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StarBiochem

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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.

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StarBiochem

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PROTEINSTRUCTUREBASICSEachproteinhasthefollowingthreelevelsofproteinstructure:PrimarystructureListstheaminoacidsthatmakeupaprotein’ssequence,butdoesnotdescribeitsshape.SecondarystructureDescribesregionsoflocalfoldingthatformaspecificshape,likeahelix,asheet,oracoil.TertiarystructureDescribestheentirefoldedshapeofawholeproteinchain.Inaddition,someproteinsinteractwiththemselvesorwithotherproteinstoformlargerproteinstructures.HowtheseproteinsinteractandfoldtoformalargerproteincomplexistermedQuaternarystructure.

CHEMICALSTRUCTURESOFTHEAMINOACIDSThe20aminoacidsshareacommonbackboneandaredistinguishedbydifferent‘R’groups,highlightedinvariouscolorsbelow.

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StarBiochem

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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.

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StarBiochem

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• 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)!

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StarBiochem

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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.

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StarBiochem

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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