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估蛋白质的结晶性。我们还试图探索当蛋白质制剂表现出较差的结晶性时改变结晶性的实用策略。最后，我们将该策略应用于一些尚未结晶的蛋白质。我们做了所有我们试验的蛋白质的结晶。以下结果被存档。我们改变了病原体相关蛋白5d(PR-5d)的结晶性，并在1.8Å分辨率下测定了它的晶体结构。我们结晶了两个托皮酮还原酶，并用多种同象置换方法独立地求解了它们的三维结构。这些结果暗示了它们立体特异性反应的结构基础。我们还用定点突变的方法研究了它们的立体特异性。我们还成功地合成了其过渡态类似物抑制剂，并测定了与其络合的酶的晶体结构。我们从病原菌紫草中成功地合成了内切多聚半乳糖醛酸酶，从玉米中成功地合成了丙酮酸磷酸二激酶。内切多聚半乳糖醛酸酶的晶体经X-射线衍射仪测定其分辨率为0.95Å。通过将谷氨酰半胱氨酸合成酶表面的半胱氨酸残基转变为丝氨酸残基，我们成功地结晶了谷氨酰半胱氨酸合成酶。我们还合成了其过渡态类似物抑制剂。使用这些抑制剂进行的动力学分析表明，该酶的活性部位构型具有一定的结构动机。

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.