Anomalous Surplus Energy Transfer Observed with Multiple FRET Acceptors 英文参考文献.docVIP

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Anomalous Surplus Energy Transfer Observed with Multiple FRET Acceptors 英文参考文献.doc

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Anomalous Surplus Energy Transfer Observed with Multiple FRET Acceptors 英文参考文献

AnomalousSurplusEnergyTransferObservedwith MultipleFRETAcceptors SrinageshV.Koushik1,PaulS.Blank2,StevenS.Vogel1* 1National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, Rockville, Maryland, United States of America, 2Eunice Kennedy Shriver National InstituteofChildHealthandHumanDevelopment,NationalInstitutesofHealth,Bethesda,Maryland,UnitedStatesofAmerica Abstract Background:Fo¨rsterresonanceenergytransfer(FRET)isamechanismwhereenergyistransferredfromanexciteddonor fluorophore to adjacent chromophores via non-radiative dipole-dipole interactions. FRET theory primarily considers the interactions of a single donor-acceptor pair. Unfortunately, it is rarely known if only a single acceptor is present in a molecularcomplex.Thus,theuseofFRETasatoolformeasuringprotein-proteininteractionsinsidelivingcellsrequiresan understandingofhowFRETchangeswithmultipleacceptors.WhenmultipleFRETacceptorsarepresentitisassumedthata quantumofenergyiseitherreleasedfromthedonor,ortransferredintototoonlyoneoftheacceptorspresent.Therateof energytransferbetweenthedonorandaspecificacceptor(kDRA)canbemeasuredintheabsenceofotheracceptors,and theseindividualFRETtransferratescanbeusedtopredicttheensembleFRETefficiencyusingasimplekineticmodelwhere thesumofallFRETtransferratesisdividedbythesumofallradiativeandnon-radiativetransferrates. Methodology/PrincipalFindings:ThegeneralityofthisapproachwastestedbymeasuringtheensembleFRETefficiencyin two constructs, each containing a single fluorescent-protein donor (Cerulean) and either two or three FRET acceptors (Venus). FRET transfer rates between individual donor-acceptor pairs within these constructs were calculated from FRET efficiencies measured after systematically introducing point mutations to eliminate all other acceptors. We find that the amountofenergytransferobservedinconstructshavingmultipleacceptorsissignificantlygreaterthantheFRETefficiency predictedfromthesumoftheindividualdonortoacceptortransferrates. Conclusions