Minimum set of mutations needed to optimize cyan fluorescent proteins for live cell imaging
Minimum set of mutations needed to optimize cyan fluorescent proteins for live cell imaging
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DOI：
10.1039/c2mb25303h
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发表时间：
2013-01-01
影响因子：
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通讯作者：
Merola, Fabienne
中科院分区：
文献类型：
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作者：
Erard, Marie;Fredj, Asma;Merola, Fabienne
青色荧光蛋白 (CFP) 广泛用作用于活细胞成像的基因编码生物传感器中的 FRET 供体。最近，通过大规模随机诱变开发了荧光量子产率大大提高的青色变体。我们证明，仅引入 T65S 和 H148G 两种突变就能够赋予流行形式 ECFP 同等的性能，从而产生海蓝宝石（QY = 0.89，tau(f) = 4.12 ns）。除了令人印象深刻的 pH 稳定性 (pK(1/2) = 3.3）外，海蓝宝石对其环境的总体敏感性非常低，并且光开关反应无法检测到。海蓝宝石在不同的哺乳动物细胞系统中提供高效、明亮的表达，具有长且单指数的细胞内荧光寿命，对蛋白质的融合或亚细胞位置几乎不敏感。 Aquamarine 还能够有利地取代 FRET 生物传感器 AKAR 中的 CFP 供体，用于蛋白激酶 A 活性的比率测量。海蓝宝石的性能表明，只需两轮简单的单点诱变就可以快速有效地优化基于 FRET 的生物传感器中的供体特性。
Cyan fluorescent proteins (CFPs) are widely used as FRET donors in genetically encoded biosensors for live cell imaging. Recently, cyan variants with greatly improved fluorescence quantum yields have been developed by large scale random mutagenesis. We show that the introduction of only two mutations, T65S and H148G, is able to confer equivalent performances on the popular form ECFP, leading to Aquamarine (QY = 0.89, tau(f) = 4.12 ns). Besides an impressive pH stability (pK(1/2) = 3.3), Aquamarine shows a very low general sensitivity to its environment, and undetectable photoswitching reactions. Aquamarine gives efficient and bright expression in different mammalian cell systems, with a long and single exponential intracellular fluorescence lifetime mostly insensitive to the fusion or the subcellular location of the protein. Aquamarine was also able to advantageously replace the CFP donor in the FRET biosensor AKAR for ratiometric measurements of protein kinase A activity. The performances of Aquamarine show that only two rounds of straightforward single point mutagenesis can be a quick and efficient way to optimize the donor properties in FRET-based biosensors.