Detecting Intramolecular Dynamics and Multiple Forster REsonance Energy Transfer States by Fluorescence Correlation Spectroscopy.pdf

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1、J.Phys.Chem.B2010,114,5895–59025895DetectingIntramolecularDynamicsandMultipleFo¨rsterResonanceEnergyTransferStatesbyFluorescenceCorrelationSpectroscopyE.ShanePrice,MatthewS.DeVore,andCareyK.Johnson*DepartmentofChemistry,UniVersityofKansas,Lawrence,Kans

2、as66045ReceiVed:December23,2009;ReVisedManuscriptReceiVed:March18,2010Fluorescencecorrelationspectroscopy(FCS)isarobustmethodforthedetectionofintramoleculardynamicsinproteinsbutisalsosusceptibletointerferencefromotherdynamicprocessessuchastripletkineti

3、csandphotobleaching.WedescribeanapproachforthedetectionofintramoleculardynamicsinproteinslabeledwithaFRETdyepairbasedonglobalfittingtothetwoautocorrelationfunctions(green-greenandred-red)andthetwocross-correlationfunctions(green-redandred-green).Weappli

4、edthemethodtodetectintramoleculardynamicsintheCa2+signalingproteincalmodulin.Dynamicsweredetectedonthe100µstimescaleinCa2+-activatedcalmodulin,whereasinapocalmodulindynamicswerenotdetectedonthistimescale.ControlmeasurementsonapolyprolineFRETconstruct(G

5、ly-Pro15-Cys)demonstratethereliabilityofthemethodforisolatingintramoleculardynamicsfromotherdynamicprocessesonthemicrosecondtimescaleandconfirmtheabsenceofintramoleculardynamicsofpolyproline.WefurthershowthesensitivityoftheinitialamplitudesoftheFCSauto-

6、andcross-correlationfunctionstothepresenceofmultipleFRETstates,staticordynamic.TheFCSmeasurementsalsoshowthatthediffusionofCa2+-calmodulinisslowerthanthatofapocalmodulin,indicatingeitheralargeraveragehydrodynamicradiusorshapeeffectsresultinginaslowertr

7、anslationaldiffusion.Introductioncorrelationsfromintramoleculardynamics.Inthesecond(nondynamic)system,weexpectFRETstatestobestaticonFluorescencecorrelationspectroscopy(FCS)revealsfluctua-theFCStimescale.SatisfactoryfittingofFCScurvesforationsinthefluoresc

8、enceintensityofasmallnumberofnondynamicsystemwillserveasacontrolforFCScurvesinmoleculesastheypassthroughtheprobevolume.FCSdetectscaseswheredynamicsmaybepresent.bothconcentrationfluctuationswithinthefocalvolumeandkineticorphotophysicalpro