Development of practical strategies to support protein crystallization for X-ray crystallography

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In this study, we have applied dynamic light scattering analysis to evaluate crystallizability of proteins. We also tried to explore practical strategy to alter the crystallizability when a protein preparation showed poor crystallizability. Finaly, we applied the strategy to some proteins that have not been crystallized yet. We did to crystallize all proteins that we tried.The following results were archived.1. We altered the crystallizability of pathogen related protein 5d (PR-5d) and determined its crystal structure at 1.8Å resolution.2. We crystallized two tropinone reductases and solved their three-dimensional structures by multiple isomorphous replacement methods independently. The results implicated the structural basis for their stereospecific reaction. We also investigated their stereospecificity by site-directed mutagenesis.3. We also succeed to synthesize its transition-state analogue inhibitor and the crystal structure of the enzyme complexed with the inhibitor was determined.4. We succeed to crystallize endopolygalacturonase from a pathogenic fungus, Stereum purpureum and pyruvate phosphate dikinase from maiz. The crystals of the endopolygalacturonase diffracted X-ray to 0.95Å resolution.5. We succeed to crystallize g-glutamylcysteine synthetase by alteration of its surface cysteine residues into serine residues. We also synthesized its transition state analogue inhibitors. Kinetic analysis using the inhibitors suggested some structural motives of the active site architecture of this enzyme.