Matsui Yasuhiro, Sakai Keisuke, Murakami Midori, Shiro Yoshitsugu, Adachi Shin ichi, Okumura Hideo, Kouyama Tsutomu
Graduate School of Science, Nagoya University, Chikusa, 464-8602, Nagoya, Japan.
J Mol Biol. 2002 Nov 29;324(3):469-81. doi: 10.1016/s0022-2836(02)01110-5.
Bacteriorhodopsin, the sole membrane protein of the purple membrane of Halobacterium salinarum, functions as a light-driven proton pump. A 3-D crystal of bacteriorhodopsin, which was prepared by the membrane fusion method, was used to investigate structural changes in the primary photoreaction. It was observed that when a frozen crystal was exposed to a low flux of X-ray radiation (5 x 10(14)photons mm(-2)), nearly half of the protein was converted into an orange species, exhibiting absorption peaks at 450 nm, 478 nm and 510 nm. The remainder retained the normal photochemical activity until Asp85 in the active site was decarboxlyated by a higher flux of X-ray radiation (10(16)photons mm(-2)). The procedure of diffraction measurement was improved so as to minimize the effects of the radiation damage and determine the true structural change associated with the primary photoreaction. Our structural model of the K intermediate indicates that the Schiff base linkage and the adjacent bonds in the polyene chain of retinal are largely twisted so that the Schiff base nitrogen atom still interacts with a water molecule located near Asp85. With respect to the other part of the protein, no appreciable displacement is induced in the primary photoreaction.
细菌视紫红质是盐生盐杆菌紫膜的唯一膜蛋白，起着光驱动质子泵的作用。通过膜融合法制备的细菌视紫红质三维晶体，用于研究初级光反应中的结构变化。观察到，当冷冻晶体暴露于低通量X射线辐射（5×10¹⁴ 光子·mm⁻²）时，近一半的蛋白质转化为橙色物种，在450nm、478nm和510nm处呈现吸收峰。其余部分保留正常光化学活性，直到活性位点的天冬氨酸85被高通量X射线辐射（10¹⁶ 光子·mm⁻²）脱羧。改进了衍射测量程序，以尽量减少辐射损伤的影响，并确定与初级光反应相关的真实结构变化。我们的K中间体结构模型表明，视黄醛多烯链中的席夫碱键和相邻键发生了很大程度的扭曲，因此席夫碱氮原子仍与位于天冬氨酸85附近的水分子相互作用。关于蛋白质的其他部分，在初级光反应中没有引起明显的位移。
