Crystal structure of the L intermediate of bacteriorhodopsin: Evidence for vertical translocation of a water molecule during the proton pumping cycle
Crystal structure of the L intermediate of bacteriorhodopsin: Evidence for vertical translocation of a water molecule during the proton pumping cycle
复制标题
DOI：
10.1016/j.jmb.2003.10.068
复制
发表时间：
2004-01-09
影响因子：
5.6
通讯作者：
Okumura, H
中科院分区：
文献类型：
--
作者：
Kouyama, T;Nishikawa, T;Okumura, H
为了研究细菌视紫红质的L中间体的结构，在160 K下用绿色光照射属于空间群P622的3D晶体，随后在100 K下用红光照射。这产生了类似于1：4的L中间体和基态的混合物。用低通量的X射线（类似于每个晶体2 × 10（15）光子/mm（2））收集这些晶体的衍射数据，并将它们的合并数据与未光解晶体的数据进行比较。这些结构数据，连同我们以前的数据，表明视网膜发色团，这是在很大程度上扭曲的K-中间体，需要一个更平面的13-顺式，15-反的配置在L中间体。这种构型变化伴随着席夫碱N-H键朝向细胞内侧的重新取向，与氨基酸残基（Leu 93）的侧链的大旋转相结合，从而与视黄醛的C13甲基接触。在这些运动之后，一个水分子，首先与希夫碱和Asp 85氢键结合，被拖到最初由Leu 93占据的空间。含有M中间体的晶体的衍射数据表明，该水分子在L至M过渡中进一步向细胞内侧移动。该水分子从质子化的席夫碱上脱离很可能导致席夫碱的pK（a）显著降低，从而促进质子向Asp 85的转移。在这些观察的基础上，我们认为，在K-到-L过渡的水分子的垂直运动是一个关键的事件，确定质子易位的蛋白质的方向性。(C)2003 Elsevier Ltd.保留所有权利。
For structural investigation of the L intermediate of bacteriorhodopsin, a 3D crystal belonging to the space group P622 was illuminated with green light at 160 K and subsequently with red light at 100 K. This yielded a similar to1:4 mixture of the L intermediate and the ground-state. Diffraction data from such crystals were collected using a low flux of X-rays (similar to2 x 10(15) photons/mm(2) per crystal), and their merged data were compared with those from unphotolyzed crystals. These structural data, together with our previous data, indicate that the retinal chromophore, which is largely twisted in the K-intermediate, takes a more planar 13-cis, 15-anti configuration in the L intermediate. This configurational change, which is accompanied by re-orientation of the Schiff base N-H bond towards the intracellular side, is coupled with a large rotation of the side-chain of an amino acid residue (Leu93) making contact with the C13 methyl group of retinal. Following these motions, a water molecule, at first hydrogen-bonded to the Schiff base and Asp85, is dragged to a space that is originally occupied by Leu93. Diffraction data from a crystal containing the M intermediate showed that this water molecule moves further towards the intracellular side in the L-to-M transition. It is very likely that detachment of this water molecule from the protonated Schiff base causes a significant decrease in the pK(a) of the Schiff base, thereby facilitating the proton transfer to Asp85. On the basis of these observations, we argue that the vertical movement of a water molecule in the K-to-L transition is a key event determining the directionality of proton translocation in the protein. (C) 2003 Elsevier Ltd. All rights reserved.