Quantum yield is a determinant for fluorescent protein (FP) applications and enhancing FP brightness through gene engineering is still a challenge. Green2, our de novo FP 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 (t_1/2, 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.

中文翻译：

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通过 F145Y 取代，Green2 向光稳定性和量子产率增强的结构引导演化。

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 亮度仍然是一个挑战。Green2 是我们通过微流控微阵列和克隆合成的从头FP，其量子产率明显低于增强型绿色 FP，尽管它们具有高同源性并共享相同的发色团。为了提高其量子产率，我们基于合理的结构分析将 F145Y 替代引入 Green2。Y145 显着提高了量子产率（0.22 对 0.18）并提高了光稳定性（t _1/2, 73.0 s vs. 46.0 s)，但不影响激发和发射光谱。进一步的结构分析表明，F145Y 取代导致发色团的直接环境发生显着的电场变化。发色团周围静电荷的扰动导致基态和激发态之间的能垒变化，从而导致量子产率和光稳定性的提高。我们的结果说明了仅基于荧光效率优化来设计 FP 的典型示例，并为 FP 的合理演化提供了新的见解。