教育部化学生物与资讯人才培育衔接计画

教育部化學生物與資訊人才培育銜接計畫蛋白質結構與功能之化學基礎PartI錢嘉琳5/14/2008AminoAcids,Peptides,andProteins•AminoAcids•PeptidesandProteins•ProteinStructure•ProteinSequencesGeneralstructureofaminoacid-carbon-carboxylgroup-aminogroupSidechain*Forallthestandardaminoacidsexceptglycine,the-carboniscovalentlybondedtofourdifferentgroups(-COO-,-NH3+,-Hand-R).The-carbonisachiralcenter.*Allmoleculeswithachiralcenterarealsoopticallyactive.Thetwostereoisomersofalinine,L-andD-alanine,arenonsuperimposablemirrorimagesofeachother(enantiomers).StericrelationshipofthestereoisomersofalaninetotheabsoluteconfigurationofL-andD-glyceraldehyde.TheaminoacidresiduesinproteinsareLstereoisomersAminoacidscanbeclassifiedbyRgroupTryptophanandtyrosineabsorbultravioletlight.Theabsorbancemaximaforbothoccursnearawavelengthof280nm.Phenylalaninegenerallycontributeslittletotheabsorbancepropertiesofproteins,Reversibleformationofadisulfidebondbytheoxidationoftwomoleculesofcysteine.DisulfidebondsbetweenCysresiduesstabilizethestructuresofmanyproteinsTheadditionalcarbonsinanRgrouparecommonlydesignated,,,,andsoforth,proceedingoutfromthecarbon,PeptidesandProteinsThepolymersofaminoacidsthatarelinkedbypeptidebonds.Theproteinisapolypeptide.FormationofapeptidebondbycondensationThepentapeptideSer-Gly-Tyr-Ala-LeuBiologicallyactivepeptidesandpolypeptidesoccurinavastrangeofsizesOxytocin:nineaminoacidresidues,secretedbytheposteriorpituitaryandstimulatesuterinecontractions.Glucagon:29residues,apancreatichormone,opposingtheactionofinsulin.LengthsofproteinsvaryconsiderablyDifferentproteinscontaindifferentaminoacidcompositionsSomeproteinscontainchemicalgroupsotherthanaminoacidsLevelsofstructureinproteins•ColumnChromatography3.Ion-exchangeChromatography4.Size-exclusionchromatography5.Affinitychromatography1.Electrophoresis:atechniquefortheseparationofproteinsisbasedonthemigrationofchargedproteinsinanelectricfield.2.Electrophoresisofproteinsisgenerallycarriedoutingelsmadeupofthecross-linkedpolymerpolyacrylamide.3.Thepolyacrylamidegelactsasamolecularsieve.Separationofproteinsisbasedontheircharge-tomassratio.4.ElectrophoresisinthepresenceofSDSseparatesproteinsonthebasisofmolecularweight.•SDSbindstomostproteinsinamountroughlyproportionaltothemolecularweightoftheprotein.•TheboundSDScontributesalargenetnegativecharge,renderingtheintrinsicchargeoftheproteininsignificant.•ThenativeconformationofaproteinisalteredwhenSDSisbound.Differentsamplesareloadedinwellatthetopofthepolyacrylamidegel.Afterelectrophoresis,theproteinsarevisualizedbyaddingadyesuchasCoomassieblue,whichbindstoproteinsSDS-PAGEcanestimatethemolecularweightofaprotein•Isoelectricfocusingatechniqueusedtodeterminetheisoelectricpoint.•ApHgradientisestablishedbyallowingamixtureofampholytestodistributethemselvesinanelectricfieldgeneratedacrossthegel.•Whenaproteinmixtureisapplied,eachproteinmigratesuntilitreachesthepHthatmatchesitspI.•IsoelectricfocusingTwo-dimensionalelectrophoresisMorethan1,000differentproteinsfromEcolicanberesolvedusingthistechniqueTheFunctionofaproteindependsonitsaminoacidsequence•ThebacteriumE.coliproducesmorethan3,000differentproteins;ahumanproduces25,000to35,000.•Eachtypeofproteinhasauniquethree-dimensionalstructureandthisstructureconfersauniquefunction.•Eachtypeofproteinhasauniqueaminoacidsequence.•Thousandsofhumangeneticsdiseaseshavebeentracedtotheproductionofdefectiveproteins.•Perhapsone-thirdoftheseproteinsaredefectivebecauseofasinglechangeintheiraminoacidsequence;hence,iftheprimarystructureisaltered,thefunctionoftheproteinmayalsobechanged.•Oncomparingfunctionallysimilarproteinsoftenhavesimilaraminoacidsequences,asanextremecaseisubiquitin.Istheaminoacidsequenceabsolutelyfixed,orinvariant,foraperticularprotein?•NO;someflexibilityispossible.•Anestimated20%to30%oftheproteinsinhumanarepolymorphic,havingaminoacidsequencevariantsinthehumanpopulation.•Manyofthesevariationsinsequencehavelittleornoeffectonthefunctionoftheprotein.•Althoughtheaminoacidsequenceinsomeregionsoftheprimarystructuremightvaryconsiderablywithoutaffectingbiologicalfunction,mostproteinscontaincrucialregionsthatareessentialtotheirfunctionandwhosesequenceisthereforeconserved.Methodsofproteinsequencing1.AutomatedEdmandegradation2.DNAsequencing3.MassspectrometryAminoacidsequenceofproteincanbededucedbyitsDNAsequenceJ.B.Fenn(1917~)K.Tanaka(1959~)KurtWüthrichESISLDNMRTheNobelPrizeWinnersinChemistry2002forthedevelopmentofmethodsforidentificationandstructureanalysisofbiologicalmacromoleculesProteinSequencesandEvolution•Proteinsequencesarearichsourceofinformationaboutproteinstructureandfunction,aswellastheevolutionoflifeonthisplanet.Abacterialevolutionaltree,basedonthesequencedivergenceobservedintheGroELfamilyofprotein