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Research and Development for Purification of Membrane Proteins

膜蛋白纯化的研究与开发

基本信息

批准号：
07458176
负责人：
KOUYAMA Tsutomu
金额：
$ 4.54万
依托单位：
Nagoya University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1995
资助国家：
日本
起止时间：
1995 至 1996
项目状态：
已结题

项目摘要

Crystallization of membrane proteins requires hard work, primarily because of difficulty in preparation of a stable, highly-purified and concentrated protein sample. In order to simplify the purification step of membrane proteins, we have tried to develop a novel procedure for selective isolation of a membrane protein by inducing self-association in a biological membrane and subsequent membrane vesicularization.Bacteriorhodopsin, a transmembrane protein found in halobacterium halobium, forms a two-dimensional crystal (called purple mebrane) under physiological condition. When purple membrane was incubated at high temperature with a small amount of detergent (octylthioglucoside) in the presence of a high concentration of precipitant, uniformly-sized spherical vesicles (polyhedral assembly) of bacteriorhodopsin was produced. The stability of the polyhedral assembly decreased at low temperature. By promoting fusion processes of the polyhedral assembly at low temperature, we obtained a new … More three-dimensional that diffracts X-ray diffract beyond 3.0 angstrom. This crystal belongs to the space group P622 with cell dimensions of a=b=104.7*, and c=114.1*, and it is shown to be made up of stacked planar membranes, in each of bacteriorhodopin trimers are arranged on a honey-comb lattice. The crystal contains native lipids (5 phospholipids per bR) and one phospholipid is bound firmly to a crevice between adjacent monomers in the trimeric unit. This lipid is suggested to act as a glue for formation of the trimeric structure.Light-harvesting chlorophyll-protein complex from pea is shown to form a polyhedral structure with a diameter of 27 nm under crystallization condition. It is assembled into an octahedral crystal that belongs to the space group of P2_13 with cell dimensions of a=b=c=390*.Another purification procedure was developed for bovine rhodopsin, a protein with a 7-fold transmembrane alpha-helices. The disk membrane of the photoreceptor cell was purified by a 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 a highly-purified sample of rhodopsin. Using this purified sample, we obtained 3D crystals of rhodopsin by the vapor diffusion hanging drop method. Less

膜蛋白的结晶需要艰苦的工作，主要是因为难以制备稳定、高度纯化和浓缩的蛋白质样品。为了简化膜蛋白的纯化步骤，我们尝试开发一种通过诱导生物膜自结合和随后的膜囊泡化来选择性分离膜蛋白的新方法。细菌视紫红质是在盐杆菌中发现的跨膜蛋白，在生理条件下形成二维晶体（称为紫色膜）。当紫膜在高温下与少量去污剂（辛基硫代葡萄糖苷）在高浓度的沉淀剂存在下孵育时，产生均匀大小的细菌视紫红质球形囊泡（多面体组装体）。在低温下，多面体组装体的稳定性下降。通过促进低温下多面体组装的熔合过程，我们得到了一种新的 ...更多信息三维的，能衍射超过3.0埃的X射线衍射。该晶体属于空间群P622，晶胞尺寸为a=B=104.7*，c=114.1*，并且显示出由堆叠的平面膜组成，其中每个细菌视紫红质三聚体排列在蜂窝晶格上。晶体含有天然脂质（每bR 5个磷脂），一个磷脂牢固地结合到三聚体单元中相邻单体之间的缝隙中。豌豆捕光叶绿素蛋白复合物在结晶条件下形成直径为27 nm的多面体结构。它组装成一个八面体晶体，属于空间群P2_13，晶胞尺寸为a=B=c=390*。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存在下用去污剂（烷基葡糖苷）处理纯化的膜。对混合物进行离心的单个步骤产生了高度纯化的视紫红质样品。利用此纯化样品，我们通过气相扩散悬滴法获得了视紫红质的三维晶体。少