Name________________________
StarBiochem
Ver.1‐LMA
1
DNAGlycosylase:acasestudyinproteinevolutionLearningObjectives• Proteinswithsimilarfunctionshavesimilar3Dstructures• Proteinswithsimilarfunctionand3Dstructuresdonotneedtohaveidenticalaminoacidssequences.
BackgroundInthisexercise,youwilluseStarBiochem,aprotein3‐Dviewer,toexplorethestructureofaDNArepairproteinfoundinmostspecies.DNArepairproteinsmovealongDNAstrands,checkingformistakes.DNAglycosylases,aspecifictypeofDNArepairproteins,recognizeDNAbasesthathavebeenchemicallyalteredandremovethem,leavingasiteintheDNAwithoutabase.OtherproteinsthencomealongtofillinthemissingDNAbase.
WewillbeginthisexercisebyexploringthestructureofoneofthehumanDNAglycosylasescalledhOGG1.WewillthenexplorethestructureofarelatedDNAglycosylaseproteinfoundinarchaebacteriacalledPa‐AGOG.
hOGG1 Pa‐AGOG
GettingstartedwithStarBiochem• TogettoStarBiochem,pleasenavigateto:http://web.mit.edu/star/biochem.• ClickontheStartbuttontolaunchtheapplication.• ClickTrustwhenapromptappearsaskingifyoutrustthecertificate.• UnderFile,clickonOpen/Importandselect“1EBM”(thefourletterIDforhOGG1)andclickOpen.
YouarenowviewingthestructureofthehumanDNAglycosylasehOGG1(1EBM),witheachbondintheproteindrawnasaline.
Practicechangingtheviewpointofthisproteinintheviewwindow:
Mac PCTOROTATE
clickanddragthemouse left‐clickanddragthemouse
TOMOVEUP/DOWNRIGHT/LEFT
apple‐clickanddragthemouse right‐clickanddragthemouse
TOZOOM
option‐clickanddragthemouse Alt‐left‐clickanddragthemouse
TakeamomenttolookatthestructureofhOGG1(1EBM)fromvariousanglesinthis"bondsonly"view.Beforeproceedingtoanswerthequestions,reviewthebasicstructuresandtermsonthenextpagewhichyoucanrefertoduringthislesson.
Name________________________
StarBiochem
Ver.1‐LMA
2
PROTEINSTRUCTUREBASICSEachproteinhasthefollowingthreelevelsofproteinstructure:PrimarystructureListstheaminoacidsthatmakeupaprotein’ssequence,butdoesnotdescribeitsshape.SecondarystructureDescribesregionsoflocalfoldingthatformaspecificshape,likeahelix,asheet,oracoil.TertiarystructureDescribestheentirefoldedshapeofawholeproteinchain.Inaddition,someproteinsinteractwiththemselvesorwithotherproteinstoformlargerproteinstructures.HowtheseproteinsinteractandfoldtoformalargerproteincomplexistermedQuaternarystructure.
CHEMICALSTRUCTURESOFTHEAMINOACIDSThe20aminoacidsshareacommonbackboneandaredistinguishedbydifferent‘R’groups,highlightedinvariouscolorsbelow.
Name________________________
StarBiochem
Ver.1‐LMA
3
ProteinStructureQuestions1TheproteinhOGG1(1EBM)interactswithDNAtohelprepairdamagedDNAbases.Inthisparticularstructure,thehOGG1proteinisboundtoasegmentofDNAthathasbeendamaged.Whatcoloristheprotein?WhatcoloraretheDNAstrands?• TohelpdistinguishtheDNAsegmentfromthehOGG1proteinclickonStructure.• ClickontheforwardarrowuntilyouseeQuaternary.• ClickonQuaternaryandthenclickonChain.
Answer
2ThehOGG1(1EBM)proteinconsistsof325aminoacids.Listthe13aminoacidsnumbered105through117inorder.• WithinStructure,clickonPrimary.• Scrolldownthroughtheaminoacidlistifnecessary.
Answer 105_______ 108_______ 111_______ 114_______ 117_______106_______ 109_______ 112_______ 115_______107_______ 110_______ 113_______ 116_______
3Withinaprotein,aminoacidssequencesformlocalstructurescalledsecondarystructures(referencepage2).Secondarystructuresincludehelices,sheetsandcoils.
a)ExplorethesecondarystructuresfoundinhOGG1(1EBM).Arehelices,sheets,and/orcoilspresentinhOGG1(1EBM)?• UnderStructureclickonSecondary.• ClickonHelices,SheetsandCoilsoneatatimemakingsuretoclickeachoneoffbeforeclickingthenextone.
AnswerHelices_______(YesorNo)Sheets_______(YesorNo)Coils_______(YesorNo)
b)WhichsecondarystructurepredominatesinthehOGG1(1EBM)protein?
Answer
c)Aminoacids105through117foldintooneofthethreesecondarystructures.Whichsecondarystructuredotheyfoldinto?• WithinSecondary,clickonAllRibbons.• ClickonPrimary.
Name________________________
StarBiochem
Ver.1‐LMA
4
• Selectallaminoacidsbetween105through117byclickingonaminoacids105and117whileholdingdownShift(Mac/PC).
• Rotatetheproteintolocatetheselectedaminoacids(white).
Answer
4DNAiscomposedoffourbases:A,T,C&G.TheDNAsequenceillustratedinthisstructure,containsadamagedDNAbase• UnderView,clickonResetmolecule.• ClickonPDBTree,IEBMandthenclickon8OG_25.• Thehighlightedstructuralelement(white),8OG_25(8‐oxoguanine),isatypeofdamagefortheDNAbaseguanine(G).
CertainaminoacidswithinhOGG1(1EBM)formcontactswiththeDNAandareabletorecognizeifaDNAbasehasbeenchemicallyalteredordamaged.WhereareyoumorelikelytofindtheaminoacidsthatrecognizedamagedDNAbaseswithinhOGG1(1EBM),inHelix1orHelix16?Explainwhy.• ClosePDBTreeandclickonStructure.• ClickonSecondaryandthenonTrackSelection.• WithinHelixSelectionclickonHelix1andHelix16.• CloseStructure.• TovisualizealltheatomswithinthehelicesandthedamagedbasedclickonViewControls.• UnderAtoms,clickonDraw.Underthisview,eachatomintheproteinisshown.[Carbonisgrey,Nitrogenisblue,Oxygenisred,andSulfurisyellow.Note:hydrogenisnotshown.]
• TohelpisolatetheaminoacidswithintheproteinandthedamagedDNAbasemovetheUnselectedtransparencyslidertotheleftuntilyouseemostoftheaminoacidsthatwereNOTselecteddisappear.
• Optional:underAtomsclickonFillspacetohaveamorerealisticviewofthespaceoccupiedbytheselectedatoms.ThisviewallowsyoutoseewhetherornotthedamagedDNAiscontactingeitheroneofthehelices.
Answer
ProteinEvolutionQuestionsThehOGG1proteinispartoflargesuperfamilyofDNAglycosylases.Proteinfamiliesrepresentgroupsofproteinsfromdifferentorganismswhosefunction(forinstance,repairingDNAdamage)hasbeenconservedthroughoutevolution.Typically,proteinfamilymembersnotonlyhaveconservedfunction,butinmanyinstancestheir3Dstructure(orpartsoftheirstructure)havealsobeenconservedthroughoutevolution.WewillnowexploreanothermemberoftheDNAglycosylaseproteinfamily:Pa‐AGOG.Pa‐AGOGisfoundinaspeciesofarchaebacteria,Pyrobaculumaerophilum,thatliveinhotmarinewaterholes.Pyrobaculumaerophilumpreferslivinginextremeenvironment.It’soptimalgrowingtemperatureis100˚C(212˚F)!
Name________________________
StarBiochem
Ver.1‐LMA
5
WewillexplorethedifferencesandsimilaritiesbetweenthehumanDNAglycosylaseprotein,hOGGanditsarchaebacteriaproteincounterpart,Pa‐AGOG.
OpenanotherviewerwindowofStarBiochem.(DonotclosetheviewerthatcontainsthehOGG1(1EBM)protein.)• LaunchanotherwindowofStarBiochem.• InthetopmenuunderFileclickonOpen/Importandselect“1XQP”,thefourletterIDforthePa‐AGOGprotein,andclickOpen.
YouarenowviewingthestructureofPa‐AGOG(1XQP),witheachbondintheproteindrawnasaline.NotethatthisstructureonlycontainstheproteinPa‐AGOG(1XQP)boundtoasingledamagedDNAbase(noDNAhelicesarepresent).TakeaminutetolookatthestructureofPa‐AGOGandthenanswerthefollowingquestions.5Pa‐AGOG(1XQP)andhOGG1(1EBM)havesimilar3Dstructures.InparticularPa‐AGOG(1XQP)sharessomeofthesamesecondarystructuresashOGG1(1EBM).
a)Whichsecondarystructure(s)arepresentinbothPa‐AGOG(1XQP)andhOGG1(1EBM)?• WithinthehOGG1(1EBM)viewer,underViewclickonResetmolecule.• ClickonStructure.• WithinSecondary,clickonAllRibbons.• GotothePa‐AGOG(1XQP)viewer.• ClickonStructure.• WithinSecondary,clickonAllRibbons.
Answer
b)Whichsecondarystructure(s)aremissinginPa‐AGOG(1XQP),butpresentinhOGG1(1EBM)?
AnswerInthearcheabacteriaproteinPa‐AGOG(1XQP)thephenylalanineaminoacid144,Phe_144,isinvolvedinrecognizingdamagedDNAbases.FirsttakealookatPhe_144inthePa‐AGOG(1XQP)protein.NoteitslocationwithintheproteinandwithrespecttothedamagedDNAbase(yellow).• InthePa‐AGOG(1XQP)viewer,underViewclickonResetmolecule.• WithinStructureclickonPrimary.• ClickonPhe_144.Theselectedaminoacidisnowshowninwhite.• ZoominandrotatethemoleculetoobservetheorientationofPhe_144withrespecttothedamagedDNAbase(yellow).NotebothitsangleandthedistancefromthedamagedDNAbase.
Name________________________
StarBiochem
Ver.1‐LMA
6
6InthehumanproteinhOGG1(1EBM),aphenylalanineaminoacidisalsoinvolvedinrecognizingdamagedDNAbases.AlthoughhOGG1(1EBM)containsaphenylalanineatposition144,thisoneisNOTtheoneinvolvedintherecognitionofdamagedDNAbases.WewillexaminetheorientationandclosenessofPhe_144withrespecttothedamagedDNAbaseinhOGG1(1EBM).
a)WhatisdifferentabouttheanglebetweenPhe_144andthedamagedDNAbaseinPa‐AGOG(1XQP)andinhOGG1(1EBM)?• InthehOGG1(1EBM)viewer,clickonResetmoleculeunderViewinthemainmenu.• WithinStructureclickonPrimary.• ClickonPhe_144.Theselectedaminoacidisnowshowninwhite.• ZoominandrotatethemoleculetoobservetheorientationofPhe_144withrespecttothedamagedDNAbase(yellow).
Answer
b)WhatelseisdifferentintheplacementofPhe_144inPa‐AGOG(1XQP)andinhOGG1(1EBM),withrespecttothedamagedDNAbase?
Answer
c)GiventhatinthehumanDNAglycosylase,hOGG1(1EBM),Phe_144isNOTresponsibleforrecognizingdamagedDNAbases,findthephenylalanineinhOGG1(1EBM)thatwouldrecognizedamagedDNAbases.UsetheplacementandorientationofPhel_144withrespecttothedamagedbaseinPa‐AGOGasareference.WhatistheaminoacidnumberofthisphenylalanineinhOGG1(1EBM)?Why?• InthehOGG1(1EBM)viewer,clickonResetmoleculeunderViewinthemainmenu.• ZoomintoseeaminoacidssurroundingthedamagedDNAbasedcoloredinyellow.• ClickonSelectionControls.• UnderSelectbymouseclickclickonResidue.ThiswillallowyoutoclickonaparticularaminoacidwithintheactualstructureinthevieweranddeterminewhichoneitisbylookingatthehighlightedaminoacidinPrimarywithintheStructurewindow.
Answer
