Structure-guided evolution of Green2 toward photostability and quantum yield enhancement by F145Y substitution.
Structure-guided evolution of Green2 toward photostability and quantum yield enhancement by F145Y substitution.
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Green2 的结构引导演化通过 F145Y 取代实现光稳定性和量子产率增强。
DOI：
10.1002/pro.3917
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发表时间：
2020
期刊：
影响因子：
8
通讯作者：
Gao Xiaolian
中科院分区：
文献类型：
--
作者：
Sun Tingting;Li Tianpeng;Yi Ke;Gao Xiaolian
量子产率是荧光蛋白(FP)应用的决定因素，通过基因工程提高FP的亮度仍然是一个挑战。微流控微阵列克隆合成的绿色荧光蛋白Green2与增强型绿色荧光蛋白具有较高的同源性和相同的发色团，但其量子产率明显低于增强型绿色荧光蛋白。为了增加其量子产率，我们在合理的结构分析的基础上，在Green2中引入了F145Y取代。Y145显著提高了量子产率(0.22vs.0.18)和光稳定性(t1/2，73.0 S vs.46.0 S)，但对激发光谱和发射光谱没有影响。进一步的结构分析表明，F145Y取代导致发色团周围环境的电场发生了显着变化。生色团周围静电电荷的扰动导致基态和激发态之间的势垒变化，从而提高了量子产率和光稳定性。我们的结果展示了一个完全基于荧光效率优化的FP工程的典型例子，并为FP的合理进化提供了新的见解。
Quantum yield is a determinant for fluorescent protein (FP) applications and enhancing FP brightness through gene engineering is still a challenge. Green2, ourde novoFP synthesized by microfluidic picoarray and cloning, has a significantly lower quantum yield than enhanced green FP, though they have high homology and share the same chromophore. To increase its quantum yield, we introduced an F145Y substitution into Green2 based on rational structural analysis. Y145 significantly increased the quantum yield (0.22 vs. 0.18) and improved the photostability (t1/2, 73.0 s vs. 46.0 s), but did not affect the excitation and emission spectra. Further structural analysis showed that the F145Y substitution resulted in a significant electrical field change in the immediate environment of the chromophore. The perturbation of electrostatic charge around the chromophore lead to energy barrier changes between the ground and excited states, which resulted in the enhancement of quantum yield and photostability. Our results illustrate a typical example of engineering an FP based solely on fluorescence efficiency optimization and provide novel insights into the rational evolution of FPs.