Time-Resolved Crystallographic Studies of of Photoreceptor Membrane Proteins

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One major purpose of this research project is to develop time-resolved crystallographic techniques for inverstigation of reaction intermediates of bacteriorhodopsin, a light-driven proton pump found in halobacterium halobium. This membrane protein consists of 7 transmembrane α-helioes and contains retinai as the choromophone. Photoisomerization of retinal initiates a reaction cycle during which one proton is activery translocated across the membrane. For better understanding of its proton pumping mechanism, it is crucial to obtain structural information of the photoreaction intermediates. In this work, we developed a novel crystallization to obtain a high-quality three-dimensional crystal of bR.Briefly, purple membrane fragments (two-dimensional crystals of bR) were converted into uniformly-sized spherical vesicles and, subsequently, the resultant vesicles were fused to each other so as to form a crystal belonging to the space group P622 and diffracting X-rays up to 2.5 Å resolution. T … More he crystal is made up of stacked planar membranes, in each of bacteriorhodopsin trimers are arranged on a honey-comb lattice. Five native lipids per protein are identified in the crystal. One phospholipid is bound firmly to a crevice between adjacent monomers in the trimeric unit, and this lipid is suggested to play an important role the proton translocation during the photocycle of bR.To investigate light-induced conformational changes in the protein, we developed a flash-cooling technique by which the transphotocycle M intermediate was trapped efficiently. From structural comparison between the M intermediate and the ground state, it is shown that one of 7 transmembrane helices moves vertically during the photocycle. on the basis of this observation, we propose a piston model to explain the uni-directional transport of proton in bR.We also investigated the conformational change taking place just after the photoisomerization of retinal. For this purpose, the frozen crystal was irradiated to red or green light from a diode laser and structural difference between the ground state and the K intermediate was calculated from X-ray diffraction data collected under different illumination conditions. It is indicated that the conformational change in the primary reaction is restricted around the retinal Schiff base, which moves toward the extracelluar side.The crystallographic techniques developed for structural analysis of bacteriorhodopsin were applied to another photoreceptor membrane protein, light-harvesting chlorophyll-protein complex (LHC-II). We extracted LHC-II forms pea chloroplasts and crystallized it using nonylglucoside as detergent and potassium chloride as precipitant. Under similar conditions, two crystal from were obtained. One is an octahedral crystal belonging to the space group of P2_13 with cell dimensions of a=b=c=390Å. this crystal is made up of icosahedral of LHC-II with a diameter of 27 nm. the other crystal belongs to the space group P6_3 or P_322. The latter crystal diffracts X-rays up to 2.2 Å resolution and, therefore, we hope that further study will provide atomic coordinates of LHC-II.Our crystallization tachnique was applied to bovine rhodopsin, a protein with a 7-fold transmembrane α-helices. Briefly, the disc membrane of the photoreceptor cell was purified by density-gradient centrifugation and the purified membrane was treated with detergent (alkylglucoside) in the presence of a high concentration of divalent cation. A single step of centrifugation of the mixture yielded highly-purified sample of rhodopsin. Using this purified sample, one of the project member (Dr.Okada) obtained a high-quality 3D crystal. His work has enabled determination of the atomic coordinates of rhodopsin. Less