Name________________________

StarBiochem

Ver.5‐M.Rokop,D.SinhaandL.Alemán

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Hemoglobin&SickleCellAnemiaExerciseLearningObjectivesInthisexercise,youwilluseStarBiochem,aprotein3Dviewer,toexplore:• thestructureofthehemoglobin(Hb)protein• thestructureoftheabnormalformofhemoglobin(HbS)thatresultsinsicklecellanemia,ageneticallyinheritedblooddisorder

• thespecificaminoacidsubstitutioninHbSthatcausessicklecellanemiaBackgroundHemoglobin(Hb)isaproteinthatfunctionsbybindingtotheoxygenmolecules(O2)intheO2‐richenvironmentofthelungs,travelingtotherestofthebodywithinredbloodcellsinthecirculatorysystem,andthenreleasingO2rapidlyintherelativelyO2‐poorenvironmentofvariousbodytissues.Hemoglobinhasthecapacitytobindbetween1to4O2molecules.ThebindingofeachO2moleculetohemoglobinincreasesitsaffinityforthenextO2molecule.

TheHbproteiniscomprisedofpolypeptidechainscalled“globin“chains.Eachoftheseglobinchainsisattachedtotheironcontaining“heme”group.

AsingleaminoacidsubstitutioninaspecificglobinchainofHbresultsintheHbSformoftheprotein.TheHbSmoleculesmayadheretoeachother,forminglargecomplexesthatcandistortnormalredbloodcells(RBC)intosickleshapedcells.ThesickledRBCshaveareducedlifespan.Additionally,thesickledRBCscanclogbloodvessels,whichcanleadtoorgandamageandpaininindividualswithsicklecellanemia.

Sicklecellanemiaisageneticdisorderthatshowsanautosomalrecessivemodeofinheritance.TheprevalanceofthisdisorderinUnitedStatesisapproximately1in5000individuals,anditmostlyaffectsAfricanAmericans,SouthAsiansandHispanics.GettingstartedwithStarBiochem• TobeingusingStarBiochem,pleasenavigateto:http://web.mit.edu/star/biochem.• ClickontheStartbuttontolaunchtheapplication.• ClickTrustwhenapromptappearsaskingifyoutrustthecertificate.• UnderFile,clickonOpen/Importandselect“1A3N”andclickOpen.

Youarenowviewingthestructureofhumanhemoglobin(1A3N),witheachbondintheproteindrawnasaline(“bondsonly”view).

Normalredbloodcell Sickleredbloodcellwww.carnegieinstitution.org

Name________________________

StarBiochem

Ver.5‐M.Rokop,D.SinhaandL.Alemán

2

Practicechangingtheviewpointofthisproteinintheviewwindow:

Mac PCTOROTATE

clickanddragthemouse left‐clickanddragthemouse

TOMOVEUP/DOWNRIGHT/LEFT

apple‐clickanddragthemouse right‐clickanddragthemouse

TOZOOM

option‐clickanddragthemouse Alt‐left‐clickanddragthemouse

Takeamomenttolookatthestructureofhumanhemoglobin(1A3N)fromvariousanglesinthis"bondsonly"view.Beforeproceedingtoanswerthequestions,youshouldreviewthebasicstructuresandtermsonthenextpagewhichyoumayrefertoduringthisexercise.

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StarBiochem

Ver.5‐M.Rokop,D.SinhaandL.Alemán

3

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

CHEMICALSTRUCTURESOFTHEAMINOACIDSThe20aminoacidsshareacommonbackboneandaredistinguishedbydifferent‘R’groups,highlightedinvariouscolorsbelow.

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StarBiochem

Ver.5‐M.Rokop,D.SinhaandL.Alemán

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ProteinStructureQuestions‐Level11Howmanyaminoacidscomprisetheprimarystructureofhemoglobin(1A3N)?• ClickonStructure.• ClickonPrimarywhichshowstheaminoacidsthataresequentiallyjoinedthroughpeptidebondstomaketheamino/polypeptidechain.Theaminoacidsofeachchainarehighlightedbyaspecificcolorandcanbedistinguishedfromthoseofotherchains.

Answer

2Howmanymonomerglobinchain(s)doyouseeinthecurrentviewofhemoglobin(1A3N)?Giventhis,whichtermbestdescribesthestructureofhemoglobin:amonomer,dimer,trimer,tetramer,orpentamer?• Todistinguishbetweenthedifferentmonomersthatmakeup1A3N,underStructureclickonQuaternary.• ClickonChain.

Answer

3Brieflylookattheprimarysequenceofeachmonomer/proteinchain.Aretheproteinchainswithinhemoglobin(1A3N)likelytobeidenticalordifferent?AnswerYes/Noandprovideabriefexplanationforyourchoice.• WithinStructure,clickonPrimary.

Answer

4Inadditiontocontainingaminoacids,hemoglobinalsocontainsfourchemicalgroupscalledhemes,whichbindtotheoxygeninourbloodstream.Whichelementscomprisethestructureofhemegroups?Howmanyatomsofeachoftheseelementsarepresentperhemegroup?

• ClickonViewandchooseResetMolecule.• ClickonPDBTreeandthenclickonthefilelabeled“1A3N”.• ClickonallHemegroupswhileholdingdownshifttoselectthematthesametime.• InViewControls,settheUnselectedtransparencysliderto“0.2”.• WithintheAtomsbox,clickonDrawtoseewhatatomsarepresent.Eachatomiscolor‐coded:Carbonisgrey,Nitrogenisblue,Oxygenisredand,inthisstructure,Ironisorange.

Answer

Name________________________

StarBiochem

Ver.5‐M.Rokop,D.SinhaandL.Alemán

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ProteinStructureQuestions‐Level25Followtheinstructionsprovidedbelowtoanswerthenextsetofquestions.• UnderSelectionControls,clickonResidues.• GotoMeasurementToolsandclickonEnableRadius.• FromthepulldownmenuchooseResidues.• SlidetheWithinRadiusslideruntilitreads“7.61”andclickonSelectWithinRadius.• GobacktoStructureandunderPrimarylookattheaminoacidresiduesthatarebeinghighlighted.• ClickonViewControlsandbringtheUnselectedsliderto“0”whilekeepingtheSelectedsliderat“1”.Youmayzoomintheselectedaminoacidforabetterview.

a)Identifytheglobinchain(s)(1,2,3and/or4)thatcontainthehighlightedaminoacids.

Answer

b)Inthe1stglobinchain,nametheaminoacidthatisclosesttotheN‐terminusend.Explainwhyyouselectedthisaminoacid.

Answer

c)Fromthechoicesprovidedbelow,selectthelevelofproteinstructurethatisrepresentedbythehighlightedaminoacids.Yourchoicesare‘primary’,‘secondary’,‘tertiary’and‘quaternary’.Selectallthatapplyandexplainwhyyouselectedaspecificoption.

Answer

d)Whichofthesehighlightedaminoacidscanpairtogetherto….• formhydrogenbonds?• exhibithydrophobicinteractions?

Answer

6Tertiaryandquaternarystructureareformedbythebendingandfoldingofpeptidechains.Thesetwolevelsofstructurearestabilizedbyvariouscovalentandnon‐covalentinteractionsbetweentheside‐chainsofdifferentaminoacidresidues.Wewillnowtakeadeeperlookattheaminoacidsinvolvedinthetertiarystructureofhemoglobin:aminoacids#85&#88inthe2ndglobinchain.Basedonthenatureoftheirside‐chains,howwouldyoucharacterizetheseaminoacids?Yourchoicesare‘ionic’,‘hydrogenbonding,‘vanderWaalsforces’,‘hydrophobic’or‘covalent’.

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StarBiochem

Ver.5‐M.Rokop,D.SinhaandL.Alemán

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• UnderStructure,clickonPrimary.• SelecttheaminoacidsbyindividuallyclickingonthemandsimultaneouslypressingControlandApplekey(Mac)/right‐click(PC).

• GotoTertiaryandwithintheColorbyResiduewindowclickoneachoptiononeatatime.

Answer

Structure‐>Function‐>DiseaseQuestions7Wewillnowtakealookatthestructureofsicklehemoglobin,HbS(2HBS),andcompareitsstructuretothatofnormal(wildtype)hemoglobin,Hb(1A3N),tounderstandhowasingleaminoacidchangeinhemoglobinleadstosicklecellanemia.• ClickonViewandchooseResetMolecule.• OpenanewwindowofStarBiochemwhilekeepingthestructureofHb(1A3N)open.• InthetopmenuunderFileclickonOpen/Import.• Clickon“2HBS”andclickOpen.

a)ComparingthecrystalstructuresofthetwoPDBfiles,2HbSand1A3N,howdoestheoverallstructureofnormal(wildtype)hemoglobindifferfromthatofsicklehemoglobin?

Answer

b)CarefullylookatthePDBstructureofthetwomoleculeswithin2HBSandthemoleculewithin1A3N.Circlethecorrectstatement(s)fromtheoptionsbelow.Thesingleaminoacidsubstitutioninsicklehemoglobin:

Answer• influencestheoverallstructureofindividualHbmolecules.

• doesnotinfluencetheoverallstructureofindividualHbmolecules.

• createsstickyregionsbetweentwoindividualHbmolecules.

8Thesingleaminoacidsubstitutionofvalineatposition#6inaspecificglobinchainofhemoglobinresultsinsicklecellanemia.

a)Identifytheglobinchain(s)inHbS(2HBS)whereyouobservethisaminoacidsubstitution.

Answer

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StarBiochem

Ver.5‐M.Rokop,D.SinhaandL.Alemán

7

b)Nametheaminoacidpresentinnormalhemoglobin,Hb(1A3N),thatisbeingsubstitutedbyvaline6insicklehemoglobin,HbS(2HBS).

Answer

9InHbS(2HBS),valine6inaspecificglobinchaininteractswithphenylalanine85andleucine88locatedintheglobinchainofanotherHbSmolecule.

a)IntheHbSstructure(2HBS),identifytheglobinchains(i.e.1,2,3,4)thatcontainthesethreeaminoacidsinaconfigurationthatallowsthemtointeractwitheachother.• UnderStructureclickonPrimary.• SelectmorethanoneaminoacidresiduebybyindividuallyclickingonthemandsimultaneouslypressingControlandApplekey(Mac)/right‐click(PC).

• Theaminoacidsyouselectgethighlightedinthestructure(white).ForabetterviewyoucangotoViewControlsandmovetheUnselectedtransparencysliderto“0”.

Answer

b)Whatisthemostlikelyinteractionbetweenvaline6andphenylalanine85andleucine88?Pleaseexplain.Answer

c)Inquestion8(b)ofthisexerciseyouhaveidentifiedtheaminoacidlocatedatposition#6innormalhemoglobin,Hb(1A3N).Thisaminoacid,unlikevaline6insicklehemoglobin(2HBS),doesnotinteractwithphenylalanine85andleucine88.Proposeanexplanationforthisobservation.

Answer

d)Basedonwhatyouhavelearnedfromthisexercise,explainwhyanaminoacidsubstitutiontovalineatposition6resultsinsicklecellanemia.

Answer

Name________________________

StarBiochem

Ver.5‐M.Rokop,D.SinhaandL.Alemán

8

Keywords:Sicklecellanemia,essentialaminoacids,oxyhemoglobinorsaturatedhemoglobin,deoxyhemoglobinordesaturatedhemoglobin,andautosomalrecessivegeneticdisorder.ThoughtQuestions1Sicklecellpatientsareveryoftenaskedtoavoiddehydrationbysignificantlyincreasingtheirfluidintake.Explainhowthisrecommendationmayhelpthesepatients.

2Abnormalitiesinthehemoglobinproteinaccountforavarietyofgeneticallyinheriteddisorderssuchassicklecellanemiaandthalassemia.Thegeneticmutationsresponsibleforthesediseasesaremuchmorecommonincertainregionsoftheworld,i.e.Africa,EasternEuropeandSouthEastAsia.Proposehownaturecouldhaveselectedforthemutantcopyofthehemoglobingeneincertainregionsoftheworld.

3Worldclasstennisplayer,PeterSampras,andfootballstarZinedineZidanearethalassemiacarriers.Theseplayersperformmuchbetterinshortversuslonglastingmatches.Basedonwhatyouhavelearnedabouthemoglobinfromthisexercise,explainwhythismaybeso.