Luecke H
Departments of Molecular Biology and Biochemistry and Physiology and Biophysics, UCI Program in Macromolecular Structure, University of California, 92697-3900, Irvine, CA, USA.
Biochim Biophys Acta. 2000 Aug 30;1460(1):133-56. doi: 10.1016/s0005-2728(00)00135-3.
High-resolution X-ray crystallographic studies of bacteriorhodopsin have tremendously advanced our understanding of this light-driven ion pump during the last 2 years, and emphasized the crucial role of discrete internal water molecules in the pump cycle. In the extracellular region an extensive three-dimensional hydrogen-bonded network of protein residues and seven water molecules leads from the buried retinal Schiff base via water 402 and the initial proton acceptor Asp85 to the membrane surface. Near Lys216 where the retinal binds, transmembrane helix G contains a pi-bulge that causes a non-proline kink. The bulge is stabilized by hydrogen bonding of the main chain carbonyl groups of Ala215 and Lys216 with two buried water molecules located in the otherwise very hydrophobic region between the Schiff base and the proton donor Asp96 in the cytoplasmic region. The M intermediate trapped in the D96N mutant corresponds to a late M state in the transport cycle, after protonation of Asp85 and release of a proton to the extracellular membrane surface, but before reprotonation of the deprotonated retinal Schiff base. The M intermediate from the E204Q mutant corresponds to an earlier M, as in this mutant the Schiff base deprotonates without proton release. The structures of these two M states reveal progressive displacements of the retinal, main chain and side chains induced by photoisomerization of the retinal to 13-cis,15-anti, and an extensive rearrangement of the three-dimensional network of hydrogen-bonded residues and bound water that accounts for the changed pK(a)s of the Schiff base, Asp85, the proton release group and Asp96. The structure for the M state from E204Q suggests, moreover, that relaxation of the steric conflicts of the distorted 13-cis,15-anti retinal plays a critical role in the reprotonation of the Schiff base by Asp96. Two additional waters now connect Asp96 to the carbonyl of residue 216, in what appears to be the beginning of a hydrogen-bonded chain that would later extend to the retinal Schiff base. Based on the ground state and M intermediate structures, models of the molecular events in the early part of the photocycle are presented, including a novel model which proposes that bacteriorhodopsin pumps hydroxide (OH(-)) ions from the extracellular to the cytoplasmic side.
在过去两年中，对细菌视紫红质的高分辨率X射线晶体学研究极大地推进了我们对这种光驱动离子泵的理解，并强调了离散的内部水分子在泵循环中的关键作用。在细胞外区域，由蛋白质残基和七个水分子构成的广泛三维氢键网络从埋藏的视黄醛席夫碱经由水402和初始质子受体天冬氨酸85延伸至膜表面。在视黄醛结合的赖氨酸216附近，跨膜螺旋G包含一个导致非脯氨酸扭结的π-凸起。该凸起通过丙氨酸215和赖氨酸216的主链羰基与位于席夫碱和细胞质区域中质子供体天冬氨酸96之间原本非常疏水区域的两个埋藏水分子形成氢键而得以稳定。被困在D96N突变体中的M中间体对应于转运循环中的晚期M状态，即天冬氨酸85质子化并向细胞外膜表面释放一个质子之后，但去质子化的视黄醛席夫碱重新质子化之前。来自E204Q突变体的M中间体对应于较早的M状态，因为在该突变体中席夫碱去质子化而不释放质子。这两种M状态的结构揭示了视黄醛光异构化为13-顺式、15-反式所诱导的视黄醛、主链和侧链的逐步位移，以及氢键连接的残基和结合水的三维网络的广泛重排，这解释了席夫碱、天冬氨酸85、质子释放基团和天冬氨酸96的pK(a)变化。此外，来自E204Q的M状态结构表明，扭曲的13-顺式、15-反式视黄醛的空间冲突的缓解在天冬氨酸96对席夫碱的重新质子化中起关键作用。现在另外两个水分子将天冬氨酸96与残基216的羰基相连，这似乎是一条氢键链的开始，该氢键链随后将延伸至视黄醛席夫碱。基于基态和M中间体结构，提出了光循环早期分子事件的模型，并提出了一个新模型，该模型认为细菌视紫红质将氢氧根（OH(-)）离子从细胞外泵向细胞质一侧。
