chapter4 protein properties classification and research methods四章蛋白质性质分类及研究方法

Chapter4ProteinProperties,Classification&ResearchMethodsOutlinePhysicalandChemicalPropertiesofProteinsResearchmethodsofproteinsClassificationofproteins(self-study)Solid-phasesynthesisofpolypeptides(self-study)UVabsorption:theabsorbancepeakat280nmpIvalueandsolubilityDenatureandRe-natureHydrolysisOtherpropertiesSomePropertiesofProteinsDisruptionofthenormal3Dstructureofaprotein,suchthatitlosesbiologicalactivity.Usuallycausedbyheat,ChemicalsorchangesinpH.Usuallyirreversible.Acookedeggcannotbe“uncooked.”

DenaturationLossofBiologicalActivityLossofSolubilityIncreasedProteolysisMoresensitivetoproteaseIncreasedviscosity

MeasuringProteinDenaturationAcid-catalyzedhydrolysis:candestroyTrp;Gln→Glu,Asn→AspBase-catalyzedhydrolysis:cancauseracemationofaminoacidsEnzymatichydrolysisProteinHydrolysisN-terminalhydrolysisC-terminalhydrolysisCarboxypeptidaseAcleavesallexceptforP,R,K;CarboxypeptidaseBcleavesonlyR,andK;CarboxypeptidaseYcleavesall;CarboxypeptidaseCcleavesall;InternalhydrolysisTrypsin-cleavesontheC-sideofKorR;Chymotrypsin-C-sideofF,Y,WPepsisn-N-sideofF,Y,WEnzymaticHydrolysisSpecificitiesofVariousEndopeptidasesLet'ssayyoudiscoveranewprotein:proteinXYoumightaskthefollowingquestions:Howbigistheprotein?Whatisitsisoeletricpoint?Isitpresentinothercells/organisms?Whatisitssequence?Whatisitsstructure?Experimentingwithproteins…Method:electrophoresisSeparatesproteinmoleculesbasedontheirsizes

1.HowbigisproteinX?Procedure:IncubateproteinXwithSDSsothatitesnegativelychargedLoadontogelApplyvoltagetothegelStaingelwithaproteindye(e.g.CoomasieBlue)ThisparticularkindofelectrophoresisisalsocalledSDS(PolyacrylamideGelElectrophoresis)ElectrophoresisMolecularWeightMarkersProteinXElectrophoresis2.WhatisproteinX'sisoelectricpoint?Method:isoelectricfocusingSeparatesproteinmoleculesbasedontheirisoelectricpoint

RememberproteinshavedifferentchargesatdifferentpH?

Procedure:MakeagelwithpHgradientLoadproteinFindthepHwheretheproteinstopsmovingHighIfyoucombineSDSandisoelectricfocusing,youget2-dimensionalgelelectrophoresis(2Dgel)Method:WesternblottingCellscontainmanydifferentprotein,howdoesoneidentifyaparticularprotein-of-interestfromagel?3.IsproteinXfoundinothercell?AntibodiesaredefenseproteinsmadebyimmunecellstorecognizeforeignproteinsinthebodyWiththehelpofantibodies!HowareantibodiesagainstproteinXobtained?DetectionofProteinXMethod:ProteinsequencingAminoacidanalysis;Proteolyticdigestion;Peptidefingerprinting;Peptidesequencing;Multiplesequencealignment;Massspectrometry4.WhatisthesequenceofproteinX?Overviewofproteinsequencing(1)Overviewofproteinsequenci