权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

Time-Resolved Crystallographic Studies of of Photoreceptor Membrane Proteins

光感受器膜蛋白的时间分辨晶体学研究

基本信息

批准号：
10680630
负责人：
KOUYAMA Tsutomu
金额：
$ 2.3万
依托单位：
Nagoya University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1998
资助国家：
日本
起止时间：
1998 至 1999
项目状态：
已结题

项目摘要

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

本研究计划的主要目的之一是发展时间分辨晶体学技术，用于研究盐生盐杆菌中发现的光驱动质子泵细菌视紫红质的反应中间体。这种膜蛋白由7个跨膜α-helioes组成，并含有视网膜作为色素。视黄醛的光异构化引发了一个反应循环，在此过程中，一个质子被激活并跨膜移位。为了更好地理解其质子泵机制，获得光反应中间体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，以获得高品质的三维晶体的bR。简而言之，紫膜碎片（二维晶体的bR）被转换成均匀大小的球形囊泡，随后，所得到的囊泡相互融合，以形成一个晶体属于空间群P622和衍射X射线高达2.5毫米分辨率。不 ...更多信息该晶体由堆叠的平面膜组成，在每个细菌视紫红质三聚体中排列在蜂窝晶格上。在晶体中鉴定了每种蛋白质五种天然脂质。一个磷脂被牢固地绑定到三聚体单元中相邻单体之间的缝隙，并且这种脂质被认为在bR的光循环过程中的质子易位中发挥重要作用。为了研究光诱导的蛋白质构象变化，我们开发了一种快速冷却技术，通过该技术，transphotocycle M中间体被有效地捕获。从M中间体和基态之间的结构比较，它表明，7个跨膜螺旋之一，在光循环过程中垂直移动。在此基础上，我们提出了一个活塞模型来解释质子在bR中的单向输运，并研究了retinal光异构化后的构象变化。为此，冷冻晶体被照射到红色或绿色光从二极管激光器和基态和K中间体之间的结构差异计算从X-射线衍射数据收集在不同的照明条件下。结果表明，初始反应中的构象变化主要集中在视网膜席夫碱附近，并向细胞外方向移动.将细菌视紫红质的晶体学结构分析技术应用于另一种光感受器膜蛋白--捕光叶绿素蛋白复合物（LHC-II）.我们从豌豆叶绿体中提取LHC-Ⅱ，用壬基葡萄糖苷作洗涤剂，氯化钾作沉淀剂进行结晶。在相似条件下，得到两种晶型。一种是八面体晶体，属于P2_13空间群，晶胞尺寸为a=B=c=390 μ m。该晶体由LHC-II的二十面体组成，直径为27 nm。另一个晶体属于空间群P6_3或P_322。后者晶体衍射X射线的分辨率高达2.2 μ m，因此，我们希望进一步的研究将提供LHC-II的原子坐标。我们的结晶技术被应用于牛视紫红质，一种具有7倍跨膜α-螺旋的蛋白质。简而言之，通过密度梯度离心纯化感光细胞的盘膜，并在高浓度二价阳离子的存在下用去污剂（烷基葡糖苷）处理纯化的膜。对混合物进行一步离心即可获得高度纯化的视紫红质样品。利用这种纯化的样品，项目成员之一（冈田博士）获得了高质量的3D晶体。他的工作使得能够确定视紫红质的原子坐标。少