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

蛋白质结晶是蛋白质三级结构晶体学分析中的一个瓶颈。特别是膜蛋白很难结晶。在这个研究项目中，我们挑战了许多膜蛋白的结晶，旨在建立膜蛋白三维结晶的一般规律。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（牛心脏）、光合反应中心（一种红藻）、钙++结合膜蛋白、鞭毛马达Mling蛋白（沙门氏菌）、H+- atp酶（一种嗜热细菌PS3）。肌动蛋白和recA蛋白，已知自组装形成丝状聚集体，也进行了研究。制备高质量膜蛋白或疏水蛋白晶体的困难主要来自于它们在溶解状态或单体状态下的不稳定性。更多地了解稳定膜蛋白三级结构的物理力量；例如，在洗涤剂和化学品（如酒精）存在的情况下，研究了蛋白质变性和蛋白质-蛋白质相互作用。项目团队由长期从事膜蛋白研究的年轻生物物理学家组成。但只有少数成员熟悉蛋白质晶体学。因此，为了相互启发和鼓励，我们举行了几次公开会议，讨论了蛋白质结晶试验中遇到的困难。我们很惊讶地发现很多人参加了会议，因为我们一直认为蛋白质晶体学在日本不是很活跃。现在我们了解到，大量的研究人员对蛋白质结晶感兴趣。我们相信，未来日本的蛋白质晶体学将变得非常活跃。在结晶技术的讨论中，提出了以下几点：1)当蛋白质的溶解度与温度显著相关时，通过缓慢地降低或增加温度可以生长出高质量的晶体；2)两种洗涤剂可以混合使用，当其中一种太强而不能保持蛋白质结构，而另一种太弱而不能溶解膜；在温度梯度存在的情况下，蒸汽扩散允许蛋白质局部浓缩，从而在有限的区域结晶。此外，还讨论了影响蛋白质结晶的其他因素，如重力、蛋白质纯度、蛋白质的自氧化等。结晶过程的视频记录被证明是了解蛋白质-蛋白质结合机制的有力手段。少

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