Delaney J K, Schweiger U, Subramaniam S
Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.
Proc Natl Acad Sci U S A. 1995 Nov 21;92(24):11120-4. doi: 10.1073/pnas.92.24.11120.
Bacteriorhodopsin is a membrane protein that functions as a light-driven proton pump. Each cycle of proton transport is initiated by the light-induced isomerization of retinal from the all-trans to 13-cis configuration and is completed by the protein-driven reisomerization of retinal to the all-trans configuration. Previous studies have shown that replacement of Leu-93, a residue in close proximity to the 13-methyl group of retinal, by alanine, resulted in a 250-fold increase in the time required to complete each photocycle. Here, we show that the kinetic defect in the photocycle of the Leu-93-->Ala mutant occurs at a stage after the completion of proton transport and can be overcome in the presence of strong background illumination. Time-resolved retinal-extraction experiments demonstrate the continued presence of a 13-cis intermediate in the photocycle of the Leu-93-->Ala mutant well after the completion of proton release and uptake. These results indicate that retinal reisomerization is kinetically the rate-limiting step in the photocycle of this mutant and that the slow thermal reisomerization can be bypassed by the absorption of a second photon. The effects observed for the Leu-93-->Ala mutant are not observed upon replacement of any other residue in van der Waals contact with retinal or upon replacement of Leu-93 by valine. We conclude that the contact between Leu-93 and the 13-methyl group of retinal plays a key role in controlling the rate of protein conformational changes associated with retinal reisomerization and return of the protein to the initial state.
细菌视紫红质是一种膜蛋白，其功能是作为光驱动质子泵。质子运输的每个循环由视黄醛从全反式到13-顺式构型的光诱导异构化启动，并由蛋白质驱动视黄醛重新异构化为全反式构型而完成。先前的研究表明，将紧邻视黄醛13-甲基的亮氨酸-93残基替换为丙氨酸，导致完成每个光循环所需的时间增加了250倍。在这里，我们表明亮氨酸-93→丙氨酸突变体光循环中的动力学缺陷发生在质子运输完成后的一个阶段，并且在强背景光照下可以克服。时间分辨视黄醛提取实验表明，在质子释放和摄取完成后很久，亮氨酸-93→丙氨酸突变体的光循环中仍持续存在13-顺式中间体。这些结果表明，视黄醛重新异构化在该突变体的光循环中在动力学上是限速步骤，并且缓慢的热重新异构化可以被吸收第二个光子所绕过。在视黄醛范德华接触的任何其他残基被替换或亮氨酸-93被缬氨酸替换时，未观察到亮氨酸-93→丙氨酸突变体所观察到的效应。我们得出结论，亮氨酸-93与视黄醛13-甲基之间的接触在控制与视黄醛重新异构化和蛋白质返回初始状态相关的蛋白质构象变化速率方面起关键作用。
