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.
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We thank A. Rizzo for helpful suggestions. Funding for this work was provided by US National Institutes of Health grants DK60275 (M.A.R.), and DK53434 and CA86283 (both to D.W.P.), the US National Science Foundation grant BBI-9871063 (D.W.P.), and the US Department of Defense Medical Free-Electron Laser program grant F49620-01-1-0429 (D.W.P.).
Vanderbilt University has applied for a patent covering the fluorescent proteins described in the article.
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Rizzo, M., Springer, G., Granada, B. et al. An improved cyan fluorescent protein variant useful for FRET. Nat Biotechnol 22, 445–449 (2004) doi:10.1038/nbt945
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