Time-Resolved Crystallographic Studies of of Photoreceptor Membrane Proteins

这项研究项目的一个主要目的是开发时间分辨结晶学技术，用于研究细菌视紫红质的反应中间产物，细菌视紫红质是盐生细菌中发现的一种光驱动质子泵。该膜蛋白由7个跨膜α-Heli组成，含有视黄醇作为色调。视网膜的光异构化启动了一个反应周期，在这个反应周期中，一个质子被非常活跃地转移到膜上。为了更好地理解其质子泵浦机理，获取光反应中间产物的结构信息是至关重要的。在这项工作中，我们开发了一种新的结晶方法来获得高质量的BR三维晶体。简单地说，紫膜碎片(BR的二维晶体)被转化为大小均匀的球形小泡，然后将生成的小泡相互融合，形成属于P622空间群的晶体，并对X射线进行高达2.5Å分辨率的衍射。T…更多的He晶体是由堆叠的平面膜组成，每个细菌视紫红质三聚体排列在蜂窝状的晶格上。晶体中每种蛋白质含有五种天然脂类。在三聚体单元中，一个磷脂牢固地结合在相邻单体之间的缝隙中，这种磷脂被认为在BR的光循环中起着重要的质子转运作用。为了研究光诱导蛋白质构象的变化，我们开发了一种闪冷技术，通过这种技术有效地捕获了跨光环M中间体。M中间态和基态的结构比较表明，在光循环过程中，7个跨膜螺旋中有1个垂直运动。在此基础上，我们提出了一个活塞模型来解释BR中质子的单向传输，并研究了视网膜光异构化后发生的构象变化。为此，冻结晶体被半导体激光器的红光或绿光照射，并根据在不同光照条件下收集的X射线衍射数据计算基态和K中间体之间的结构差异。结果表明，初级反应的构象变化局限于视网膜Schiff碱基周围，该碱基向细胞外侧移动。用于细菌视紫红质结构分析的结晶学技术被应用于另一种光感受器膜蛋白-捕光叶绿素-蛋白复合体(LHC-II)。我们从豌豆叶绿体中提取了LHC-II，并以壬基葡萄糖苷为洗涤剂、氯化钾为沉淀剂进行了结晶。在相似的条件下，得到了两种晶型。一种是八面体晶体，属于P2_13空间群，晶胞尺寸为a=b=c=390Å。该晶体由直径为27 nm的LHC-II二十面体组成。另一种晶体属于P6_3或P_322空间群。后者的晶体对X射线的衍射分辨率高达2.2LHC，因此，我们希望进一步的研究将提供LHC-II的原子坐标。我们的结晶技术应用于牛视紫质，一种具有7倍跨膜α-螺旋的蛋白质。简单地说，用密度梯度离心法纯化光感受器细胞的盘膜，并在高浓度二价阳离子存在的情况下用洗涤剂(烷基葡萄糖苷)处理纯化的膜。混合物只需一步离心法即可得到高纯度的视紫红质样品。使用这个纯化的样本，项目成员之一(冈田博士)获得了高质量的3D晶体。他的工作使测定视紫红质的原子坐标成为可能。较少

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