Subramaniam S, Lindahl M, Bullough P, Faruqi A R, Tittor J, Oesterhelt D, Brown L, Lanyi J, Henderson R
MRC Laboratory for Molecular Biology, Cambridge, England.
J Mol Biol. 1999 Mar 19;287(1):145-61. doi: 10.1006/jmbi.1999.2589.
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 wild-type bacteriorhodopsin, the single mutants D38R, D96N, D96G, T46V, L93A and F219L, and the triple mutant D96G/F171C/F219L 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 A resolution to define light-driven changes in protein conformation. Our experiments demonstrate that in wild-type bacteriorhodopsin, a large protein conformational change occurs within approximately 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 light-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 M1) are well approximated by one protein conformation, while the structures of the later intermediates (M2, 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 M1 and M2 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.
我们报道了对野生型细菌视紫红质光循环以及光循环中存在动力学缺陷的多种突变蛋白构象变化的全面电子晶体学分析。在野生型细菌视紫红质、单突变体D38R、D96N、D96G、T46V、L93A和F219L以及三突变体D96G/F171C/F219L的光循环后期积累的特定中间体，通过在光照后不同时间将二维晶体冷冻在液态乙烷中而被捕获。从这些晶体记录的电子衍射图用于构建分辨率为3.5埃的投影差分傅里叶图，以确定光驱动的蛋白质构象变化。我们的实验表明，在野生型细菌视紫红质中，光照后约1毫秒内会发生大的蛋白质构象变化。对野生型和突变型细菌视紫红质在优先积累M或N中间体的条件下的结构变化分析表明，被困在同一蛋白质中的M和N中间体之间的结构差异很小。然而，当相同的光学中间体被困在不同的突变体中时，会观察到相当大的差异。在一些突变体中，甚至在光照之前就存在部分构象变化，光照后会发生额外的变化。选定的突变，如D96G/F171C/F219L三突变体中的突变，可充分破坏野生型结构的稳定性，从而在黑暗中产生几乎全部程度的构象变化，而光诱导的额外变化最小。我们得出结论，在显示长寿命M、N或O中间体的突变体中观察到的结构变化差异，最好描述为一种基本构象变化类型的变体，而不是代表所积累的光学中间体特有的结构变化。因此，我们的观察结果支持了一种简化的野生型细菌视紫红质光循环观点，即初始状态和早期中间体（K、L和M1）的结构可以由一种蛋白质构象很好地近似，而后期中间体（M2、N和O）的结构可以由另一种蛋白质构象很好地近似。我们提出，在野生型细菌视紫红质和大多数突变体中，M1和M2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膜的细胞外侧切换到细胞质侧做出重要贡献。
