Light-driven decarboxylation of wild-type green fluorescent protein
Light-driven decarboxylation of wild-type green fluorescent protein
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DOI：
10.1021/ja034588w
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发表时间：
2003-06-11
影响因子：
15
通讯作者：
Tonge, PJ
中科院分区：
文献类型：
--
作者：
Bell, AF;Stoner-Ma, D;Tonge, PJ
使用稳态吸收、荧光和拉曼光谱研究野生型GFP对UV和可见光的响应。如先前报道的[货车Thor，Nat.Struct.Biol.2002，9，37-41]，GFP的照射导致E222的脱羧。本文报道了光驱动脱羧反应的速率强烈地依赖于激发波长，以254 nm > 280 nm > 476 nm的顺序减小。脱羧的相对效率解释的Kolbe型机制，其中激发态的发色团作为氧化剂接受电子从E222。具体而言，有人提出，254 nm激发填充的S2（或更高）激发态的发色团，而404和476 nm激发填充的S1激发态的中性和阴离子形式，分别和三个激发态的相对氧化能力控制的脱羧反应的速率。此外，W57在GFP的生物物理学中的作用已经通过将该残基突变为苯丙氨酸来探索。这些研究表明，虽然W57不影响脱羧，但该残基参与与发色团的共振能量转移，从而部分解释了野生型GFP在UV照射后观察到的绿色荧光。最后，比较拉曼光谱从nonilluminated和脱羧形式的野生型绿色荧光蛋白提供了进一步的振动带内的中性和阴离子形式的发色团的分配。此外，这些光谱提供了有价值的洞察蛋白质和生色团之间的特定相互作用，控制野生型GFP的光学特性。
The response of wild-type GFP to UV and visible light was investigated using steady state absorption, fluorescence, and Raman spectroscopies. As reported previously [van Thor, Nat. Struct. Biol. 2002, 9, 37-41], irradiation of GFP results in decarboxylation of E222. Here it is reported that the rate of the light-driven decarboxylation reaction strongly depends on the excitation wavelength, decreasing in the order 254 nm > 280 nm > 476 nm. The relative efficiencies of decarboxylation are explained in terms of the Kolbe-type mechanism in which the excited state of the chromophore acts as an oxidant by accepting an electron from E222. Specifically, it is proposed that 254 nm excitation populates the S2 (or higher) excited state of the chromophore, whereas 404 and 476 nm excitation populate the S1 excited state of neutral and anionic forms, respectively, and that the relative oxidizing power of the three excited states controls the rate of the decarboxylation reaction. In addition, the role of W57 in the photophysics of GFP has been probed by mutating this residue to phenylalanine. These studies reveal that while W57 does not affect decarboxylation, this residue is involved in resonance energy transfer with the chromophore, thereby partially explaining the green fluorescence observed upon UV irradiation of wild-type GFP. Finally, comparison of Raman spectra obtained from nonilluminated and decarboxylated forms of wild-type GFP has provided further vibrational band assignments for neutral and anionic forms of the chromophore within the protein. In addition, these spectra provide valuable insight into the specific interactions between the protein and the chromophore that control the optical properties of wild-type GFP.