跨膜蛋白的同源建模流程(DS)Modeling-Transmembrane-Proteins

TutorialsMacromoleculetoolstutorialsModelingTransmembraneProteinsModelingTransmembraneProteinsRequiredfunctionalityandmodules:DiscoveryStudioClient,DSSequenceAnalysis,DSMODELER,DSProteinFamiliesRequireddatafiles:2rh1.pdbandADRB1_HUMAN.fastaTime:30minutesIntroductionTransmembraneproteinsareveryimportantinpharmaceuticalresearchsincetransmembraneproteinssuchastheGprotein-coupledreceptors(GPCR),arethetargetformanyexistingdrugsaswellasformanydrugcandidatesindevelopment.ThistutorialdemonstrateshowthesetoftoolsandprotocolsavailableinDiscoveryStudiocanbecombinedinaworkflowtoconstructahomologymodelofthebeta-1-adrenergicreceptorbasedontherecentlysolvedX-raystructureforthebeta-2-adrenergicreceptor.Thefollowingtasksarecoveredinthistutorial:LocatingandopeningtheinputfilesPredictingTransmembraneregionsofaproteinAligningsequencetotemplatesBuildingahomologymodelAddanimplicitmembranetothemodelstructureAdjustthepositionofthemembraneLocatingandopeningtheinputfilesThesequenceofthehumanbeta-1-adrenergicreceptorisprovidedinthesamplesfolder.Toopenthesequenceinasequencewindow.ChooseFile|Open...fromthemenubarandopenSamples|Tutorials|ProteinModeling|ADRB1_HUMAN.fasta.ThisopensthefileinanewSequenceWindowlabeledADRB1_HUMAN.Thefirststepinbuildingahomologymodelforatargetistoidentifyasuitabletemplate.Ifatemplatewithhighsequenceidentityisavailable,itcanoftenbeidentifiedwithasimpleBLASTsearch.Inthecaseoftransmembraneproteins,thenumberofX-raystructuresavailableisratherlimited,sothistutorialdoesnotincludethisstep.Formoredetailsonidentifyingtemplates,seetheCreatinghomologymodelsfromaproteinsequencetutorial.YouwillusearecentlysolvedstructureoftheX-raystructureofthebeta-2-adrenergicreceptor,PDBcode2rh1asatemplate.ChooseFile|Open...fromthemenubarandopenSamples|Tutorials|ProteinModeling|2rh1.pdb.ThisopensthefileinanewMoleculeWindowlabeled2rh1.Note.ABLASTsearchonthePDB_nr95databaserevealsanotherpossibletemplate,theX-raystructurefortheturkeybeta-1-adrenergicreceptor.Thatstructure,at70.5%sequenceidentitytothetargetsequencewouldbeasuperiortemplate.However,thistutorialuses2rh1tohighlightDiscoveryStudiofunctionalitythatwouldnotberelevantinthecaseofatemplatewithsuchhighsequenceidentity.Thenextstepinthemodelbuildingprocessistoalignthetargetsequencewiththetemplatesequence.ThefirststepistocombinebothsequencesinoneSequencewindow.ClicktheADRB1_HUMANSequenceWindowtabtomakeitactive.Right-clickinthewindowandchooseInsertSequence|FromWindows....ThiswillopentheInsertSequencefromWindowsdialog.Select2rh1andclickOK.1Thiswillinsertthesequenceof2rh1intotheADRB1_HUMANSequenceWindow.Thestatusbardisplaysthesimilarityandidentitybetweenthetwosequences.NotethatthesequencesarenotalignedandtheSequenceIdentityandSequenceSimilarityscores(~7.0%,~27.4%)areverylow.PredictingTransmembraneregionsofaproteinTheAlignSequencesProtocolallowsuseofsecondarystructureinformationtoimprovethequalityofthealignment.Secondarystructurepredictionmethods,suchasDSC,arebasedonthesolventexposurepatternsofglobularproteins.Alargepartofthesurfaceoftransmembraneproteinsisexposedtothehydrocarbonenvironmentofthemembraneinterior,ratherthanwater.Thisresultsinadifferentpatternofhydrophilicandhydrophobicresiduesintheaminoacidsequencewhichcausesstandardsecondarystructurepredictionmethodstobecomeunreliable.InthistutorialtheTransMemmethod,whichisdesignedtopredicthelicalregionsintransmembraneproteins,isused.OpentheMacromolecules|AnalyzeTransmembraneProteinstools.ClickPredictTransmembraneHelices.ThisaddssecondarystructurecartoonsintheADRB1_HUMANSequenceWindow.NoticethattheTRANSMEMpredictionfor2rh1hasalongregion(residuesA:ASN1002-A:TYR1161)forwhichTRANSMEMpredictsnohelicalregions.Thisregioncorrespondstothesequenceoflysozyme,whichwasfusedintothebeta-2-adrenergicreceptorinordertofacilitatecrystallization.Standardstructurepredictionmethodswillpredictsecondarystructureinthisregion.Suchalargeinsertionwillconfoundthealignmentalgorithmifsecondarystructurepredictionsareusedtoguidethealignment.AligningsequencetotemplatesOpentheMacromolecules|AlignSequencesandStructurestoolsandclickAlignSequences...toopentheAlignSequencesdialog.VerifythattheInputSequenceSetparameterissettoADRB1_HUMAN:All.ClicktheUseSecondaryStructuresparameterandchooseTRANSMEM.ClickRunandwaitforthejobtocomplete.Thistakesabout1.5minutesonaPentium4,1GbRAM,3GHzmachine.Whenthejobcompletes,theADRB1_HUMANSequenceWindowisautomaticallyupdatedwiththesequencealignmentandadialogreportsthetheSequenceIdentityandSequenceSimilarityscores(~42.1%,~53.3%)forthecalculatedalignment.ClickOKtoclosethedialog.TheSequenceIdentityandSequenceSimilarityscores(~42.1%,~53.3%)arenowhigher.Youshouldalsonotethattherearenowmoreresiduescoloreddarkturquoise,indicatingstrongsimilarityinthoseregions.BuildingahomologymodelOpentheMacromolecules|CreateHomologyModelstoolsandclickBuildHomologyModels...toopentheBuildHomologyModelsdialog.ClicktheInputSequenceAlignmentparameterandchooseADRB1_HUMAN:Allfromthelist.ClicktheInputTemplateStructuresparameterandselect2rh1.ClicktheInputModelSequenceparameterandch