Fluorescence lifetime readouts of Troponin-C-based calcium FRET sensors: a quantitative comparison of CFP and mTFP1 as donor fluorophores.

ABSTRACT: We have compared the performance of two Troponin-C-based calcium FRET sensors using fluorescence lifetime read-outs. The first sensor, TN-L15, consists of a Troponin-C fragment inserted between CFP and Citrine while the second sensor, called mTFP-TnC-Cit, was realized by replacing CFP in TN-L15 with monomeric Teal Fluorescent Protein (mTFP1). Using cytosol preparations of transiently transfected mammalian cells, we have measured the fluorescence decay profiles of these sensors at controlled concentrations of calcium using time-correlated single photon counting. These data were fitted to discrete exponential decay models using global analysis to determine the FRET efficiency, fraction of donor molecules undergoing FRET and calcium affinity of these sensors. We have also studied the decay pr

SUBMITTER: Laine R 

PROVIDER: S-EPMC3494685 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature