Structural investigation of the active site in bacteriorhodopsin: Geometric constraints on the roles of Asp-85 and Asp-212 in the proton-pumping mechanism from solid state NMR
Structural investigation of the active site in bacteriorhodopsin: Geometric constraints on the roles of Asp-85 and Asp-212 in the proton-pumping mechanism from solid state NMR
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DOI：
10.1021/bi991106d
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发表时间：
2000-01-18
期刊：
影响因子：
2.9
通讯作者：
Griffin, RG
中科院分区：
文献类型：
--
作者：
Griffiths, JM;Bennett, AE;Griffin, RG
利用固体核磁共振光谱法确定了细菌视紫红质（bR）的bR（555）、bR（568）和M-412状态下，Asp-85和Asp-212侧链的羧基碳与视网膜发色团的14碳之间的距离。这些距离是通过二维rf驱动重耦合（RFDR）和自旋扩散实验中观察到的C-13-C-13磁化交换来检测的。相对RFDR交叉峰强度与核磁共振实验模拟的比较得出了bR（568）和M-412中[14-C-13]视网膜距离的距离测量值分别为4.4 +/- 0.6和4.8 +/- 1.0埃（,）。自旋扩散数据与这些结果一致，表明Asp-212到14- c -视网膜距离在转化为m态后增加了16 +/- 10%。在所有状态下[14-C-13]视网膜到[4-C-13]Asp-85之间没有交叉峰，bR（555）中任何[4-C-13]Asp残基与[14-C-13]视网膜之间没有交叉峰，表明这些距离超过6.0埃。对于bR(568)，核磁共振距离约束与最近对完整膜的衍射研究结果一致，而对于M态，核磁共振结果与在Asp-85和Asp-212残基区域使用两个结合水的理论模拟结果一致。核磁共振提供的结构信息将有助于完善目前对天冬氨酸残基在bR质子泵送机制中的作用的理解。
Constraints on the proximity of the carboxyl carbons of the Asp-85 and Asp-212 side chains to the 14-carbon of the retinal chromophore have been established for the bR(555), bR(568), and M-412 states of bacteriorhodopsin (bR) using solid-state NMR spectroscopy. These distances were examined via C-13-C-13 magnetization exchange, which was observed in two-dimensional RF-driven recoupling (RFDR) and spin diffusion experiments. A comparison of relative RFDR cross-peak intensities with simulations of the NMR experiments yields distance measurement and 4.4 +/- 0.6 and 4.8 +/- 1.0 Angstrom for the [14-C-13]retinal distances in bR(568) and M-412, respectively(,) The spin diffusion data are consistent with these results and indicate that the Asp-212 to 14-C-retinal distance increases by 16 +/- 10% upon conversion to the M-state. The absence of cross-peaks from [14-C-13]retinal to [4-C-13]Asp-85 in all states and between any [4-C-13]Asp residue and [14-C-13]retinal in bR(555) indicates that these distances exceed 6.0 Angstrom. For bR(568) the NMR distance constraints are in agreement with the results fi om recent diffraction studies on intact membranes, while for the M state the NMR results agree with theoretical simulations employing two bound waters in the region of the Asp-85 and Asp-212 residues. The structural information provided by NMR should prove useful for refining the current understanding of the role of aspartic acid residues in the proton-pumping mechanism of bR.