Balance between ultrafast parallel reactions in the green fluorescent protein has a structural origin
Balance between ultrafast parallel reactions in the green fluorescent protein has a structural origin
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DOI：
10.1529/biophysj.108.129957
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发表时间：
2008-08-15
影响因子：
3.4
通讯作者：
Sage, J. Timothy
中科院分区：
文献类型：
--
作者：
van Thor, Jasper J.;Ronayne, Kate L.;Sage, J. Timothy
绿色荧光蛋白的荧光光循环在功能上依赖于特定的结构蛋白环境。谷氨酸222的平衡蛋白质侧链构象和反应性之间的直接关系被建立，特别是在荧光光循环中的超快质子转移反应的速率。我们表明，在光循环的平行变换有一个结构的起源，我们报告的振动特性的响应氨基酸的超快时间尺度。绿色荧光蛋白的蓝色激发驱动两个平行的，激发态的氘核转移反应与10 ps和75 ps的时间常数，通过氢键网络的谷氨酸222的掩埋的羧酸侧链。通过分析E222 D突变体的瞬态吸收数据，可以将1456 cm（-1）和1441 cm（-1）模式分别指定为nsym，将1564 cm（-1）和1570 cm（-1）特征分别指定为nu（asym）。与野生型相比，E222 D的测量可以用仅一个差谱来描述，其中nsym模式在1435 cm（-1）处，nasym模式在1567 cm（-1）处，还将大的Δ nu（非对称-对称）与缓慢的激发态质子转移动力学相关联。密度泛函理论计算和已发表的模型化合物和理论研究将Δ nu（非对称）的差异与通过羧酸盐的平衡几何形状和COO-伸缩频率差异检测到的氢键相互作用的强度和数量相关。光循环动力学与受体侧链构象的相关性表明，从S205到E222的质子转移控制了整个激发态质子转移过程的速率，这与最近的理论预测一致。光选择性测量显示协议本地化的C = O振动的发色团，Q69，和E222与密度泛函理论和从头计算放置在X-射线几何，并提供其振动响应的中间体中的光循环。
The fluorescence photocycle of the green fluorescent protein is functionally dependent on the specific structural protein environment. A direct relationship between equilibrium protein side-chain conformation of glutamate 222 and reactivity is established, particularly the rate of ultrafast proton transfer reactions in the fluorescence photocycle. We show that parallel transformations in the photocycle have a structural origin, and we report on the vibrational properties of responsive amino acids on an ultrafast timescale. Blue excitation of GFP drives two parallel, excited-state deuteron transfer reactions with 10 ps and 75 ps time constants to the buried carboxylic acid side chain of glutamate 222 via a hydrogen-bonding network. Assignment of 1456 cm(-1) and 1441 cm(-1) modes to nsym and assignment of 1564 cm(-1) and 1570 cm(-1) features to nu(asym) of E222 in the 10 ps and 75 ps components, respectively, was possible from the analysis of the transient absorption data of an E222D mutant and was consistent with photoselection measurements. In contrast to the wild-type, measurements of E222D can be described with only one difference spectrum, with the nsym mode at 1435 cm(-1) and the nasym mode at 1567 cm(-1), also correlating a large Delta nu(asym-sym) with slow excited-state proton transfer kinetics. Density Functional Theory calculations and published model compound and theoretical studies relate differences in Delta nu(asym-sym) to the strength and number of hydrogen-bonding interactions that are detected via equilibrium geometry and COO-stretching frequency differences of the carboxylate. The correlation of photocycle kinetics with side-chain conformation of the acceptor suggests that proton transfer from S205 to E222 controls the rate of the overall excited-state proton transfer process, which is consistent with recent theoretical predictions. Photoselection measurements show agreement for localized C = O vibrations of chromophore, Q69, and E222 with Density Functional Theory and ab initio calculations placed in the x-ray geometry and provide their vibrational response in the intermediates in the photocycle.