The Fritz Haber Research Center, Institute of Chemistry, The Hebrew University of Jerusalem , Jerusalem 91904, Israel.
J Chem Theory Comput. 2017 Jan 10;13(1):353-369. doi: 10.1021/acs.jctc.6b00939. Epub 2016 Dec 8.
Inside proteins, protons move on proton wires (PWs). Starting from the highest resolution X-ray structure available, we conduct a 306 ns molecular dynamics simulation of the (A-state) wild-type (wt) green fluorescent protein (GFP) to study how its PWs change with time. We find that the PW from the chromophore via Ser205 to Glu222, observed in all X-ray structures, undergoes rapid water molecule insertion between Ser205 and Glu222. Sometimes, an alternate Ser205-bypassing PW exists. Side chain rotations of Thr203 and Ser205 play an important role in shaping the PW network in the chromophore region. Thr203, with its bulkier side chain, exhibits slower transitions between its three rotameric states. Ser205 experiences more frequent rotations, slowing down when the Thr203 methyl group is close by. The combined states of both residues affect the PW probabilities. A random walk search for PWs from the chromophore reveals several exit points to the bulk, one being a direct water wire (WW) from the chromophore to the bulk. A longer WW connects the "bottom" of the GFP barrel with a "water pool" (WP1) situated below Glu222. These two WWs were not observed in X-ray structures of wt-GFP, but their analogues have been reported in related fluorescent proteins. Surprisingly, the high-resolution X-ray structure utilized herein shows that Glu222 is protonated at low temperatures. At higher temperatures, we suggest ion pairing between anionic Glu222 and a proton hosted in WP1. Upon photoexcitation, these two recombine, while a second proton dissociates from the chromophore and either exits the protein using the short WW or migrates along the GFP-barrel axis on the long WW. This mechanism reconciles the conflicting experimental and theoretical data on proton motion within GFP.
在蛋白质内部，质子在质子通道（PW）上移动。从现有的最高分辨率 X 射线结构开始，我们对（A 态）野生型（wt）绿色荧光蛋白（GFP）进行了 306 ns 的分子动力学模拟，以研究其 PW 如何随时间变化。我们发现，从发色团经 Ser205 到 Glu222 的 PW，在所有 X 射线结构中都有观察到，在 Ser205 和 Glu222 之间会快速插入水分子。有时，会存在替代的 Ser205 旁路 PW。Thr203 和 Ser205 的侧链旋转在发色团区域 PW 网络的形成中起着重要作用。由于其较大的侧链，Thr203 显示出其三个构象态之间较慢的转变。Ser205 经历更频繁的旋转，当 Thr203 甲基靠近时会减慢。这两个残基的组合状态会影响 PW 的概率。从发色团开始的 PW 随机游走搜索揭示了几个通向主体的出口点，其中一个是从发色团到主体的直接水通道（WW）。一个较长的 WW 将 GFP 桶的“底部”与位于 Glu222 下方的“水池”（WP1）连接起来。这两个 WW 在 wt-GFP 的 X 射线结构中没有观察到，但在相关的荧光蛋白中已经报道了它们的类似物。令人惊讶的是，本文所使用的高分辨率 X 射线结构表明，Glu222 在低温下质子化。在较高温度下，我们建议阴离子 Glu222 与 WP1 中质子形成离子对。光激发后，这两个重新结合，而第二个质子从发色团中脱离，要么使用短 WW 离开蛋白质，要么沿 GFP 桶轴沿着长 WW 迁移。这种机制协调了 GFP 内质子运动的相互矛盾的实验和理论数据。
