Stalling chromophore synthesis of the fluorescent protein Venus reveals the molecular basis of the final oxidation step.
Stalling chromophore synthesis of the fluorescent protein Venus reveals the molecular basis of the final oxidation step.
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DOI：
10.1039/d0sc06693a
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发表时间：
2021-03-31
期刊：
影响因子：
8.4
通讯作者：
Jones DD
中科院分区：
文献类型：
--
作者：
Auhim HS;Grigorenko BL;Harris TK;Aksakal OE;Polyakov IV;Berry C;Gomes GDP;Alabugin IV;Rizkallah PJ;Nemukhin AV;Jones DD
荧光蛋白（FP）已经彻底改变了生命科学，但发色团成熟的机制仍然没有完全理解。在这里，我们表明，在发色团内的光响应的非规范氨基酸的掺入失速成熟的金星，黄色FP，在中间阶段;晶体结构表明存在O2位于上面的脱水烯醇化形式的咪唑啉酮环，接近严格保守的Gly 67，占据扭曲的构象。His 148采用“开放”构象，从而形成允许O2进入未成熟发色团的通道。吸收光谱支持QM/MM模拟表明，第一个氧化步骤涉及的氢过氧中间体的形成与脱氢的亚甲基桥。在体外和体内，通过释放H2 O2形成完全缀合的成熟发色团。中断和光化学重新启动发色团成熟的可能性和机制的见解开辟了新的方法，工程光学控制的荧光蛋白。荧光蛋白（FP）已经彻底改变了生命科学，但生色团成熟机制仍然没有完全理解。在这里，我们光化学陷阱成熟在一个关键阶段和结构的中间体。
Fluorescent proteins (FPs) have revolutionised the life sciences, but the mechanism of chromophore maturation is still not fully understood. Here we show that incorporation of a photo-responsive non-canonical amino acid within the chromophore stalls maturation of Venus, a yellow FP, at an intermediate stage; a crystal structure indicates the presence of O2 located above a dehydrated enolate form of the imidazolone ring, close to the strictly conserved Gly67 that occupies a twisted conformation. His148 adopts an “open” conformation so forming a channel that allows O2 access to the immature chromophore. Absorbance spectroscopy supported by QM/MM simulations suggests 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, both in vitro and in vivo. The possibility of interrupting and photochemically restarting chromophore maturation and the mechanistic insights open up new approaches for engineering optically controlled fluorescent proteins. Fluorescent proteins (FPs) have revolutionised the life sciences, but the chromophore maturation mechanism is still not fully understood. Here we photochemically trap maturation at a crucial stage and structurally characterise the intermediate.
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DOI：
10.1107/s0907444905036693
发表时间：
2006-01-01
影响因子：
2.2
作者：
Evans, P
通讯作者：
Evans, P
影响因子：
3.7
作者：
Arpino JA;Baldwin AJ;McGarrity AR;Tippmann EM;Jones DD
通讯作者：
Jones DD
影响因子：
2.9
作者：
Barondeau, DP;Kassmann, CJ;Getzoff, ED
通讯作者：
Getzoff, ED
DOI：
10.1107/s0907444904019158
发表时间：
2004-12-01
影响因子：
2.2
作者：
Emsley, P;Cowtan, K
通讯作者：
Cowtan, K
影响因子：
3.3
作者：
Grigorenko, Bella L.;Polyakov, Igor, V;Nemukhin, Alexander, V
通讯作者：
Nemukhin, Alexander, V