Name________________________

StarBiochem

Ver.5‐D.SinhaandL.Alemán 1

GreenFluorescentProtein(GFP)ExerciseLearningObjectivesInthisexercise,youwillusetheStarBiochemandStarORFsoftwaretoolstoexplore:• thesequenceoftheGFPgeneandthestructureofitsprotein• thefluorophorethataccountsforGFP’sfluorescence

BackgroundGFPisafluorescentproteinisolatedfromseveralorganismsincludingthepacificjellyfishAequoriaVictoria.GFPconvertstwospecificwavelengthsofbluelightintogreenfluorescentlightbyenergytransfer.Proteinsthatfluorescecontaincomponentscalledfluorophores.InGFPthefluorophoreoriginatesfromaninternaltripeptidesequence(Ser‐Tyr‐Gly)whichispost‐translationallymodifiedtothestructureshownbelow,a4‐(p‐hydroxybenzylidene)‐imidazolidin‐5‐one.ItisworthnotingthattheformationofGFP’sfluorophoreisanautocatalyticprocessthatrequiresnocofactororenzymaticreaction.ThisfluorophoreishighlystableevenathightemperaturesandvariouspHconditions.ThegeneforGFPhasbeenisolatedandhasbecomeausefultoolformakingfusionproteinsinwhichGFPislinkedtootherproteinsandfunctionsasafluorescentproteintag.GFPtaggingcanbeusedinawiderangeofapplications:asatracerforcelllineage,asareporterofgeneexpression,orasareporterofprotein‐proteininteractions.GettingstartedwithStarORFStarORFisasix‐frametranslationsoftwaretoolforexploringgenesequences.• TogettoStarORF,pleasenavigatetohttp://web.mit.edu/star/ORF.• ClickontheStartbuttontolaunchtheapplication.• ClickTrustwhenapromptappearsaskingyouifyoutrustthecertificate.• IntheInputbox,pastethesequenceprovidedbelow.

Name________________________

StarBiochem

Ver.5‐D.SinhaandL.Alemán 2

ThecDNAsequenceoftheGFPgene(5'→3'direction)isshownbelow.tacacacgaataaaagataacaaagatgagtaaaggagaagaacttttcactggagttgtcccaattcttgttgaattagatggcgatgttaatgggcaaaaattctctgtcagtggagagggtgaaggtgatgcaacatacggaaaacttacccttaaatttatttgcactactgggaagctacctgttccatggccaacacttgtcactactttctcttatggtgttcaatgcttttcaagatacccagatcatatgaaacagcatgactttttcaagagtgccatgcccgaaggttatgtacaggaaagaactatattttacaaagatgacgggaactacaagacacgtgctgaagtcaagtttgaaggtgatacccttgttaatagaatcgagttaaaaggtattgattttaaagaagatggaaacattcttggacacaaaatggaatacaactataactcacataatgtatacatcatggcagacaaaccaaagaatggaatcaaagttaacttcaaaattagacacaacattaaagatggaagcgttcaattagcagaccattatcaacaaaatactccaattggcgatggccctgtccttttaccagacaaccattacctgtccacacaatctgccctttccaaagatcccaacgaaaagagagatcacatgatccttcttgagtttgtaacagctgctgggattacacatggcatggatgaactatacaaataaatgtccagacttccaattgacactaaagtgtccgaacaattactaaattctcagggttcctggttaaattcaggctgagactttatttatatatttatagattcattaaaattttatgaataatttattgatgttattaataggggctattttcttattaaataggctactggagtgtatUsingtheStarORFsoftwaretoolanswerthefollowingquestions.1WhatisthelengthoftheGFPcDNA?

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2WhatisthepercentageofeachnucleotidebaseintheGFPcDNA?

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3WhatisthesequenceofthefirsttenbasesofGFP’snoncodingDNAstrand(5'→ 3'direction).

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4Whatisthesequenceofthefirst15basesofGFP’smRNA(5'→ 3'direction)?

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5UsingthecDNAsequenceprovidedinthisexercise,youestimateGFP’smRNAlength.Inyourlaboratory,youthenisolatetotalGFPRNAfromjellyfishandresolveitonagelbasedontheRNAsizedifference.YoufindtwodifferentGFPRNAs:oneoftheRNAsisbiggerthanyourestimateandtheotherRNAisthesamesizeasyourestimate.Howdoyouexplainthisresult?

Name________________________

StarBiochem

Ver.5‐D.SinhaandL.Alemán 3

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6ThecDNAsequenceprovidedinthisexercisehasonlyoneopenreadingframe(ORF).Explainwhy• ORFsarevisuallyrepresentedbygreenlineswithintheSix‐frametranslationbox.

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7Whatarethefirst10aminoacidsoftheGFPprotein?• UndertheSixframetranslationbox,clickonthegreenline,whichindicatesapotentialORFwithinthesequenceprovided.

• Thefulltranslatedaminoacidsequence,representedbythegreenline,isindicatedwithinthePutativeORFproteinsequencewindow.

• ThetotalnumberofaminoacidsofthetranslatedsequenceisindicatedbelowthePutativeORFproteinsequencewindow.

• TheaminoacidsequencecanberepresentedwithinStarORFinthe1letteror3‐letteraminoacidcode.

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WewillnowcomparetheGFPproteinsequencededucedfromthecDNAsequenceprovidedinthisexercisewiththeproteinsequenceyouwillobtainfromGFP’sproteinstructure.ToexplorethestructureofGFPanditsprimarysequence,wewilluseStarBiochem,aprotein3D‐viewer.• TogettoStarBiochem,pleasenavigatetohttp://web.mit.edu/star/biochem.• ClickontheStartbuttontolaunchtheapplication.• ClickTrustwhenapromptappearsaskingyouifyoutrustthecertificate.• UnderFile,clickonOpen/Importandselect“1EMA”.• ClickOpen.

YouarenowviewingthestructureofGFP(structureID:“1EMA”)witheachbondintheproteindrawnasaline(“bondsonly”view).

Name________________________

StarBiochem

Ver.5‐D.SinhaandL.Alemán 4

Practicechangingtheviewpointofthisproteinintheviewwindow.

Mac PCTOROTATE

clickanddragthemouse

left‐clickanddragthemouse

TOMOVEUP/DOWNRIGHT/LEFT

apple‐clickanddragthemouse right‐clickanddragthemouse

TOZOOM

option‐clickanddragthemouse

Alt‐left‐clickanddragthemouse

TakeamomenttolookatthestructureofGFP(1EMA)fromvariousanglesinthis“bondsonly”view.8WhatisthetotallengthoftheGFPproteinsequencethatyouobtainedusingStarORFandStarBiochemsoftwaretools?Isitthesameordifferent?Explainyouranswerandreasoning.• InStarBiochem,underStructure,clickonPrimarywhichshowstheprotein’saminoacidsequence.

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9ThefluorophoreinGFPoriginatesfromtheSer65‐Tyr66‐Gly67tripeptide.Forthistripeptideprovidethefollowing:

a)ApossibleDNAsequencewithlabeled5'and3'ends.

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b)ApossiblemRNAsequence(5'→ 3'direction).

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c)Thecorrespondinganti‐codonsequence(5'→ 3'direction).

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Name________________________

StarBiochem

Ver.5‐D.SinhaandL.Alemán 5

10YoucomeacrossfourGFPcDNAs.Eachofthesehasoneofthefollowingpointmutations.Completethefollowingtable.

Mutation(mRNAsequence)

WhatisthecorrespondingmutationinthecDNA?

Doesthefluorophoreform?Explainbriefly.

IstheresultingGFPproteinfunctional(fluoresces)?Explainbriefly.

G#27toU#27

Extra“A”addedafterthe896thnucleotide,priorto“G”

A#740toG#740

U#220toG#220

U#222toC#222

Name________________________

StarBiochem

Ver.5‐D.SinhaandL.Alemán 6

11WewillnowtakeacloserlookatthestructureofGFP(1EMA).

a)DrawthechemicalstructureofthefluorophoreinGFP(1EMA).IndicatetheatomswithinthestructureandthepartsthatarecontributedbySer65,Tyr66,andGly67.Labelthealphacarboncontributedbyeachofthesethreeaminoacids.• InStarBiochem,underPDBtree,clickonthe1EMAfolderandthenclickon“Cro_66”.• IntheViewControlspanel,settheUnselectedtransparencysliderto“0”.• ClickonDrawwithintheAtomsboxtoseewhatatomsarepresent.Eachatomiscolor‐coded:Carbonisgrey,Nitrogenisblue,andOxygenisred.

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b)StatethemostlikelyinteractionbetweenSer65,Tyr66andGly67thatcontributestotheformationofthefluorophore.Yourchoicesare‘hydrogenbond’,‘ionicbond’,‘hydrophobicinteraction’,‘covalentbond’,‘peptidebonds’,or‘vanderWaalsforces’.

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c)Whichsecondarystructuresurroundsthefluorophore:helices,sheetsorcoils?• WithinthemainmenugotoView.• ClickonResetMolecule.• UnderStructure,clickonSecondary.• ExplorethedifferentsecondarystructuresbyindividuallyclickingonHelices,SheetsorCoilswithintheShowRibbonsbox.Alternatively,youcanclickonAllRibbonswithintheShowRibbonsboxtoviewallstructuresatthesametime.

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d)Howmaythissecondarystructurebeimportantforfluorophorefunction?

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