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

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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