Crystallographic structure of the retinal and the protein after deprotonation of the schiff base: the switch in the bacteriorhodopsin photocycle.

摘要

We illuminated bacteriorhodopsin crystals at 210K to produce, in a photostationary state with 60% occupancy, the earliest M intermediate (M(1)) of the photocycle. The crystal structure of this state was then determined from X-ray diffraction to 1.43A resolution. When the refined model is placed after the recently determined structure for the K intermediate but before the reported structures for two later M states, a sequence of structural changes becomes evident in which movements of protein atoms and bound water are coordinated with relaxation of the initially strained photoisomerized 13-cis,15-anti retinal. In the K state only retinal atoms are displaced, but in M(1) water 402 moves also, nearly 1A away from the unprotonated retinal Schiff base nitrogen. This breaks the hydrogen bond that bridges them, and initiates rearrangements of the hydrogen-bonded network of the extracellular region that develop more fully in the intermediates that follow. In the M(1) to M(2) transition, relaxation of the C(14)-C(15) and C(15)z.dbnd;NZ torsion angles to near 180 degrees reorients the retinylidene nitrogen atom from the extracellular to the cytoplasmic direction, water 402 becomes undetectable, and the side-chain of Arg82 is displaced strongly toward Glu194 and Glu204. Finally, in the M(2) to M(2)' transition, correlated with release of a proton to the extracellular surface, the retinal assumes a virtually fully relaxed bent shape, and the 13-methyl group thrusts against the indole ring of Trp182 which tilts in the cytoplasmic direction. Comparison of the structures of M(1) and M(2) reveals the principal switch in the photocycle: the change of the angle of the C(15)z.dbnd;NZ-CE plane breaks the connection of the unprotonated Schiff base to the extracellular side and establishes its connection to the cytoplasmic side.