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PAGE6Chapter3-AminoAcidsandPri__ryStructureofProteinsFunctionsofproteins:1-catalysts-enzymesformetabolicpathways2-storageandtransport-e.g.myoglobinandhemoglobin3-structural-e.g.actinmyosin4-mechanicalwork-movementofflagellaandciliamicrotubulemovementduringmitosismusclecontraction5-decodinginfor__tion-translationandgeneexpression6-hormonesandhormonere__ptors7-specializedfunctions-e.g.antibo___sStructureofaminoacidsThereare20commonaminoacidscalled-aminoacidsbecausetheyallh__eanaminoNH3+groupandacarboxylgroupCOOHattachedtoC-2carboncarbon.AtpHof7aminogroupisprotonated-NH3+andcarboxylgroupisionizedCOO-.Theaminoacidiscalledazwitterion.pKaofacarboxylgroup=
1.8-
2.5pKaofaaminogroup=
8.7-
10.7Thecarbonischiralorasymmetric4differentgroupsareattachedtothecarbon;ex__ptionisglycine.Aminoacidsexistasstereoisomerssamemolecularformulabutdifferinarrangementofgroups.DesignatedDrightorLleft.AminoacidsusedinnatureareofLconfiguration.carboxylategroupattop--pointsawaysidechainatbottomaminogrouporientationdeterminesNH3+onleft=LNH3+onright=DCanalsouseRSsystemofnomenclature.Structuresof20commonaminoacids:Aminoacidsaregroupedbaseduponthepropertiesandstructuresofsidechains.1aliphaticRgroupsconsistofcarbonsandhydrogensglycine-R=H__allesta.a.withnochiral__nteralanine-R=CH3methylgroupvalineR=branched;hydrophobic;importantinproteinfoldingleucineR=4carbonbranchedsidechainisoleucineR=2chiral__ntersprolineR=ring;putsbendsorkinksinproteins;containsasecondaryaminogroup2aro__ticRgroupsh__ephenylringphenylalanine-veryhydrophobictyrosine-hydrophobicbutnotasmuchbecauseofpolargroupstryptophan-“AbsorbUVlightat280__--usedtoesti__te[protein]3sulfur-containingRgroupsmethionine-sulfurisinternalhydrophobiccysteine-sulfuristerminal--highlyreactive;canformdisulfidebonds4sidechainswithalcoholsserine--hydroxylgroups--hydrophilicthreonine-“5basicRgroupshistidine-hydrophilicsidechains-+chargedatneutralpHlysine-“arginine-strongbase6acidicRgroupsandamidederivativesaspartate-carboxylgroup-confer-chargesonproteinsgluta__te-carboxylgroupasparagine-amideofaspartate-sidegroupsuncharged--polarglutamine-amideofgluta__te-“AmidegroupscanformHbondswithatomsofotherpolaraminoacids.IonizationofAminoAcidsAllaminoacidsareh__eaneutralnetchargeatphysiologicalpH
7.
4.Thecarboxylandaminogroupsandanyotherionizablegroupsdeterminecharge.Eachaminoacidhas2or3pKavalues7aminoacidsh__esidechainsthatareionizableseeTable
3.
2.Thiscomplicatesthebasictitrationcurvesothatthereare3inflectionpointsratherthan
2.AtagivenpHaminoacidsh__edifferentnetcharges.Canusetitrationcurvesforaminoacidstoshowionizablegroups.TheisoelectricpointpIisthepHatwhichtheaminoacidhasnonetcharge=zwitterion.IfpHpIaminoacidwouldbenegativelycharged.IfpHpIaminoacidwouldbepositivelycharged.IfpH=pIaminoacidwouldh__enocharge.CanuseHenderson-HasselbalchequationtocalculatethefractionofgroupionizedatagivenpH.acarboxylgrouppKa
1.8-
2.5aaminogrouppKa
8.7-
10.7IfpHpKaagreateramountofthegroupisprotonatedNH3+orCOOHIfpHpKathereisagreaterionizationandagreateramountofunprotonatedoranionformNH2orCOO-.IfpH=pKathen[conjugatebase]=[weakacid].FortheionizationofthecarboxylgroupofalaninepH=pKa+log[conjugatebase][weakacid]7=
2.4+log[RCOO-][RCOOH]
4.6=log[RCOO-][RCOOH]39810:1meaningtheanionpredominatesgreatlyalmostallCOOHgroupsareionized.Fortheionizationoftheaminogroupofalanine7=
9.9+log[NH2]-NH3+---NH2=H+[NH3+]-
2.9=log[NH2][NH3+]
0.001:1or1in1000moleculesundissociatedgrouppredominatesAtpIof
6.15thereisnonetchargeallofthecarboxylgroupsareunprotonatedandnoneoftheaminogroupisunprotonated.pI=pKa1+pKa2/2ForRgroupsthatareionizablethepIisnotsimplythe__erage!!!PeptideBondsThepri__rystructureofaproteinisthelinearsequen__ofaminoacidsthatarecovalentlybondedtoformapolypeptidechain.Formedbycondensationreactioninwhichamoleculeofwaterisremoved.Eachaminoacidresidueiscalledbyreplacing-ineor-atewith-ylglycine---glycylThepeptidebondisaplanarbondwithnorotationaroundC-Naxis.Ifisalsointhetransform.Willtalkabouttheconsequen__slater.ProteinPurificationTechniquesAllworkdoneat4oCtominimizedegradation.1-preparationoftheproteinsolutionWithappropriatebuffermustfirstdisrupt__llsbymechanicalhomogenizationwitheitherdetergentand/orenzymetreatment.Usea__ntrifugetoseparateintopelletandsupernatant.2-fractionationreliesonproteinsolubilitydifferen__sUseammoniumsulfatesalt--interfereswithnoncovalentbondsbetweenproteinandothermolecules.Remembersolubilityisbaseduponinteractionsofmoleculewithwatermoleculesviahydrogenbonds.Differentproteinsprecipitateoutatdifferent[salt].Use__ntrifugetoremoveprecipitatedprotein--resuspendinbuffer.UsedialysistochangeoutsolventandgetridofNH4SO
4.3-chro__tographyFurtherfractionatesproteinsbaseduponprotein’sinteractionwith__trix.Mostcommonlyusediscolumnchro__tography.Usesbeadsor__llulosefibers.Proteinsolutioniswashedthroughcolumn.Eluatecollectedandassayedforprotein.Therearethreetypesofcolumnchro__tography:1ion-exchangeanionorcationSeparatesbaseduponproteincharge.Elutebychanging[salt].2gelfiltrationUsesporousresin.Separatesbaseduponproteinsize.3affinitychro__tographyAttachligandto__trix.Canbesubstrateantibodyetc.Eluateusinghigh[ligand]orhigh[salt].Resultsin1000-10000foldpurification.4-electrophoresisSeparatesproteinsbaseduponmigrationinanelectricfield.PAGE-polyacrylamidegelelectrophoresisUsesacrylamideasgel__trix.Separatesbaseduponsizeandchargebufferisslightlybasicsomostproteinsh__enegativecharge.SDS-PAGE-sodiumdodecylsulfatepolyacrylamidegelelectrophoresisUsesSDSand2-mercaptoethanol.Separationbaseduponsizeonly.Forbothmuststaingeltovisualizeproteins.Bandscanbecutoutofgelproteinelectroelutedandpurified.AminoAcidCompositionofProteins1-aminoacid____ysisGivesrelativequantitiesofeachaminoacidbutnothingaboutorder.Firstmustboilproteinin6NHClfor24hourstobreakpeptidebonds.Su__ecthydrolysatetoion-exchangechro__tographyatdifferentpH’sORusephenylisothiocyanatePITCtogeneratederivationsandsu__ectresulttoHPLC.Problems:1-acidhydrolysisconvertsasparaginetoasparticacidandglutaminetoglutamicacid2-alsolosesomeserinethreonineandtyrosine.3-sidechainoftryptophanisdestroyed.AminoAcidResidueSequen__UseEd__ndegradationUsesPITCreagentthatreactswiththefreeN-terminustoformaPTC-peptide---treatwithtrifluoroa__ticacid---lastaminoacidiscle__ed---extractwithorganicsolventbutylchloride---PTH-aminoacid---____yzedbychro__tography.Thentakere__iningpeptideremovenexttolastaminoacidetc.Isnowauto__ted--sequenator.Goodfor__allpeptideof50aminoacidresidues.Ifproteinisgreaterthan50aminoacidresiduesmustuseproteasesorchemicalreagentstocle__esomeofthepeptidebonds.1-cyanogenbromide-reactswithmethionineresidues-cutsonCOOHside2-proteasestrypsin-cle__estocarboxylsideoflysineandargininechymotrypsin-cle__esataro__ticandbulksnonpolarsidechainsphenylalaninetyrosinetryptophanStaphylococcusaureusV8protease-cle__estocarboxylsideofgluta__teandaspartateNeedtouseatleast2differentcle__agetechniquestoobtainoverlappingsequen__susingEd__ndegradation.PAGE12。
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