Chromophores absorb light in photosensitive proteins and thereby initiate fundamental biological processes such as photosynthesis, vision and biofluorescence. An important goal in their understanding is the provision of detailed structural descriptions of the ultrafast photochemical events that they undergo, in particular of the excited states that connect chemistry to biological function. Here we report on the structures of two excited states in the reversibly photoswitchable fluorescent protein rsEGFP2. We populated the states through femtosecond illumination of rsEGFP2 in its non-fluorescent off state and observed their build-up (within less than one picosecond) and decay (on the several picosecond timescale). Using an X-ray free-electron laser, we performed picosecond time-resolved crystallography and show that the hydroxybenzylidene imidazolinone chromophore in one of the excited states assumes a near-canonical twisted configuration halfway between the trans and cis isomers. This is in line with excited-state quantum mechanics/molecular mechanics and classical molecular dynamics simulations. Our new understanding of the structure around the twisted chromophore enabled the design of a mutant that displays a twofold increase in its off-to-on photoswitching quantum yield.

中文翻译：

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通过时间分辨串联飞秒晶体学捕获的光可转换荧光蛋白在激发态下的发色团扭曲

发色团吸收光敏蛋白中的光，从而启动基本的生物学过程，例如光合作用，视觉和生物荧光。他们理解的一个重要目标是提供它们所经历的超快速光化学事件，特别是将化学与生物学功能联系起来的激发态的详细结构描述。在这里我们报告可逆光开关荧光蛋白rsEGFP2中两个激发态的结构。我们通过飞秒照射rsEGFP2处于非荧光关闭状态来填充状态，并观察到它们的建立（在不到1皮秒的时间内）和衰减（在几皮秒的时间尺度上）。使用X射线自由电子激光，反式与顺式异构体。这符合激发态量子力学/分子力学和经典分子动力学模拟。我们对扭曲发色团周围结构的新理解使得突变体的设计得以实现，该突变体的开关光开关量子产率显示出两倍的增长。