Weidlich O, Schalt B, Friedman N, Sheves M, Lanyi J K, Brown L S, Siebert F
Institut für Biophysik und Strahlenbiologie, Albert-Ludwigs-Universität, Freiburg, Germany.
Biochemistry. 1996 Aug 20;35(33):10807-14. doi: 10.1021/bi960780h.
The hypothesis was tested whether in bacteriorhodopsin (BR) the reduction of the steric interaction between the 9-methyl group of the chromophore all-trans-retinal and the tryptophan at position 182 causes the same changes as observed in the photocycle of 9-demethyl-BR. For this, the photocycle of the mutant W182F was investigated by time-resolved UV-vis and pH measurements and by static and time-resolved FT-IR difference spectroscopy. We found that the second half of the photocycle was similarly distorted in the two modified systems: based on the amide-I band, the protonation state of D96, and the kinetics of proton uptake, four N intermediates could be identified, the last one having a lifetime of several seconds; no O intermediate could be detected; the proton uptake showed a pronounced biphasic time course; and the pKa of group(s) on the cytoplasmic side in N was reduced from 11 in wild type BR to around 7.5. In contrast to 9-demethyl-BR, in the W182F mutant the first part of the photocycle does not drastically deviate from that of wild type BR. The results demonstrate the importance of the steric interaction between W182 and the 9-methyl group of the retinal in providing tight coupling between chromophore isomerization and the late proton transfer steps.
我们对一个假说进行了验证，即在细菌视紫红质（BR）中，发色团全反式视黄醛的9-甲基与182位色氨酸之间的空间相互作用减弱，是否会导致与9-去甲基-BR光循环中观察到的相同变化。为此，我们通过时间分辨紫外可见光谱和pH测量以及静态和时间分辨傅里叶变换红外差光谱，研究了突变体W182F的光循环。我们发现，在这两个修饰系统中，光循环的后半部分同样发生了扭曲：基于酰胺-I带、D96的质子化状态和质子摄取动力学，可以识别出四种N中间体，最后一种的寿命为几秒；未检测到O中间体；质子摄取呈现出明显的双相时间进程；并且N中细胞质侧基团的pKa从野生型BR中的11降低到了约7.5。与9-去甲基-BR不同，在W182F突变体中，光循环的第一部分与野生型BR相比没有明显偏差。结果表明，W182与视黄醛的9-甲基之间的空间相互作用对于在发色团异构化和后期质子转移步骤之间提供紧密耦合至关重要。
