分子生物學(xué)48742850課件

2024/4/16分子生物學(xué)48742850NoncovalentinteractionsTheRandomcoil纏繞Linearpolypeptideandpolynucleotidechainscontainbondsaboutwhichthereisfreerotation.Intheabsenceofanyintrastrand（分子內(nèi)）

interactions,eachmonomerwouldbefreetorotatewithrespecttoitsadjacentmonomersFew,ifany,nucleicacidorproteinmoleculesexistinnatureasrandomcoilsbecauseofmanyinteractionsbetweenelementsofthechainsNoncovalentinteractionsHydrogenbonds氫鍵Thehydrophobicinteraction疏水作用Ionicbonds離子鍵Vanderwaalsattraction范德華力effectsofnoncovalentinteractionsDeterminethethreedimensionalstructuresofmacromolecules.Constrain(約束)alinearchaintofoldinsuchawaythatdifferentregionsofthechain,arebroughttogether.Disulfidebonds(二硫鍵;covalent)alsoaffectsthemolecularpropertiesofproteins.MacromoleculeisolationandcharacterizationProteinsMolecularsieves分子篩:molecularweightChromatography層析:charge(positivelyornegativelycharged)oraffinitypropertiesElectrophoresis電泳：SDS,chargeandsize;mostconvenientmethodUltracentrifugation超離心AminoacidsequencingX-raycrystallographyx射線晶體學(xué)Electronmicroscopy電子顯微鏡MacromoleculeisolationandcharacterizationNucleicacidGenecloningPCR:polymerasechainreactionMolecularsieves:molecularweightElectrophoresis:Agarosegel,mostconvenientmethodUltracentrifugationNucleotidesequencingElectronmicroscopySummaryStructuralcharacteristicsofmacromoleculesComponentsofmacromoleculesNoncovalentinteractionsCovalentbondsBondsfordeterminingthree-dimensionalstructureMethodsforisolationandcharacterizationofproteinandnucleicacidsImportantconceptsandaspectsyouarerequiredtounderstandSmallMoleculesvsMacromoleculesMonomers,PolymersandMacromoleculesCommonMonomersinCellsWhatAAhaveincommon?ExceptionWhatmakeAAunique?DifferentcategoriesofAAandtheirspecialfeatures:formHbonds,saltbridges,disulfidebond,responsibleforUVabsorbance,etcDandLformsofAA4differentlevelsofproteinstructuresandtheirfeatures:basisofstructures,bondsinvolvedChemicalcomponentsofDNAandtheirpositionsindoublehelixstructureImportantforcesinmaintainingDNAstibilizationDifferencebetweenDNAandRNADandLsugarsMonosaccharidesDisaccharidesPolysaccharidesPolysaccharides’majorrolesPeptidebondDisulfidebondglycosidicbondStructuralcharacteristicsoflipidsNoncovalentinteractionsMethodsforproteinisolationandcharacterizationMethodsfornucleicacidisolationandcharacterizationTheendofchapter2Chapter6

TheGeneticMaterial遺傳物質(zhì)StudyobjectivesBackgroundinformationonthemechanismsofheredity.ExperimentalevidencethatidentifiedDNAasthegeneticmaterial.PropertiesofDNAasthegeneticmaterial,includingstorage,transmission,stability,andmutation.TheKeyExperimentsContributestoIdentifyingDNAasthePrimaryGeneticMaterial1928F.Griffith/Transformation1944O.T.Avery/FurtherConfirmation1952A.D.HersheyandM.Chase/PhageLabeling1928F.GriffithTransformationStreptococcuspneumoniae(pneumococci)growingascoloniesonthesurfaceofaculturemedium.Left:Thepresenceofacapsule(包膜）aroundthebacterialcellsgivesthecoloniesaglistening（發(fā)亮）,smooth(S)appearance.Right:Pneumococcilackingcapsuleshaveproducedtheserough(R)colonies.TheExperimentsSomethingintheheat-killedScellstransformedtheR-typebacteriaintoS-typecells.TheConclusionsTherewasa"transformingagent"inthetheheattreatedwildtypebacteria(smooth)whichtransformedthelivemutant(rough)typebacteriatobeabletoproducewildtypebacteriacapsule.O.T.Avery’sTestifyBackground:In1944,OswaldAveryandhiscolleaguesatTheRockefellerInstituteinNewYorkCityeventuallyshowedthatthe"something"wasDNA.Averyextractedrelativelypurenucleicacids(DNA+RNA)frompneumococcustodeterminewhatwasthe"transformingagent"observedinGriffith'sexperimentsAvery’sTransformationExperiment-1944DeterminedthatDNAwasthegeneticmaterialresponsibleforGriffith’sresults(notRNA).In1952,A.D.HersheyandM.ChaseperformedanotherexperimentthatprovidedfurtherevidencethatgenesweremadeofDNA.PhageLabelingBackgroundT2phageisaviruswhichinfectsthebacteriaE.coli.Thephagelookslikeatinylunarlandingmodule.Inanycase,about20minafterthephageadsorbstothesurfaceofthebacteria,thebacteriaburstsopen(lysis)andreleasesamultitudeofprogeny(子代）virus.LifecycleofT2phageTheExperimentalIdeaNormalDNAcontainsonlyhydrogen,nitrogen,carbon,oxygenandphosphorousatomsIfthemediainwhichthebacteriagrew(andwereinfected)included32PlabeledATP,progenyphagecouldberecoveredwiththisisotopeincorporatedintoitsDNA.Normalproteinscontainonlyhydrogen,nitrogen,carbon,oxygen,andsulfuratomsLikewise,ifthemediacontained35Slabeledmethioninetheresultingprogenyphagecouldberecoveredwiththisisotopepresentonlyinitsproteincomponents.Phageweregrowninthepresenceofeither32Por35Sisotopiclabels

InfectedE.colibacteriawithtwotypesoflabeledT232Pisdiscoveredwithinthebacteriaandprogenyphages,whereas35Sisnotfoundwithinthebacteriabutreleasedwithphageghosts.

Hershey-ChaseBacteriophageExperiment-19531969:AlfredHersheyCONCLUSIONThematerialwhichwasbeingtransferredfromthephagetothebacteriaduringinfectionappearedtobemainlyDNA.Theresultsprovidedadditionalsupportfortheviewthat

DNAwasthehereditarymaterialandproteinwasNOTthehereditarymaterial.FurtherproofforDNAasthegeneticmaterialcameafewyearslaterItwasdemonstratedthatthenaked,purifiedDNAofthesmallbacteriophageφX-174couldsuccessfullyinfectbacterialprotoplasts(原生質(zhì)體，cellslackingaportionoftheircellwall)intheabsenceofanyviralprotein,andproducenormal,infectiousphageprogeny.RNAasGeneticMaterialIn1955,H.Fraenkel-ConratandR.Williamsshowedthataviruscanbeseparated,invitro,intoitscomponentpartsandreconstitutedasaviablevirus.ThisfindingletFraenkel-ConratandB.Singertoreconstitutetobaccomosaicvirus(煙草花葉病毒）withpartsfromdifferentstrains.

PlantsinfectedbyTMVdevelopcharacteristicmosaic（嵌花式）-likelesions（病灶）ontheirleaves.Used2viralstrainstodemonstrateRNAisthegeneticmaterialofTMVReconstitutedTMVinvitroConclusionsabouttheseearlyexperimentsGriffith1928&Avery1944:DNA(notRNA)istransformingagent.Hershey-Chase1953:DNA(notprotein)isthegeneticmaterial.Fraenkel-Conrat&Singer1957:RNA(notprotein)isgeneticmaterialofsomeviruses.Exception?TheonlyexceptionisonetypeofdiseaseinwhichtransmissionisbyaproteinwithoutaccompanyingDNAorRNA.Prion:proteinaceousinfectionparticlelackingnucleicacidPrioncausesagroupoffataltransmissible（可遺傳的）neurodegenerative（神經(jīng)退行性的）diseasesofbothhumansandanimalsThediseasesareinfectious,inherited,orsporadicSymptoms:progressivedementia（癡呆）,motordysfunction(ataxia共濟(jì)失調(diào);myoclonus肌陣攣)Thetermprioncamefromproteinaceousinfection.Firstidentifiedwith“Spongiformencephalopathies”Characteristicsofinfection:LossofmotorcontrolDementiaParalysisEncephalitis腦炎WidespreadneuronallossWaysofinfection:Infectious(includingdiet,aftersurgicalprocedures,cornealtransplantsetc.)Hereditary(autosomalanddominant)Prion:proteinaceousinfectionparticlelackingnucleicacidTransmissiblespongiformencephalopathiesAnimalsBovinespongiformencephalopathy(BSE)ScrapieinsheepandgoatsTransmissibleminkencephalopathyChronicwastingdiseaseofdeer,elkHumansKuruCreutzfeldt-Jacobdisease(CJD)Fatalfamilialinsomnia(FFI)Gerstmann-Strausslersyndrome(GSS)TSEsarealwaysfatalTypesofTSEsInfectiouse.g.,kuru,BSE(madcowdisease),scrapieSpreadbyconsumptionofinfectedmaterialorgantransplant,esp.corneatransfusionSporadic1-2millioninfectedworldwide,lateinlifeEvidencemountingthatsomesporadicTSEisreallyresultofinfectionFamilialDuetoautosomaldominantmutationofPrPInherited–atleast10-15%oftotalhumanTSEcasesEachofthesecanbetransmittedexperimentally

MajorContributorstotheHistoryofPrions

Glassesresearchin1950’sand60’sIn1967TikvahAlperatHammersmithHospitalfound“particles”responsiblefortransmittablespongioformencephalitiscontainednonucleicacids.In1982StanleyPrusineratUCSFconcludednoNA,firstnamed“proteinaceousinfectiousparticlesthatresistinactivationbyproceduresthatmodifynucleicacids”asPRIONS-receivedNobelPrizein1997.Howdoprionsfunction?

PrPScPrPcTheprionproteinexistsintwoforms.Thenormalprotein(PrPc)canchangeitsshapetoaharmful,disease-causingform(PrPSc).TheconversionfromPrPctoPrPScthenproceedsviaachain-reaction.WhenenoughPrPScproteinshavebeenmadetheyformlongfilamentousaggregatesthatgraduallydamageneuronaltissue.TheharmfulPrPScformisveryresistanttohightemperatures,UV-irradiationandstrongdegradativeenzymes.NewFormofDiseaseTransmissionUntilthe80’s,scientiststhoughtthattransmissiblediseasesmustincludegeneticmaterialinordertoinfectahost–however,inthecaseoftheabnormalPrP–thisdoesn’tseemtoapply.Prions:PrP=PrPcPrPCandPrPSchaveidentical1ostructureEncodedbysinglecopygeneonchrom.20Prions:PrP=PrPcnormalcellsexpresscellularPrP(glycoprotein)ontheirsurfaceintheplasmamembraneAnormalPrPcproteinisrecycledfromtheendosomalcompartmenttothecellsurface–thiscyclingbetweenthecellsurfaceandanendocyticcompartmentisaprocessthatismediatedbyclathrin-coatedpitsandaPrPCreceptorPrions:PrP=PrPcPrPcismajorlyinthe"alphahelixconformation“withsmallbetasheets.normalcellularprionscanbebrokendownquiteeasilybynormalmeansasmostproteins-byheat,orproteases,etc.PrPPhysiology?functionisstillunknown-maybeinvolvedincell-to-cellcommunication?researchershavefoundthatPrPconthesurfaceofnervecells,caninteractwithothermoleculestorelaysignalsarrivingfromoutsidethecell,avitalprocessinthenervoussystemknownas``signaltransduction.''Someresearchersspeculatethatprionsarenotneededfor“routine”functionsbutsomehowenablethenervoussystemto“fine-tune”itselfatthecellularlevelOtherstudies:prion-likepropertiestoamechanisminvolvedinmaintainingmemoryinvolvementintheimmunesystemfunctionincircadianrhythmandsleepregulation

AbnormalPrion:PrPscsameaminoacidsequenceasnormalprioni.e.theirprimarystructureisthesamebuttheirsecondarystructureisdominatedbybetaconfirmationresistsnormaldegradationtechniquese.g.formaldehyde,temperature,UVlight,proteases&nucleasesStimulatesnoimmuneresponseinthehostThekeyeventinthepathogenesisNormalPrionscontainmajorlyαhelixAbnormalPrionshavemoreβsheetconformationalconversionofanormalcellsurfaceglycoprotein(PrPC)intoapathogenicisoform(PrPSc)PrionbiologyForaprion(PrPSc)toinfectahost,thehostmusthavearecognizablecellularform(PrPc)ofthatprionGenerally,thecloserthephylogeneticrelationshipbetweenthedonorhostandtherecipient,thegreaterthechanceforinfection,andthemorerapidlysymptomsoccurLevelofaccumulationofpriondoesnotnecessarilycorrespondtolevelofdiseasePathologiceffectoccurswhenanormalPrPcproteinisconvertedtoPrPscproteinintheendosomalcompartmentofthecell,andisnolongerrecycledbacktothesurfaceofthecellwhenthePrPscproteinbeginstoaccumulateintheendosomalcompartments,amyloid（淀粉樣）depositsformandcausethecell(neuron)tolooseviability,resultingindeathConversionSequenceChangeinstructuralconfiguration–foldingFormationofinsolubleaggregates(amyloidplaques)NeuronaldamageleadingtoneuronaldeathDeathofthehostNormal=alphahelicalconfigurationAbnormal=betasheetconfiguration=markerforinfectivityFutureFocusinPrionResearchAlzheimer'sdiseaseandParkinson'sdisease?BecausesuggestionsthatPrPprotectsthebrainagainstdementiaandotherdegenerativeproblemsassociatedwitholdage–prionsmaypreventthePurkinjecellsfromdyingPropertiesoftheGeneticMaterialDNAhastheabilitytostoregeneticinformation,whichcanbeexpressedinthecellasneeded.Thisinformationcanbetransmittedtodaughtercellswithminimalerror.(Thisprocessrequirescomplexenzymesandrepairmechanisms.)DNApossessesbothphysicalandchemicalstabilitysoinformation(presentinduplicate，二倍體)isnotlostoverlongp