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Name________________________
StarBiochem
Ver.5‐M.Rokop,D.SinhaandL.Alemán
1
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.
Name________________________
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.
Name________________________
StarBiochem
Ver.5‐M.Rokop,D.SinhaandL.Alemán
4
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
5
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#85Xinthe2ndglobinchain.Basedonthenatureoftheirside‐chains,howwouldyoucharacterizetheseaminoacids?Yourchoicesare‘ionic’,‘hydrogenbonding,‘vanderWaalsforces’,‘hydrophobic’or‘covalent’.
Name________________________
StarBiochem
Ver.5‐M.Rokop,D.SinhaandL.Alemán
6
• 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
Name________________________
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.