An alternative excited-state proton transfer pathway in green fluorescent protein variant S205V
An alternative excited-state proton transfer pathway in green fluorescent protein variant S205V
复制标题
DOI：
10.1110/ps.073112007
复制
发表时间：
2007-12-01
期刊：
影响因子：
8
通讯作者：
Remington, S. James
中科院分区：
文献类型：
--
作者：
Shu, Xiaokun;Leiderman, Pavel;Remington, S. James
野生型绿色荧光蛋白(wt-GFP)在395 nm附近有一个明显的吸收带，属于中性发色团形式。激发该带时产生的绿色发射是由发色团羟基通过由水分子和Ser205组成的氢键网络到Glu222的激发态质子转移(ESPT)引起的。虽然已经获得了Glu222作为末端质子受体的证据，但没有证据表明Ser205参与了质子转移过程。为了研究Ser205在质子转移中的作用，我们将Ser205突变为valine。然而，衍生的GFP变异体S205V在400 nm激发时仍然产生绿色荧光。时间分辨发射光谱表明，ESPT对绿色荧光有贡献，质子转移的速度比wt-GFP慢30倍。S205V的晶体结构揭示了Glu222和Thr203的重排，形成了一个新的氢键网络。我们认为这个网络是一种替代的ESPT途径，具有独特的特征，可以解释质子转移速度显著减慢的原因。为了支持这一提议，S205V/T203V双突变体被证明是一种新的蓝色荧光蛋白，含有一个基于酪氨酸的发色团，但不能进行ESPT。这些结果对ESPT的详细机理和wt-GFP的光循环，特别是对循环中光谱鉴定的中间体的结构具有重要意义。
Wild-type green fluorescent protein (wt-GFP) has a prominent absorbance band centered at similar to 395 nm, attributed to the neutral chromophore form. The green emission arising upon excitation of this band results from excited-state proton transfer (ESPT) from the chromophore hydroxyl, through a hydrogen-bond network proposed to consist of a water molecule and Ser205, to Glu222. Although evidence for Glu222 as a terminal proton acceptor has already been obtained, no evidence for the participation of Ser205 in the proton transfer process exists. To examine the role of Ser205 in the proton transfer, we mutated Ser205 to valine. However, the derived GFP variant S205V, upon excitation at 400 nm, still produces green fluorescence. Time-resolved emission spectroscopy suggests that ESPT contributes to the green fluorescence, and that the proton transfer takes place similar to 30 times more slowly than in wt-GFP. The crystal structure of S205V reveals rearrangement of Glu222 and Thr203, forming a new hydrogen-bonding network. We propose this network to be an alternative ESPT pathway with distinctive features that explain the significantly slowed rate of proton transfer. In support of this proposal, the double mutant S205V/T203V is shown to be a novel blue fluorescent protein containing a tyrosine-based chromophore, yet is incapable of ESPT. The results have implications for the detailed mechanism of ESPT and the photocycle of wt-GFP, in particular for the structures of spectroscopically identified intermediates in the cycle.