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Development of Crystallization Techniques for Membrane Proteins : on the Bese of Understanding of Protein-Structure Determining Forces

膜蛋白结晶技术的发展:基于对蛋白质结构决定力的理解

基本信息

批准号：
01304061
负责人：
KOUYAMA Tsutomu
金额：
$ 6.91万
依托单位：
The Institute of Physical and Chemical Research
依托单位国家：
日本
项目类别：
Grant-in-Aid for Co-operative Research (A)
财政年份：
1989
资助国家：
日本
起止时间：
1989 至 1990
项目状态：
已结题

项目摘要

Protein crystallization is a bottle-necked process in crystallographic analyses of the tertiary structure of proteins. Especially membrane proteins are very difficult to crystallize. In this research project, we challenged to crystallize many membrane proteins, aiming at establishing general rules for the three-dimensional crystallization of membrane proteins. The membrane proteins that we examined are : bacteriorhodopsin, rhodopsin (bovine and octopus), receptor proteins of neurotransmitters, cytochrome c oxidase (bovine heart), photosynthetic reaction center (a red alga), Ca++-binding membrane proteins, the Mling protein of flagellar motors (Salmonella), H+-ATPase (a thermophilic bacterium PS3). Actin and recA protein, which are known to self-assemble to form filamentary aggregates, were also investigated. Difficulty in making high-quality crystals of membrane proteins or hydrophobic pfoteins comes primarily from their instability in solubilized or monomeric states. Thus we tried to … More understand the physical forces stabilizing the tertiary structure of membrane proteins ; e. g., protein denaturation and protein-protein interaction were investigated in the presence of man of detergents and chemicals (e. g., alcohols).The project team consisted of young biophysicists who had been long working on membrane proteins. But only a few members had been familiar to the protein crystallography. In order to enlighten and encourage each other, therefore, we hold several public meetings and discussed the difficulties that were encountered in the trials of protein crystallization. We were surprised to find a large audience in the meetings, because we had thought that the protein crystallography was not very active in Japan. Now we understand that a large number of researchers are interested to protein crystallization. We are convinced that the protein crystallography in Japan will become very active in future.In the discussion about crystallization techniques, the followings were proposed to be prominent : 1) A high-quality crystal can be grown by decreasing or increasing temperature very slowly, when the protein solubility is significantly temperature-dependent ; 2) Two kinds of detergent can be used in combination, when one of them is too strong to keep the protein structure while the other is too mild to solubilize membranes ; 3) Vapor diffusion in the presence of a temperature gradient allows proteins to be concentrated locally and thus to be crystallized in a restricted region. Also, many other factors that influence protein crystallization, i. e., the gravity, the purity of protein, the self-oxidation of protein, were discussed. Video-recording of crystallization processes was proven to be powerful in understanding the mechanism of protein-protein association. Less

蛋白质结晶是蛋白质三级结构的晶体学分析中的瓶颈过程。特别是膜蛋白质非常难以结晶。在本研究项目中，我们对许多膜蛋白进行了结晶，旨在建立膜蛋白三维结晶的一般规则。我们检测的膜蛋白有：细菌视紫红质，视紫红质（牛和章鱼），神经递质受体蛋白，细胞色素c氧化酶（牛心），光合反应中心（红色），Ca++-结合膜蛋白，鞭毛马达的Mling蛋白（沙门氏菌），H+-ATP酶（嗜热细菌PS3）。肌动蛋白和recA蛋白，这是已知的自组装，以形成粘附性聚集体，也进行了研究。制备膜蛋白或疏水性pfotein的高质量晶体的困难主要来自它们在溶解或单体状态下的不稳定性。因此，我们试图 ...更多信息理解稳定膜蛋白三级结构的物理力; e.例如，在一个实施例中，研究了蛋白质变性和蛋白质-蛋白质相互作用在多种洗涤剂和化学品（例如，例如，在一个实施例中，该项目小组由长期从事膜蛋白研究的年轻生物药理学家组成。但只有少数成员熟悉蛋白质晶体学。因此，为了互相启发和鼓励，我们举行了几次公开会议，讨论蛋白质结晶试验中遇到的困难。我们惊讶地发现会议中有很多观众，因为我们认为蛋白质晶体学在日本不是很活跃。目前，蛋白质结晶已经引起了众多研究者的兴趣。在对结晶技术的讨论中，我们认为：（1）当蛋白质的溶解度与温度有明显的相关性时，通过缓慢的降温或升温可以生长出高质量的晶体; 2）当两种去污剂中的一种太强而不能保持蛋白质结构而另一种太温和而不能溶解膜时，可以组合使用两种去污剂; 3）在存在温度梯度的情况下的蒸气扩散允许蛋白质局部浓缩并因此在受限区域中结晶。此外，还研究了影响蛋白质结晶的其他因素，如蛋白质的结晶度、蛋白质的结晶度、蛋白质的结晶度等.例如，对蛋白质的比重、纯度、自氧化等进行了讨论。结晶过程的视频记录被证明是强大的理解蛋白质-蛋白质缔合的机制。少