Fluorescent proteins (FPs) have revolutionised the life sciences but the mechanism of chromophore maturation is still not fully understood. Incorporation of a photo-responsive non-canonical amino acid within the chromophore stalls maturation of Venus, a yellow FP, at an intermediate stage; the crystal structure reveals the presence of O2 located above a dehydrated enolate imidazolone (I) ring, close to the strictly conserved Gly67 that occupies a twisted conformation. His148 adopts an open conformation, potentially allowing O2 access to the chromophore. Absorption spectroscopy supported by QM/MM simulations suggest that the first oxidation step involves formation of a hydroperoxyl intermediate in conjunction with dehydrogenation of the methylene bridge. A fully conjugated mature chromophore is formed through release of H2O2 upon irradiation of this intermediate, both in vitro and in vivo. The possibility of interrupting and photochemically restarting chromophore maturation, and the mechanistic insights opens up new approaches for engineering optically controlled fluorescent proteins.

中文翻译：

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荧光蛋白金星的停滞发色团成熟揭示了最终氧化步骤的分子基础

荧光蛋白（FPs）彻底改变了生命科学，但发色团成熟的机理仍不完全清楚。在生色团内掺入光响应性非经典氨基酸，可在中间阶段阻止黄色FP的维纳斯成熟。晶体结构揭示了O2存在于脱水的烯醇式咪唑酮（I）环上方，接近严格保守的Gly67，其具有扭曲的构象。His148采用开放构象，可能使O2进入发色团。QM / MM模拟所支持的吸收光谱表明，第一个氧化步骤包括与亚甲基桥的脱氢结合形成氢过氧中间体。通过在体外和体内照射该中间体时释放H2O2，可以形成完全共轭的成熟生色团。中断和光化学重启生色团成熟的可能性，以及对机理的见解为工程化光学控制的荧光蛋白开辟了新途径。