,withalengthof1000nm-itcanstretchto3000nm!MainProteinClassesFibrous

-extendedshape,insoluble(e.g.keratin,collagen,elastin)Globular

-compactshape,watersoluble(e.g.myoglobin,trypsin)Membranous

-oftenmultidomain–onelipidsoluble(e.g.photoreactioncentre,ATPsynthase)BiologicalFunctionsofProteinsProteinsaretheagentsofbiologicalfunctionEnzymes-RibonucleaseSignaltransduction–InsulinanditsreceptorControlofGeneexpression-TranscriptionfactorsImmunity-AntibodyTransportandStorage-HemoglobinStructuralproteins–Hair,CollagenContractileproteins-Actin,MyosinExoticproteins-Antifreeze

proteinsinfishProteinsare:Polypeptides+possibly–cofactors,coenzymes,prostheticgroups,othermodificationsPolypeptidesarecovalentlylinked-aminoacidsCofactorsarenon-aminoacidcomponentse.g.metalionslikeZn2+incarboxypeptidaseCoenzymesareorganiccofactorse.g.nucleotidesinlactatedehydrogenaseProstheticgroupsaretightlyattachedcofactorse.g.hemeinmyoglobinHierarchyofproteinstructurePrimaryStructure(1o):Uniquesequenceofaminoacids:sequenceisdeterminedbygeneticmaterialSecondaryStructure(2o):regularrepeatingstructuresofthepolypeptidechain(i.e.a-helices,b-sheets);coiling/foldingasaresultofhydrogenbondingTertiaryStructure(3o):3-DshapeduetobondingofR-groupsQuaternaryStructure(4o):associationof2ormorepolypeptides;ExHGB;notallhavethislevelHierarchyofproteinstructurePrimarySecondaryTertiaryQuaternaryPrimary-sequenceSecondary-e.g-helixor-sheetTertiary-3DshapeQuaternary-subunitorganization(>onepolypeptidechain)FourlevelsofproteinstructurePrimary:aminoacidsequenceDuetocovalentbondTheSequenceofAminoAcids

inaProteinisauniquecharacteristicofeveryproteinisencodedbythenucleotidesequenceofDNAisthusaformofgeneticinformationisreadfromtheaminoterminustothecarboxylterminus1ostructure=linearaminoacidsequenceFortheInsulinAchain,the1ostructureis:Gly-Ile-Val-Glu-Gln-Cys-Cys-Thr-Ser-Ile-Cys-Ser-Leu-Tyr-Gln-Leu-Glu-Asn-Tyr-Cys-AsnInsulinwasthefirstpolypeptidetobesequenced(byFrederickSangerin1953).Thesequencingofinsulindemonstratedforthefirsttimethatproteinsarecomposedofspecific,definedaminoacidsequences.TheaminoacidsequenceofaproteinisspecifiedbythegeneencodingthatproteinDNA:ggcattgtggaacaatgctgtaccagcmRNA:ggcauuguggaacaaugcuguaccagcProtein:Gly-Ile-Val-Glu-Gln-Cys-Cys-Thr-SerThePeptideBondhaspartial(40%)doublebondcharacterisabout0.133nmlong-shorterthanatypicalsinglebondbutlongerthanadoublebondDuetothedoublebondcharacter,thesixatomsofthepeptidebondgrouparealwaysplanar!isusuallyfoundinthetransconformation,butX-ProisanexceptionNpartiallypositive;OpartiallynegativeTheCoplanarNatureofthePeptideBondSixatomsofthepeptidegrouplieinaplane!RestrictionbyAmidePlaneAtomsinthepeptidebondlieinaplane.Resonancestabilizationenergyofthisplanarstructureisapproximately88kJ/mol;RotationcanonlyoccuraroundthetwobondsconnectedtotheCaatom;RotationaroundtheCaandcarbonylbondiscalledy(psi);RotationaroundtheCaandnitrogenbondiscalledf(phi).RotationofAmidePlanesIf(f,y)areknownforallresidues,thestructurefortheentirebackboneisknown.Some(f,y)aremorelikelythanothersinafoldedproteinPositive(f,y)valuescorrespond toclockwiserotationaroundbonds whenviewedfromtheCa.Zero isdefinedwhentheC=OorN-H bondbisectstheR-Ca-Hangle.(f,y)=(0,180),twocarbonyloxygensaretooclose;(f,y)=(180,0),twoamidegroupsareoverlapping;(f,y)=(0,0),carbonyloxygenoverlapswithamidegroup;RamachandranPlotTheplotusesfashorizontalAxisyasverticalaxis.The(f,y)angleforeachresiduecanbeenteredontheplot.Forfoldedproteins,their(f,y)anglesclusterinfewregionsoftheplot.Theupperleftcornerarebeta-sheetvaluesandmiddleleftarea-helicesvalues.Linessignifiesthenumberofaminoacidsperturnofhelix(+meansright-handed,-left-handed)ClassesofSecondaryStructureAllthesearelocalstructuresthatarestabilizedbyhydrogenbonds

AlphahelixOtherhelicesBetasheet(composedof"betastrands")Tightturns(akabetaturnsorbetabends)Betabulge

TheAlphaHelixThealphahelixisahelicalstructure.Allalphahelicesinproteinsareright-handed;H-bondpatternsofthealphahelix:Alphahelix:CarbonyloxygenoftheithresidueformsH-bondwithamideprotonofthe(i+4)thresidue.Sotherearen-4H-bondsinahelixofnaminoacids;310helix:carbonyloxygenoftheithresidueformsH-bondwithamideprotonofthe(i+3)thresidue.3residues(or10atoms)perturn;Prolineisnotfoundina-helixexceptatthebeginningofana-helix;Helixpropensityofanaminoacidisameasureofthelikelyhoodfortheaminoacidtobeinahelix;Glu,Met,Ala,Leuhavehighpropensities;Examplesofa-helicalproteinsincludea-keratin(structuralproteins)andcollagen(fibrousprotein);Linus

Pauling(NobelPrizeinChemistry,1954)figuredoutthestructureofa-keratinhelix.Thecarboxylgroupofresiduenishydrogenbondedtothebackboneamideoftheaminoacidatpositionn+4.TheAlphaHelixKnowthesenumbers

Residuesperturn:3.6Riseperresidue:1.5AngstromsRiseperturn(pitch):3.6x1.5A=5.4AngstromsThebackboneloopthatisclosedbyanyH-bondinanalphahelixcontains13atomsphi=-60degrees,psi=-45degreesThenon-integralnumberofresiduesperturnwasasurprisetocrystallographersTheAlphaHelixResiduesperturn:3.6Riseperresidue:1.5?Riseperturn:5.4?(f,y)=~(-60o,-45o)C=ON-HsidechainTotaldipolemomentInsulinchainAcontainstwoa-helices

Schematicviewofthecross-sectionofana-helix.Sidechainsareshownasgreencircles.a-helicesareoftenamphipathic

Ahelicalwheelrepresentationofanamphipathica-helixfromalcoholdehydrogenaseisshown.Inahelicalwheel,across-sectionalviewofthea-helixisdrawnasaspiralwithaminoacidsoccurringevery100oalongthespiral(360odividedby3.6aminoacidsperturngives100operaminoacid).TheBeta-PleatedSheetComposedofbetastrands

AlsofirstpostulatedbyPaulingandCorey,1951StrandsmaybeparallelorantiparallelRiseperresidue:

3.47Angstromsforantiparallelstrands3.25AngstromsforparallelstrandsEachstrandofabetasheetmaybepicturedasahelixwithtworesiduesperturnAminoacidsthattendtobefound

inb-strandsValIlePheTyrTrpThrTheBetaTurn(tightturn,orb-bend)Betaturnsconnectbetastrandsandreversethedirectionofbetastrands;Prolineandglycinehavehighpropensityforbetaturns;ThecarbonyloxygenoftheithresidueformsH-bondwiththeamideprotonofthe(i+3)thresidue;Tightturnpromotesformationofantiparallelbetasheets.TheBetaBulgeBetabulgeoccursbetweennormalb-strands.Comprisedoftworesiduesononestrandandoneontheother;Bulgescausebendingofotherwisestraightanti-parallelbetastrands;BetabulgeAnti-parallelstrandsLoopsconnectsecondary

structuralelementstogetherTertiarystructureThethreedimensionalarrangementofallatomsinagivenproteinisreferredtoastheprotein’stertiarystructure.Contrasttertiarystructurewithsecondarystructure,whichrefersprimarilywiththearrangementofaminoacidsthatareadjacentintheprimarystructure(sequence),orverycloseintheprimarystructure.Again,non-covalentforcesarethosethataremostimportant,althoughdisulfidebondscontributetotertiarystructure.Motif→domain→tertiary(conformation)ProteinConformationThetertiarystructurethataproteinassumestocarryoutitsphysiologicalroleinsideacellisknownasthenativestateorsometimesthenativeconformationNeedtodistinguishconformationfromconfiguration.Configurationdenotesthegeometricpossibilitiesforaparticularsetofatoms.Inchangingconfiguration,covalentbondsmustbebroken.Aparticularstereochemistryaboutagivencenterisconsideredtobeaconfiguration.Theprimarysequenceofaproteinisaconfiguration.Conformationdenotesthe3-Darchitectureofaprotein.Itisestablishedbyavarietyofweakforces.Incontrasttoconfiguration,aconformationcanchangereadily.Theconformationofaproteinisfirstandforemostestablishedbyitsprimarystructure(aminoacidsequence).Itsinteractionwithsolvent(generallyH2O)andthepHandioniccompositionarealsocriticalinestablishingand/ormaintainingaprotein’sconformation.Howtoinvestigate

proteintertiarystructure?X-raycrystaldiffractionNMR(lessthan120aa)ForcesthatInfluenceProtein

TertiaryStructureHydrogenbonds-Theatomsofthepeptidebondwilltendtoformhydrogenbondswheneverpossible.Aminoacidsidechainsthatarecapableofforminghydrogenbondswilltypicallybefoundonthesurfaceofproteins,sothattheymayinteractwithwater.Althoughtheenergyofthehydrogenbond(~12kJ/mol)isfairlyweakwhencomparedtocovalentinteractions,theyarenumerous,andtogethercontributeasignificantamountofenergyandstabilitytoproteinconformation.Hydrophobicinteractions-Theinteriorofareproteinsalmostexclusivelycontainaminoacidswithhydrophobicsidechains.We’llfindthattheneedtoburyhydrophobicsidechainsofaminoacidsiswhatdrivesaproteintofoldintoitsproperconformation.VanderWaalsinteractions-inducedelectricalinteractions.Contributesignificantlytoconformationalstabilityintheinterioroftheprotein.ElectrostaticInteractionsDisulfidebondMotifsMotifisasimplecombinationofafewsecondarystructuralelementswithaspecificgeometricarrangement.Somemotifsareassociatedwithaparticularbiologicalfunctionwhileothersarenotbutratherarepartofalargerstructuralassembly.

Lengthofloopisgenerallytwotofiveresiduesb-hairpinFoundinalmosteveryproteinthathasaparallelb-sheetTheHelix-Turn-HelixMotifInvolvedinDNArecognitionFoundinprokaryoticsystemsTypicallylessthan100aminoacidsIneukaryoticsystems,thereisthehomeodomainwhichisinvolvedinDNAbindingIneukaryoticsystems,thereareothermotifs-zincfingersandleucinezippersFirstobservedinCAPinEscherichiacoliCrorepressor66aminoacidslrepressor/DNAcomplexTheEFHandItisahelix-loop-helixmotifbutwithacompletelydifferentdispositionofthea-helicesandtheactualtypesofaminoacidspresent.Thistypeofmotifwasfirstobservedinparvalbuminwhichisamuscleproteinfromcarp.Thecalciumioniscoordinatedbysevenoxygens.ParvalbuminTheImmunoglobulinFold

Itisacompressedanti-parallelb-barrelbuiltupfromonethree-strandedb-sheetpackedagainstafour-strandedanti-parallelsheet.ItcontainsaGreekkeyinitsfold.Theb-helixfirstobservedinpectatelyaseCofErwiniachrysanthemiin1993hasnowbeenobservedinmorethanadozenproteins-someareenzymesandsomearenot-forexample,thefilamentoushemagglutininsecretiondomaincontainsthismotif(PNAS,Vol101,p.6194,April2004).thestructuralmotifconsistsofthreeparallelb-sheetsthatwraparoundtoformab-helix.ProteinDomainsProteindomainsaredistinct,stable,globularunitsorfoldswithinasinglepolypeptide.Frequentlydomainswillretaintheircorrectthree-dimensionalstructurewhencleavedproteolyticallyfromtheparentpeptide,orwhenexpressedasasinglyentity.Differentdomainswilloftenhavedistinctfunctionsintheparentprotein.Themuscleprotein,troponinC(shownatleft),appearslikeadumbell.Thetwolobes(blueandpurple),whichrepresenttwodomains,functiontobindcalciumions.DNApolymerasehasthreedomainsAdomainisasegmentofthepolypeptidethatformsastablestructureindependentlyofthetheremainderoftheprotein.QuaternaryStructureThemajorityofproteinsinnatureexistasoligomers,whicharecomplexes(noncovalentassociations)oftwoormoremonomersubunits.Thenumberandarrangementofthesecomplexesisreferredtotheproteinsquaternarystructure.Aspreviouslydiscussed,thesesubunitscanbeidentical(homomultimers),ornonidentical(heteromultimers).Hemoglobiniscomposedoffourchains,whicharetwoalphachainsand2betachains.Itisatetramer,oradimerofab

protomers.Aprotomeristhesimplestrepeatingstructuralunitofanoligomericprotein.Theadvantagesofoligomerizationaremultifold.1.Thereisusuallyanincreaseinstabilityoftheprotein,duetoalesssurfacetovolumeratio.2.Allowsforregulation.3.LessDNAisneedtoencodeahomomultimerthanalargepolypeptideofasi