Structure and Mechanism of the Photoactivatable Green Fluorescent Protein
Structure and Mechanism of the Photoactivatable Green Fluorescent Protein
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DOI：
10.1021/ja808851n
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发表时间：
2009-04-01
影响因子：
15
通讯作者：
Remington, S. James
中科院分区：
文献类型：
--
作者：
Henderson, J. Nathan;Gepshtein, Rinat;Remington, S. James
光激活型绿色荧光蛋白T203H变体（PA-GFP）在天然和光激活状态下的晶体结构得到了解析，在488 nm光照下，它们分别发出暗荧光和亮荧光。我们证明了PA-GFP的光激活是紫外线诱导的Glu222侧链脱羧的结果，该脱羧将发色团平衡转移到阴离子形式。加上T203H突变，稳定了天然PA-GFP中性发色团，Glu222脱羧产生了相对于野生型GFP (WT) 100倍的对比度增强。此外，这些结构提供了WT和PA-GFP之间的光谱差异、稳态荧光最大值和激发态质子转移动力学的见解。
Crystal structures of the photoactivatable green fluorescent protein T203H variant (PA-GFP) have been solved in the native and photoactivated states, which under 488 nm illumination are dark and brightly fluorescent, respectively. We demonstrate that photoactivation of PA-GFP is the result of a UV-induced decarboxylation of the Glu222 side chain that shifts the chromophore equilibrium to the anionic form. Coupled with the T203H mutation, which stabilizes the native PA-GFP neutral chromophore, Glu222 decarboxylation yields a 100-fold contrast enhancement relative to wild-type GFP (WT). Additionally, the structures provide insights into the spectroscopic differences between WT and PA-GFP steady-state fluorescence maxima and excited-state proton transfer dynamics.