Fluorescence Lifetime Readouts of Troponin-C-Based Calcium FRET Sensors A Quantitative Comparison of CFP and mTFP1 as Donor Fluorophores 英文参考文献.docVIP

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Fluorescence Lifetime Readouts of Troponin-C-Based Calcium FRET Sensors A Quantitative Comparison of CFP and mTFP1 as Donor Fluorophores 英文参考文献.doc

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Fluorescence Lifetime Readouts of Troponin-C-Based Calcium FRET Sensors A Quantitative Comparison of CFP and mTFP1 as Donor Fluorophores 英文参考文献

FluorescenceLifetimeReadoutsofTroponin-C-Based CalciumFRETSensors:AQuantitativeComparisonofCFP andmTFP1asDonorFluorophores RomainLaine1,2,3*,DanielW.Stuckey3¤,HughManning2,SeanC.Warren2,GordonKennedy2, DavidCarling3,ChrisDunsby2.,AlessandroSardini3.,PaulM.W.French2. 1Institute of Chemical Biology (ICB), Imperial College of Science, Technology Medicine, Institute of Chemical Biology (ICB), London, England, 2Photonics Group, Blackett Lab, Imperial College of Science, Technology Medicine, London, England, 3Medical Research Council (MRC) Clinical Sciences Centre, Imperial College of Science,TechnologyMedicine,London,England Abstract WehavecomparedtheperformanceoftwoTroponin-C-basedcalciumFRETsensorsusingfluorescencelifetimeread-outs. Thefirstsensor,TN-L15,consistsofaTroponin-CfragmentinsertedbetweenCFPandCitrinewhilethesecondsensor,called mTFP-TnC-Cit, was realized by replacing CFP in TN-L15 with monomeric Teal Fluorescent Protein (mTFP1). Using cytosol preparationsoftransientlytransfectedmammaliancells,wehavemeasuredthefluorescencedecayprofilesofthesesensors atcontrolledconcentrationsofcalciumusingtime-correlatedsinglephotoncounting.Thesedatawerefittedtodiscrete exponentialdecaymodelsusingglobalanalysistodeterminetheFRETefficiency,fractionofdonormoleculesundergoing FRETandcalciumaffinityofthesesensors.Wehavealsostudiedthedecayprofilesofthedonorfluorescentproteinsalone anddeterminedthesensitivityofthedonorlifetimetotemperatureandemissionwavelength.Live-cellfluorescencelifetime imaging (FLIM) of HEK293T cells expressing each of these sensors was also undertaken. We confirmed that donor fluorescenceofmTFP-TnC-Citfitswelltoatwo-componentdecaymodel,whiletheTN-L15lifetimedatawasbestfittedtoa constrained four-component model, which was supported by phasor analysis of the measured lifetime data. If the constrainedglobalfittingisemployed,theTN-L15sensorcanprovidealargerdynamicrangeoflifetimereadoutthanthe mTFP-TnC-CitsensorbuttheCFPdonorissignificantlymoresensitivetochanges