An improved cyan fluorescent protein variant useful for FRET.
Many genetically encoded biosensors use Förster resonance energy transfer (FRET) between fluorescent proteins to report biochemical phenomena in living cells. Most commonly, the enhanced cyan fluorescent protein (ECFP) is used as the donor fluorophore, coupled with one of several yellow fluorescent protein (YFP) variants as the acceptor. ECFP is used despite several spectroscopic disadvantages, namely a low quantum yield, a low extinction coefficient and a fluorescence lifetime that is best fit by a double exponential. To improve the characteristics of ECFP for FRET measurements, we used a site-directed mutagenesis approach to overcome these disadvantages. The resulting variant, which we named Cerulean (ECFP/S72A/Y145A/H148D), has a greatly improved quantum yield, a higher extinction coefficient and a fluorescence lifetime that is best fit by a single exponential. Cerulean is 2.5-fold brighter than ECFP and replacement of ECFP with Cerulean substantially improves the signal-to-noise ratio of a FRET-based sensor for glucokinase activation.
Megan A
MA
Rizzo
Gerald H
GH
Springer
Butch
B
Granada
David W
DW
Piston
D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry*; D019556:COS Cells; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D005941:Glucokinase / Q000378:metabolism; D049452:Green Fluorescent Proteins; D006863:Hydrogen-Ion Concentration; D007202:Indicators and Reagents / Q000494:pharmacology; D008027:Light; D008164:Luminescent Proteins / Q000737:chemistry* / Q000494:pharmacology; D008856:Microscopy, Fluorescence; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011994:Recombinant Proteins / Q000737:chemistry; D013053:Spectrophotometry; D013997:Time Factors
D001426:Bacterial Proteins; D007202:Indicators and Reagents; D008164:Luminescent Proteins; D011994:Recombinant Proteins; C054562:yellow fluorescent protein, Bacteria; D049452:Green Fluorescent Proteins; D005941:Glucokinase