Rizzo Megan A, Springer Gerald H, Granada Butch, Piston David W
Nat Biotechnol. 2004 Apr;22(4):445-9. doi: 10.1038/nbt945. Epub 2004 Feb 29.
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.
许多基因编码的生物传感器利用荧光蛋白之间的荧光共振能量转移（FRET）来报告活细胞中的生化现象。最常见的是，增强型青色荧光蛋白（ECFP）用作供体荧光团，与几种黄色荧光蛋白（YFP）变体之一作为受体偶联。尽管存在几个光谱学上的缺点，即量子产率低、消光系数低以及荧光寿命最适合用双指数拟合，但仍使用ECFP。为了改善用于FRET测量的ECFP的特性，我们采用定点诱变方法来克服这些缺点。产生的变体，我们将其命名为天蓝色蛋白（ECFP/S72A/Y145A/H148D），具有大大提高的量子产率、更高的消光系数以及最适合用单指数拟合的荧光寿命。天蓝色蛋白比ECFP亮2.5倍，用天蓝色蛋白取代ECFP可显著提高基于FRET的葡萄糖激酶激活传感器的信噪比。
