Cyan and yellow super fluorescent proteins with improved brightness, protein folding, and FRET Forster radius
Cyan and yellow super fluorescent proteins with improved brightness, protein folding, and FRET Forster radius
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DOI：
10.1021/bi0516273
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发表时间：
2006-05-30
期刊：
影响因子：
2.9
通讯作者：
Gadella, Theodorus W. J., Jr.
中科院分区：
文献类型：
--
作者：
Kremers, Gert-Jan;Goedhart, Joachim;Gadella, Theodorus W. J., Jr.
增强型青色和黄色荧光蛋白广泛用于基于荧光共振能量转移的双色成像和蛋白质-蛋白质相互作用研究。这些荧光蛋白的使用可能因其热敏感性、暗淡荧光和聚集倾向而受到限制。在这里，我们报告了用于改进这些荧光蛋白的定点诱变方法的结果。我们创建了 ECFP 和 EYFP 的单体优化变体，它们在 37 摄氏度下折叠得更快、更有效，并且具有卓越的溶解度和亮度。表达 SCFP3A 的细菌比表达 ECFP 的细菌亮 9 倍，比表达 Cerulean 的细菌亮 1.2 倍。 SCFP3A 具有更高的量子产率 (0.56) 和荧光寿命。表达 SYFP2 的细菌比表达 EYFP(Q69K) 的细菌亮 12 倍，几乎比表达 Venus 的细菌亮 2 倍。在 HeLa 细胞中，这种改善不太明显。尽管如此，表达 SCFP3A 和 SYFP2 的细胞分别比表达 ECFP 和 EYFP (Q69K) 的细胞亮 1.5 倍。 SCFP3A 和 SYFP2 的增强很可能是由于内在亮度的增加（与 ECFP 和 EYFP(Q69K) 相比，纯化的重组蛋白分别为 1.7 倍和 1.3 倍）以及由于增强的蛋白质折叠和成熟。后者的增强对细菌中荧光产量的增加最为显着，而对于哺乳动物细胞系统来说似乎不太显着。由于 SCFP3A 的高量子产率和更长的寿命以及 SYFP2 的高消光系数，SCFP3A 和 SYFP2 成为荧光共振能量转移的优质供体-受体对。此外，还对 SCFP1（一种荧光寿命较短但光谱与 ECFP 和 SCFP3A 相同的 CFP 变体）进行了表征。利用 SCFP1 和 SCFP3A 之间巨大的寿命差异，我们能够首次对活细胞中光谱相同的荧光物质进行双寿命成像。
Enhanced cyan and yellow fluorescent proteins are widely used for dual color imaging and protein-protein interaction studies based on fluorescence resonance energy transfer. Use of these fluorescent proteins can be limited by their thermosensitivity, dim fluorescence, and tendency for aggregation. Here we report the results of a site-directed mutagenesis approach to improve these fluorescent proteins. We created monomeric optimized variants of ECFP and EYFP, which fold faster and more efficiently at 37 degrees C and have superior solubility and brightness. Bacteria expressing SCFP3A were 9-fold brighter than those expressing ECFP and 1.2-fold brighter than bacteria expressing Cerulean. SCFP3A has an increased quantum yield (0.56) and fluorescence lifetime. Bacteria expressing SYFP2 were 12 times brighter than those expressing EYFP(Q69K) and almost 2-fold brighter than bacteria expressing Venus. In HeLa cells, the improvements were less pronounced; nonetheless, cells expressing SCFP3A and SYFP2 were both 1.5-fold brighter than cells expressing ECFP and EYFP(Q69K), respectively. The enhancements of SCFP3A and SYFP2 are most probably due to an increased intrinsic brightness (1.7-fold and 1.3-fold for purified recombinant proteins, compared to ECFP & EYFP( Q69K), respectively) and due to enhanced protein folding and maturation. The latter enhancements most significantly contribute to the increased fluorescent yield in bacteria whereas they appear less significant for mammalian cell systems. SCFP3A and SYFP2 make a superior donor-acceptor pair for fluorescence resonance energy transfer, because of the high quantum yield and increased lifetime of SCFP3A and the high extinction coefficient of SYFP2. Furthermore, SCFP1, a CFP variant with a short fluorescence lifetime but identical spectra compared to ECFP and SCFP3A, was characterized. Using the large lifetime difference between SCFP1 and SCFP3A enabled us to perform for the first time dual-lifetime imaging of spectrally identical fluorescent species in living cells.