Protein conformational changes in the bacteriorhodopsin photocycle
Protein conformational changes in the bacteriorhodopsin photocycle
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DOI：
10.1006/jmbi.1999.2589
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发表时间：
1999-03-19
影响因子：
5.6
通讯作者：
Henderson, R
中科院分区：
文献类型：
--
作者：
Subramaniam, S;Lindahl, I;Henderson, R
我们报告了一个全面的电子晶体学分析的构象变化，在光循环的野生型细菌视紫红质和各种突变蛋白质与动力学缺陷的光循环。在野生型细菌视紫红质、单突变体D38 R、D96 N、D96 G、T46 V、L93 A和F219 L以及三突变体D96 G/F171 C/3F 219 L的光循环的后期阶段中积累的特定中间体通过在用闪光灯照射后的不同时间在液体乙烷中冷冻二维晶体来捕获。从这些晶体记录的电子衍射图案用于构建3.5埃分辨率的投影差傅立叶图，以定义蛋白质构象的光驱动变化。我们的实验表明，在野生型细菌视紫红质，一个大的蛋白质构象变化发生在类似的1毫秒后照明。在野生型和突变体细菌视紫红质的条件下，当M或N中间体的结构变化的分析表明，有被困在同一蛋白质中的M和N中间体之间的结构只有很小的差异。然而，当相同的光学中间体被困在不同的突变体中时，观察到相当大的变化。在一些突变体中，甚至在光照之前就存在部分构象变化，在光照后发生额外的变化。选定的突变，如D96 G/F171 C/F219 L三重突变体中的突变，可以充分破坏野生型结构的稳定性，以在黑暗中产生几乎全部程度的构象变化，而额外的Fight诱导的变化最小。我们的结论是，在显示长寿命的M，N或O中间体的突变体中观察到的结构变化的差异，最好描述为一个基本类型的构象变化的变化，而不是代表的结构变化是独特的光学中间体的积累。因此，我们的观察结果支持了野生型细菌视紫红质的光循环的简化视图，其中初始状态和早期中间体（K，L和M）的结构很好地近似于一种蛋白质构象，而后期中间体（M-2，N和O）的结构很好地近似于另一种蛋白质构象。我们建议，在野生型细菌视紫红质和大多数突变体中，M-1和M-2状态之间的这种构象变化可能对有效地将席夫碱的质子可及性从细胞外侧切换到膜的细胞质侧做出重要贡献。(C)北京：科学出版社.
We report a comprehensive electron crystallographic analysis of conformational changes in the photocycle of wild-type bacteriorhodopsin and in a variety of mutant proteins with kinetic defects in the photocycle. Specific intermediates that accumulate in the late stages of the photocycle of wildtype bacteriorhodopsin, the single mutants D38R, D96N, D96G, T46V, L93A and F219L, and the triple mutant D96G/F171C/3F219L were trapped by freezing two-dimensional crystals in liquid ethane at varying times after illumination with a light flash. Electron diffraction patterns recorded from these crystals were used to construct projection difference Fourier maps at 3.5 Angstrom resolution to define light-driven changes in protein conformation. Our experiments demonstrate that in wild-type bacteriorhodopsin, a large protein conformational change occurs within similar to 1 ms after illumination. Analysis of structural changes in wild-type and mutant bacteriorhodopsins under conditions when either the M or the N intermediate is preferentially accumulated reveals that there are only small differences in structure between M and N intermediates trapped in the same protein. However, a considerably larger variation is observed when the same optical intermediate is trapped in different mutants. In some of the mutants, a partial conformational change is present even prior to illumination, with additional changes occurring upon illumination. Selected mutations, such as those in the D96G/F171C/F219L triple mutant, can sufficiently destabilize the wild-type structure to generate almost the full extent of the conformational change in the dark, with minimal additional Fight-induced changes. We conclude that the differences in structural changes observed in mutants that display long-lived M, N or O intermediates are best described as variations of one fundamental type of conformational change, rather than representing structural changes that are unique to the optical intermediate that is accumulated. Our observations thus support a simplified view of the photocycle of wild-type bacteriorhodopsin in which the structures of the initial state and the early intermediates (K, L, and M,) are well approximated by one protein conformation, while the structures of the later intermediates (M-2, N and O) are well approximated by the other protein conformation. We propose that in wild-type bacteriorhodopsin and in most mutants, this conformational change between the M-1 and M-2 states is likely to make an important contribution towards efficiently switching proton accessibility of the Schiff base from the extracellular side to the cytoplasmic side of the membrane. (C) 1999 Academic Press.