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Fluorescent Nucleoside Triphosphates for SingleMolecule...

来自 : 蚂蚁淘

By:ChristopherP.Toseland¹²,MartinR.Webb¹

Affiliation(s):(1)MRCNationalInstituteforMedicalResearch,London,UK(2)InstitutfürZellulärePhysiologie,PhysiologischesInstitut,LudwigMaximiliansUniversität,Munich,Germany

BookTitle:SingleMoleculeEnzymology:MethodsandProtocols

Series:MethodsinMolecularBIOLOGy|Volume:778|Pub.Date:Aug-31-2011|PageRange:161-174|DOI:10.1007/978-1-61779-261-8_11

Subject:Biochemistry

Theinterconversionofnucleosidetriphosphate(NTP)anddiphosphateoccursinsomeofthemostimportantcellularreactions.Itiscatalyzedbydiverseclassesofenzymes,suchasnucleosidetriphosphatases,kinases,andATPsynthases.Triphosphatasesincludehelicases,myosins,andG-proteins,aswellasmanyotherenergy-transducingenzymes.Thetransferofphosphatebykinasesisinvolvedinmanymetabolicpathwaysandincontrolofenzymeactivitythroughproteinphosphorylation.Tounderstandtheprocessescatalyzedbytheseenzymes,itisimportanttomeasurethekineticsofindividualelementarystepsandconformationchanges.Fluorescentnucleotidescandirectlyreportonthebindingandreleasesteps,andconformationalchangesassociatedwiththeseprocesses.Insingle-moleculestudies,fluorescentnucleotidescanallowtheirroletobeexploredbyfollowingpreciselythetemporalandspatialchangesintheboundnucleotide.Here,theselectionoffluorophoresandnucleotidemodificationsarediscussedandmethodsaredescribedtoprepareATPanalogswithexamplesoftwoalternatefluorophores,diethylaminocoumarinandCy3.

KeyWords:Fluorescentnucleotides-ATP-GTP-Motorproteins-TIRFmicroscopy

1Introduction

TheconversionsofATPtoADPandGTPtoGDParemediatedbyawiderangeofenzymes.Theseincludemotorproteinssuchasmyosins,helicases,andkinesins,alongwithproteinsfromsignalingpathways,suchaskinasesandG-proteins.Withrespecttomanymotorproteins,theenergyfromtheATPhydrolysisiscoupledtochangesinproteinconformation,and/orprotein–trackinteractions,enablingfunctionssuchasmusclecontraction,DNAunwinding,andmodulationofprotein–proteininteractions.

Fluorescencenucleotidesarewidelyappliedtoinvestigatesolutionkineticsofsuchtriphosphatasesandkinases.Forexample,theyareusedtomeasurethekineticsofindividualstepsintheenzymicreaction(binding,hydrolysis,productrelease,andassociatedstructuralchanges)andtounderstandfullyhowsuchactivitiesarecoupledtotheproteinfunction.Insuchmeasurements,achangeinthefluorescencepropertiesisrequiredtogiveasignalassociatedwiththeprocessofinterest.Mostoften,thischangeisinintensity,butotherpropertiessuchasanisotropyarealsoused.Importantly,significantfluorescencechangesaremoreimportantinthistypeofusethanoverallfluorophorebrightness.Inaddition,fluorophorephotobleachingisusuallynotamajorproblem,aslightsourcescanbeoflowerintensitythanthoseforsingle-moleculevisualization,describedbelow.

Theuseoffluorescentnucleotidesinsingle-moleculeassayshasincreasedoverthepast15years,andthishasbeenespeciallydrivenbythestudyofmotorproteins,inwhichthereisapreciserelationshipbetweenmovementandnucleotidehydrolysis.Totalinternalreflectionfluorescencemicroscopy(TIRFM)isreADIlyusedtovisualizeindividualfluorescentATPandADPmoleculesallowingthemeasurementsofATPturnoversbysinglemyosinmolecules(1–4).Forsuchmeasurements,abright,photostable,fluorophoreisamajorfactor:thelightsourcesmustbeintensetogetsufficientphotonsemittedfromsinglecomplexes.Morerecently,single-moleculefluorescencemeasurementshavebeencombinedwithtranslocationmeasurementstoshowthecouplingbetweenATPaseactivityandtranslocationalongactin(4).TIRFMselectivelyexcitesmoleculeswithin100–200nmofthesurface,dramaticallyreducingthebackgroundfluorescencefromunboundfluorophoresinthebulksolution.Thisimprovementinthesignal-to-noiseratioallowsdetectionofindividualfluorescentATPmolecules,whenboundtosurface-attachedproteins.However,thepossibilityoffurtherimprovementinthesignal-to-noiseratio,throughafluorescenceintensityincreaseonproteinbinding,couldimproveeitherthespatialortemporal,resolutionofmeasurements.Thischapterconsidersonlytheuseoffluorescenceintensitymeasurementsofasinglefluorophoreonthenucleotide.However,developmentssuchasspFRET(single-particleFörsterResonanceEnergyTransfer)(5)hasclearpotentialtoextendtheapplicationsoffluorescentnucleotides.

Inthischapter,theselectionofthefluorophoresandtypesofnucleotidemodificationsarediscussed.Twoexamplesyntheses,purifications,andcharacterizationsaredescribedthatresultinfluorescentadducts,differingbothinthetypeoffluorophoreandinthelinkagebetweenthefluorophoreandnucleotide.ThestructuresofthetwoadductsareshowninFig.1.

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Fig.1.Fluorescentnucleotideanalogs.(a)Deac-aminoATPand(b)Cy3-edAATP.

1.1SelectionoftheLabelingPosition

Fluorescentadenineandguaninenucleotideshavebeenwidelyusedtoreportuponbinding,proteinreleaseandstructuralchanges(6–11).Fluorophoresaresensitiveprobes,easilyusedatsubmicromolarconcentrations,andcanhavepropertiesthatreportrapidly,evenonsmallperturbationsintheregionofthefluorophore.Thus,ATPandanalogshavebeenmodifiedwithfluorophoresatseverallocationsinthemolecularstructureandtherangeofmodificationshasbeenreviewed(12,13).Thechoiceofattachmentsiteisimportant,bothtogetafluorophoreinapositiontoreportbutalsotomodifytheparentnucleotidewithoutsignificantperturbationofthebiochemicalproperties.First,thepurinebasecanbemadefluorescenteitherbymodificationorbyusingafluorescentanalogofthenaturalbase,asinthecaseofformycintriphosphate(FTP)(8).However,suchmodificationsmaydisrupttheprotein–nucleotideinteractions,asthereisoftenhighselectivityinthebasebindingsiteofproteins(13).Thephosphatechaincanalsobemodified,aswithγ-AMNS-ATPforinvestigationsofEscherichiacoliRNApolymerase(14),butthesemodificationsfrequentlydisruptthecleavagestepbypreventingcorrectbindingofthephosphatesorbyblockingaccesstotheγ-phosphate.Ribosemodificationsareoftenthemostsuccessful,wherebytheanalogcloselymimicstheactivityofATP.InmanyenzymesthatbindGTPorATP,the2′-and/or3′-hydroxylgroupsoftheribosearepartlyexposedtotheproteinsurface,whilethebaseandphosphatesarewellburied.Thisallowsariboselabeltositattheentrancetothebindingsiteandpotentiallyreportonchangesinthatregion,withonlyasmalleffectonthebindingandcatalyticproperties.

1.2SelectionofFluorophore

Whenfluorescentanalogsaretobeusedformicroscopy,themaincriteriaforchoicesoffluorophorearehighfluorescenceintensity(highextinctioncoefficientandfluorescencequantumyield),excitationandemissionmaximabestsuitedtotheexcitationsource,withoutinterferencefromothercomponentsinthesystem,andstABIlityagainstphotobleaching.Withsomefluorophores,theextinctioncoefficientandparticularlythequantumyieldcanchangesignificantlywiththechemicalenvironmentofthefluorophore.Suchchangescanbeproblematicinchoosingasuitablefluorophore,butiftheintensitychangesaremonitored,thenchangesinintensitycanbeharnessed,asdescribedbelowforadiethylaminocoumarin.Long-wavelengthfluorophores,suchasthecyaninedyes(e.g.,Cy3),havegoodpropertiesforfluorescencemicroscopyandhavebeenusedfordetectingfluorescencefromsinglemolecules.Typically,fluorophoresthatexciteatlongerwavelengthsarerelativelylargemoietieswithmultiringstructuresandmayhavesignificantlyhydrophobicregions.Thismayleadtononspecificbindingtoproteinsandsurfaces.Therearenowmanycommerciallyavailablefluorophore-labelingreagentsthatgivevariationsonquantumyield,photostability,andwavelengths.Adiscussionofthisvarietyisoutsidethescopeofthischapter.ForadditionalinformationandforcommercialsourcesofsuchlabelsaswellasATPanalogssee:http://www.Invitrogen.com,http://www.sigmaaldrich.com,http://www.Roche-applied-science.comandhttp://www.jenabioscience.com.Quantumdotshavesignificantpotentialforfutureuse,buttheirlargesizerelativetonucleosidetriphosphatesislikelytomakethemdifficulttoapplygenerallyhere.

1.3SelectionoftheLinkerBetweenNucleotideandFluorophore

Asdescribedabove,labelingattheribosehydroxylgroupshasbeenusefulbecausethismodificationmaynotleadtolargeperturbationsofthebiochemicalproperties.However,suchlabelingofthehydroxylgroupsleadstotheformationofamixtureof2′-and3′-isomers,whosebiochemicalandfluorescentpropertiesmaydifferwhenbound(13).Thisproblemcanbecircumventedbyusingaparentnucleotide,inwhichonlyonehydroxylisavailableformodification,forexamplethecommerciallyavailable2′-deoxyATP,orthesynthetic3′-amino-3′-deoxyATP(15).Alternatively,theisomersofsomelabelednucleotidesinterconvertonlyveryslowlyandcanbesuccessfullyseparatedbychromatographyandstoredassingleisomers(3,16).

Asmentionedintheabovesection,fluorophoresforfluorescencemicroscopyarerelativelylargeandsomaydisruptthebiochemicalcycle.Toamelioratethisproblem,severallinkersareavailabletospacethefluorophoresfurtherawayfromthecatalyticsite.Essentially,azero-lengthlinkerisachievedbydirectlabelingoftheaminegroupontheriboseringof3′-amino-3′-deoxyATP.Longerchemicallinkerscanusedifferentlengthsofdiamino-n-alkanes,suchas2′(3′)-O-[N-(2-aminoethyl)-carbamoyl]ATP(edaATP(17))and2′(3′)-O-[N-(3-aminopropyl)carbamoyl]ATP(pdaATP(15)).Insomecases,includingCy3,thecommerciallyavailablelabelsincludeaspacerchainbetweenthefluorophoreandthereactivegroupusedforattachment.Inthesecases,thefluorophorewillbepositionedfurtherawayfromtheproteinand,therefore,shouldnotinterferesignificantlywiththenucleotideassociationandcatalysis.However,movingthefluorophorefurtherawayfromtheproteinmayreduceanychangestofluorescentpropertiesonbinding.Allmodificationsmaybedeleterioustotheenzymicactivity,andtherefore,itisimportanttoassesstheimpactofthesechanges.Methodstoassesstheseeffectsaredescribedlater.

1.4OtherConsiderations

TherequirementsforthesynthesisofATPanalogsvarywidely.Thosedescribedherearerelativelysimplelabelingreactions,performedunderaqueousconditions,sopotentiallyhighyieldscanbeobtainedusingconditionsandequipmentavailableinmostlaboratories.Thesuccessofthelabelingdependsbothonthechemicalreactionsperseandonthepropertiesofthefluorophore.Forexample,veryhydrophobicgroupsmayimpairthesuccessofareactionthatoccursperfectlywellwithsimplerlabels.Thepurificationoftheproductalsomaydependonthephysicalpropertiesofthefluorescentlabel.TwospecificexamplesaredescribedforthelabelingnucleotidesattheriboseringwithCy3anddiethylaminocoumarin(Fig.1).

Thevisualizationofthebindingoffluorescentnucleotidestoproteinsbylightmicroscopehasbeenlimitedbytechnicalproblemssuchasthenonspecificbindingofthefluorescentnucleotidetothecoverslip.Thishaslimitedthemaximumnucleotideconcentrationthatcouldbeusedwithanalogssuchas2′(3′)-(Cy3-O-[N-(2-aminoethyl)carbamoyl])ATP(Cy3-edaATP,Fig.1b(3))to<100nM.Fluorescentgroupsmayalsobindtomacromoleculessuchasproteins,independentlyofthenucleotideanditsbindingsite,andparticularlyifusedathighconcentration.Acontrol,suchasdisplacingthelabeledwithunlabelednucleotide,willtestifsuchnonspecificbindingoccurs.

ThefluorescentATPanalog,(3′-(7-diethylaminocoumarin-3-carbonylamino)-3′-deoxyadenosine-5′-triphosphate(deac-aminoATP))(Fig.1a)hasalowquantumyieldwheninsolution,butthisincreasesdramaticallywhenboundtosomeproteins.Thisgenerateslargefluorescencechanges,suchasthe20-foldincreasewhenboundtomyosinVa(18).Thisenablesadistinctionbetweencoverslipbound“background”moleculesandthoseboundbyproteins(4),whichmaycompensatefortherelativelylowoptimalexcitationwavelengthandphotostability.

2Materials

2.1Labeling

1.	2′,3′-O-(2-Aminoethyl-carbamoyl)-adenosine-5′-triphosphate(edaATP)(JenaBiosciences)(seeNote1).
2.	3′-Amino-3′-deoxyATPtriethylammoniumsalt(aminoATP)(15).
3.	Cy3N-hydroxysuccinimideester(NHS-ester)(GEHealthcare).
4.	7-Diethylaminocoumarin-3-carboxylicacid(Invitrogen).
5.	20mMSodiumbicarbonate,pH8.4.
6.	Dimethylformamide(DMF).
7.	Tributylamine.
8.	Isobutylchloroformate.
9.	HPLCsystem,preferablywithbothabsorbanceandfluorescencedetectors.
10.	Stronganionexchange(SAX)Partispherecolumn(0.4 × 10cm)(Whatman).
11.	0.4M(NH₄)₂HPO₄adjustedtopH4.0withconcentratedHCl.
12.	HPLC-gradeMethanol.
13.	HPLC-gradeAcetonitrile.

2.2Purification

1.	DEAEcellulosecolumn(2 × 30cm).
2.	Triethylamine(technicalgrade).
3.	Glassdistillationapparatussuitableforupto500mlandhavinggroundglassjoint.
4.	Boilingchips.
5.	Dryice.
6.	2-LBuchnerflask,withbungandplastictubingonthesidearm,connectedtoaglass-scintergasbubbler.
7.	Chromatographysystem(fractioncollector,gradientmaker,pump,etc.)at4°Cwithabsorbanceandfluorescencedetector,ifpossIBLe.

2.3Concentration

1.	Rotaryevaporator,equippedwithacoldfingercondenserandhigh-vacuumoilpump.
2.	Methanol(highestgradeavailable).
3.	Isopropanol(technicalgrade).
4.	Dryice.

2.4Characterization

1.	Spectrophotometer.
2.	HPLCsystem,preferablywithbothabsorbanceandfluorescencedetectors.
3.	Stronganionexchange(SAX)Partispherecolumn(0.4 × 10cm)(Whatman).
4.	0.4M(NH₄)₂HPO₄,pH4withconcentratedHCl.
5.	HPLC-grademethanol.
6.	HPLC-gradeacetonitrile.
7.	Fluorescencespectrophotometer.

2.5ATPaseAssay

1.	MDCC-PBP(19).Phosphatebindingprotein(A197C)fromE.coli,labeledwith(N-[2-(1-maleimidyl)ethyl]-7-diethylaminocoumarin-3-carboxamide)(Invitrogen)(seeNote2).
2.	Rhodamine-PBP(20).Phosphatebindingprotein(A17C,A197C)fromE.coli,labeledwith6-iodoacetamidotetramethylrhodamine(seeNote2).
3.	Fluorescencespectrophotometer.
4.	Inorganicphosphatestandardsolution.

3Methods

3.1SynthesisofCy3-edaATP

ThismethodisbasedonthatdescribedbyOiwaetal.(3)andgivesmixed(2′,3′)isomers(Fig.1b).

3.1.1Labeling

1.	Mix4μmolCy3NHS-esterwith20μmoledaATPin20mMsodiumbicarbonate,pH8.4,for1hatroomtemperature(seeNote3).
2.	AnalyzethereactionmixtureusingHPLCtoconfirmtheformationofCy3-edaATP.EquilibrateaPartisphereSAXcolumnwith0.4M(NH₄)₂HPO₄with20%(v/v)methanol:flowrateof1ml/minatroomtemperature(seeNotes4and5).
3.	Addanaliquotofthereactionmixture(1–10nmol)to100μLoftherunningbuffer.
4.	Injectthesolutionontothecolumn.
5.	Followtheabsorbanceat254nmandfluorescencewithexcitationof550nmandemissionof570nm.ThechromatogramwillshowtheelutionofCy3NHS-ester,edaATP,andCy3-edaATP,respectively(seeNote6).
6.	InjectknownstandardsofCy3NHS-esterandedaATPatthesameconcentrationasthereactionmixturetoidentifypeaks.

3.1.2PreparationofTriethylammoniumBicarbonateSolution

1.	Distiltriethylamine(500ml),discardingthefirstandlast10%ofthedistillate.Usethemiddle80%ofthedistillate(seeNote7).
2.	Addcold(4°C),distilled,deionizedwaterto139.4mldistilledtriethylaminetogive1Lofa1Msolution(seeNote8).
3.	Inafumehood,putdryiceintheBuchnerflask,andwiththesolutioninice,bubbleCO₂throughthesolutionuntilthepHis7.5–7.6(approximately2h)usingthescinteredglassbubbler.KeeptheBuchnerflask,containingthedryice,raisedabovethesolutiontoreducetheriskofsuckingback.
4.	Storetriethylammoniumbicarbonate(TEAB)at4°Cinawell-stopperedcontainer.Itlastsapproximately1–2months,butthepHgraduallyriseswithtime.Inthiscase,rebubbleCO₂throughit.

3.1.3PurificationofNucleotide

1.	PreequilibratetheDEAE-cellulosecolumnwith10mMTEAB,pH7.6at1ml/minat4°C.
2.	AlterthepHofthereactionmixtureto7.6usingacidorbase,reducetheconductivitybydilutioninwatersoitisclosetothatof10mMTEABandloadontothecolumn.
3.	Washthecolumnwith10mMTEAB,pH7.6ataflowrateof1ml/minuntilnomorepinkmaterialiseluted.
4.	Elutethenucleotidewithalineargradientof10–800mMTEAB(totalvolume600ml).Followtheabsorbanceat254nm.UnreactededaATPiselutedfirstfollowedbyCy3-edaATP(seeNote9).
5.	IdentifythefractionscontainingCy3-edaATPbymeasuringtheabsorbanceat550nmand260nm.

3.1.4Concentration

1.	PoolfractionscontainingCy3-edaATPandremoveTEABbyrotaryevaporation.Useaflaskwithacapacityatleastfourtimesthevolumeofthesolution.
2.	Fillthecondenserwithdryice–isopropanol.
3.	Addthepooledfractionstotheflask,rotate,andslowlyapplythevacuumtobeginevaporation.Warmtheflaskinawaterbathat30°C.Reducethevolumeto∼5ml.Whenthesolutionvolumeisreducedto10–20%,frothingmaybegin(seeNote10).
4.	Addmethanol(∼10%ofinitialsolutionvolume)andrepeattheevaporation.
5.	Repeatmethanoladditionsandevaporationthreetimes:duringthisitshouldbepossibletoremoveessentiallyallthesolventbeforeaddingmoremethanol.Atthefinalstage,evaporateallofthemethanol.TheCy3-edaATPwillremainasagum.
6.	Dissolvein<3mlmethanolandtransfertoapearflask(10ml)andreconcentrate,withverycarefulapplicationofthevacuumtoavoidfrothing.Finally,dissolveinwaterorbufferandadjusttopH ∼ 6–7beforestoringat−80°C(seeNote11).

3.1.5Characterization

1.	MeasuretheabsorbancespectraofCy3-edaATPin50mMTris–HCl,pH7.5between220and700nm.TakingtheextinctioncoefficientfortheCy3tobe150,000M⁻¹cm⁻¹at552nm(21)andtheextinctioncoefficientforadenosinetobe15,200M⁻¹cm⁻¹at260nm,calculatetheconcentrationofthenucleotide(seeNote12).
2.	CharacterizeCy3-edaATPbyHPLCusingthesamemethodasabove.ThemajorpeakshouldbeCy3-edaATP.CheckforthepresenceofCy3-edaADP,edaATP,andedaADP.DeterminethepuritybyintegratingtheCy3-edaATPpeakwithanyotherpeaks(seeNote13).
3.	MeasurethefluorescenceexcitationandemissionspectrumofCy3-edaATPin50mMTris–HCl(pH7.5).Typically,1μMinasolutionof60μlwillbeused.Usethepeakwavelengthfromtheabsorbancemeasurementastheexcitationwavelengthtomeasuretheemission.Then,usethepeakintheemissionspectrumfortheexcitationspectrum.Addanexcessoftheproteinofinteresttothesample(e.g.,10-fold)andrepeatthemeasurement(seeNotes14and15).Comparethetwospectratodeterminethechangeinfluorescencewhenboundtoprotein.

3.1.6GeneratingCy3-edaADP

1.	Cy3-edaADPcanbeobtainedbyhydrolysisofCy3-edaATP.AddthedesiredconcentrationofCy3-edaATP(e.g.,100μM)torabbitskeletalmusclemyosin(1mg/ml)in1mMMgCl₂,0.2mMdithiothreitol(DTT),and10mMTris–HCl,pH7.0.4°Cfor2h(seeNote16).
2.	Centrifugethesampleat235,000 ´ gat4°Ctoremovethemyosin.
3.	AnalyzetheproductusingHPLC,asdescribedabove.
4.	Storethesupernatantat−80°C.

3.2SynthesisofDeac-aminoATP

ThismethodisbasedonthatdescribedbyWebbetal.(15)andgivesasingleproductasreactionoccursonlyatthe3′-amine(Fig.1a).

3.2.1Labeling

Thismethodrequiresthestartingmaterial3′-amino-3′-deoxyATP(15).

1.	Activate7-diethylaminocoumarin-3-carboxylicacid(16.4mg,62.8μmol)bydissolvingindryDMF(1ml),coolingitonice,andaddingtributylamine(25μl,103μmol)andisobutylchloroformate(10μl,77μmol).
2.	Leavethereactionmixtureonicefor50min.
3.	Add3′-amino-3′-deoxyATP(40μmol,triethylammoniumsalt)inwater(300μl)totheactivatedcoumarinandstiratroomtemperaturefor2h.
4.	AnalyzethereactionmixtureusingHPLCtoconfirmtheformationofdeac-aminoATP.EquilibrateaPartisphereSAXcolumnwith0.4M(NH₄)₂HPO₄with5%(v/v)acetonitrileataflowrateof1.5ml/minatroomtemperature.
5.	Addanaliquotofthereactionmixture(1–10nmol)to100μloftherunningbuffer.
6.	Injectthesolutionontothecolumn.
7.	Followtheabsorbanceat254nmandfluorescencewithexcitation435nmandemission465nm.Elutiontimesareapproximately1.6minfor7-diethylaminocoumarin-3-carboxylicacid,3.5minfor3′-amino-3′-deoxyATP,and13minfordeac-aminoATP(seeNote6).

3.2.2Purification

1.	ThereactionmixturewaspurifiedonaDEAE-cellulosecolumn.Equilibratethecolumnwith10mMTEAB,pH7.6at1ml/minat4°C.
2.	AlterthepHofthereactionmixtureto7.6usingacidorbase,reducetheconductivitybydilutioninwatersoitisclosetothatof10mMTEABandloadontothecolumn.
3.	Washthecolumnwith10mMTEAB,pH7.6ataflowrateof1ml/minfortwocolumnvolumes.
4.	Elutethenucleotidewithalineargradientof10–600mMTEAB(totalvolume1L).Followtheabsorbanceat254nm(seeNote17).UnreactedaminoATPiselutedfirstfollowedbydeac-aminoATP.

3.2.3Concentration

Theproductdeac-aminoATPisconcentratedasdescribedforCy3-edaATPandstoredat−80°C.

3.2.4Characterization

3.3AssesstheEffectsofModifications

ThemethoddescribeshereisforanATPaseorGTPase.ThisspecificexampleusesaDNAhelicaseBacillusstearoThermophilusPcrA.TheeasiestmethodtoprovideanoverallassessmentoftheeffectofanATPmodificationistomeasureasteady-stateATPaseassay.Shouldtherebeachangeinthesteady-stateparameters(greaterthan20%),thentheindividualstepsoftheATPcyclecouldbeinvestigated.Itiscommonforthediphosphateaffinitytoincreasewithmodificationstotheribosering(9,18,22,23).

Itisalsohighlyrecommendedthatafunctionalactivityassayisperformed,suchasmeasuringDNAunwindingbyaDNAhelicaseoraninvitromotilityassaywithmyosin.Thisisanalternateassessmentofthemodificationeffect:thelabelmayinterferewithonecriterionwhichmaynotbenoticeableintheother.

1.	Measuretheabsorbancespectraofdeac-aminoATPin50mMTris–HCl,pH7.5between220and700nm.Takingtheextinctioncoefficientforthecoumarintobe46,800M⁻¹cm⁻¹at429nmandforadenosinetobe15,200M⁻¹cm⁻¹at260nm,calculatetheconcentrationsofthenucleotide(seeNote12).
2.	Characterizedeac-aminoATPbyHPLCusingthesamemethodasabove.Themajorpeakshouldbedeac-aminoATP.Determinethepuritybyintegratingthedeac-aminoATPpeakwithanyotherpeaks.
3.	MeasurethefluorescencespectraasdescribedforCy3-edaATP,butusingthecorrespondingexcitationandemissionpeaksforthecoumarin(Fig.2). Fig.2.Fluorescencechangeuponbindingofdeac-aminoADPtomyosinS1.ExcessmyosinS1wasaddedtobindallnucleotides.Deac-aminoADP(0.3μM)wasexcitedat435nmand10μMS1wasadded.InsertshowsthetitrationofmyosinS1intoasolutionofdeac-aminoADP.Thishighlightstheneedtosaturatethenucleotidetodeterminethemaximumfluorescencechange.MyosinS1wasaddedtoasolutionof0.1μMnucleotide,andthefluorescencewasmonitoredat480nm,withexcitationat435nm.
4.	FollowthesameproceduredescribedfortheCy3-edaATPtogeneratethediphosphate.

1.	Prepareamixture(60μl)of2nMPcrAhelicase,500nMdT₂₀oligonucleotideand10μMMDCC-PBP(or6IATR-PBP)inabuffercontaining50mMTris–HCl,pH7.5,3mMMgCl₂and150mMNaCl.
2.	Recordthefluorescencebyexcitingat436nmandemissionat465nmforMDCC-PBP,orexcitation555nmandemission575nmfor6IATR-PBP.
3.	AddATPatvariousconcentrations(1mMto0.5μM)(seeNote18).
4.	Repeatthemeasurementsatthesameconcentrationsofdeac-aminoATPorCy3-edaATP(seeNote18).
5.	Performacalibrationofthefluorescencesignalusingknownconcentrationsofinorganicphosphate.
6.	ComparetheV_maxandK_mvaluesforthenativeandmodifiednucleotides.

4Notes

1.	ItisalsopossibletosynthesizeedaATP(3,15,17).
2.	MDCC-PBPisavailablecommerciallyfromInvitrogen,butcannotbeusedwithdiethylaminocoumarin-labelednucleotidesbecausethefluorophoresarethesame.Similarly,6IATR-PBPcannotbeusedifafluorophorewithsimilarwavelengthsispresent,suchasCy3orotherrhodamine.
3.	UseanexcessofnucleotideoverCy3NHS-esterduetotheexpenseofthefluorophore.
4.	Filteranddegasthe(NH₄)₂HPO₄andthenaddtheHPLCgrademethanol.
5.	Alternatively,thereactioncanbefollowedbythin-layerchromatographyonsilicaplates(13).
6.	Usingthefluorescencedetectionisapproximately100-foldmoresensitivethanabsorbance.Ifnecessary,absorbancepeakscanbecollected,theirfluorescencemeasuredinafluorimeter,andtheirfullabsorbancespectrummeasuredinaspectrometer.
7.	Donotstoredistilledtriethylamineforlaterusewithoutredistillation:itdecomposesonstorage.Itmaybepossibletousehigh-puritytriethylaminewithoutdistillation,buttriethylaminedoesformimpuritiesonstorage.ThedistillationensuresthatonlyvolatilecomponentsendupintheTEABsolution.
8.	Triethylamineitselfisonlypartiallymisciblewithwater:therewillbetwolayersinitially,whichbecomesasinglesolutionaftersomeCO₂hasbeenabsorbed.
9.	Alternatively,followthefluorescenceofCy3usinganexcitationof550nmandemissionof570nm.
10.	Applyandremovethevacuumslowlytopreventthesolutionsplashingandfrothing,andsopassingintothecondenser.
11.	AliquottheATPintosmallamountsbeforefreezingtoavoidfreeze-thawcycles.ItisadvisabletocheckthepurityofthenucleotidebyHPLCperiodicallyduringlong-termstorage.
12.	Theratiobetweenthemolaramountofthefluorophoreandadenosineshouldbe∼1.Ifnot,thereislikelytobecontaminatingfluorophoreinthepreparation.
13.	Determinethelimitofsensitivityfortheinstrumentbyinjectingknownamounts.Typically,1%contaminationshouldbedetected;forexample,if10nmolisinjected,itshouldbepossibletodetect0.1nmol.Itispossiblethatagreateramountofnucleotidewillhavetobeinjected.
14.	Ideally,theadditionofexcessproteintothenucleotidesamplewouldleadtothemaximumpotentialsignalchange.However,thisisdependentontheaffinitybetweennucleotideandprotein.
15.	Unlesstheproteinhasalowhydrolysisrateconstant,ortheproteinrequiresanactivator,itislikelythatanyfluorescencechangeoccursduetotheformationofdiphosphate.Thediphosphatefluorescencechangeshouldbemeasuredindependently.
16.	ItisalsopossibletobeginthelabelingwithedaADPandrepeatthesameprotocolasdescribeabovetoproducethefluorescentdiphosphate.Inaddition,itispossibletouseotherATPasesorcommerciallyavailableglycerolkinasewithd-glyceraldehyde(albeitthatribose-modifiednucleotidesarepoorsubstratesforthiskinase)toachievethehydrolysisofthetriphosphate(24).TheresultingADPanalogispurifiedbyadesaltingcolumn(PD10)orrepeatingtheion-exchangechromatography.
17.	Alternatively,followthefluorescenceofthediethylaminocoumarinusinganexcitationof430nmandemissionof465nm.
18.	AvoiddilutingtheATPsamplestolowconcentrations.Use60×concentratedstocks.Byadding1μloftheATPtothe60μlreactionmixture,thecorrectATPconcentrationisachieved.

AcknowledgmentsWewouldliketothankthevariouscoworkers,whohavebeeninvolvedinsynthesisanduseoffluorescentnucleotidesandarecoauthorsofpublicationscitedhere.WethanktheMedicalResearchCouncil,UK(C.P.T.andM.R.W.)andEuropeanMolecularBiologyOrganization(C.P.T)forfinancialsupport.

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