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

在这项研究中，我们应用动态光散射分析来评价蛋白质的结晶性。我们也试图探索实际的策略，以改变结晶性时，蛋白质制剂表现出较差的结晶性。最后，我们将该策略应用于一些尚未结晶的蛋白质。我们把所有的蛋白质都结晶了。存档了以下结果。我们改变了病原体相关蛋白5d （PR-5d）的结晶性，并在1.8Å分辨率下测定了其晶体结构。我们结晶了两种托品酮还原酶，并通过多种同构置换方法独立求解了它们的三维结构。结果暗示了其立体特异反应的结构基础。我们还通过定点诱变研究了它们的立体特异性。我们还成功地合成了它的过渡态类似抑制剂，并测定了与该抑制剂配合的酶的晶体结构。我们成功地从一种病原菌中结晶出了半乳糖醛酸内聚酶，从紫发菌中结晶出了紫发菌，从玉米中结晶出了磷酸丙酮酸二激酶。内聚半乳糖醛酸酶晶体的x射线衍射到0.95Å分辨率。我们通过将g-谷氨酰半胱氨酸合成酶表面的半胱氨酸残基转化为丝氨酸残基，成功地结晶了它。我们还合成了它的过渡态类似抑制剂。利用抑制剂的动力学分析表明了该酶活性位点结构的一些结构动机。

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.