CRYSTALLOGRAPHIC STUDIES OF PROTEIN STRUCTURE/FUNCTION

这项提案的总体目标是使用蛋白质结晶学来 了解酶和酶的催化效率的结构基础 蛋白质动力学如何与蛋白质功能和热稳定性相关联。 酶催化是所有生化过程的中心，其 缺陷是大多数遗传性代谢性疾病的核心。详细 了解酶是如何工作的应该有助于更好地理解 代谢性疾病及其治疗的基础生物化学。在 这一总体研究计划有八个子项目。具体的 第一个子项目的目的是了解蛋白质的结构 磷酸丙糖催化效率的影响因素 异构酶。两种来源的复合酶的晶体结构 用底物和抑制剂与突变体的结构进行比较 由定点突变产生的酶。磷酸丙糖异构酶 是碳水化合物代谢的中心酶，它的不足 导致包括神经功能障碍在内的多系统紊乱， 溶血性贫血和心源性猝死的倾向。第二 该项目旨在使用结晶学和遗传选择的组合 要了解蛋白质动力学和催化之间的关系 热稳定性。目标酶2-异丙基苹果酸脱氢酶是 对亮氨酸的生物合成是必不可少的。项目3旨在开发 酶-底物复合体的低温结晶学方法 还可以绘制蛋白质的动态特性图，作为 温度。所有被研究的酶都有一个“玻璃化”转变。 远这种情况发生在零下55摄氏度左右，导致温度显著下降 蛋白质中的集体运动。在这一过渡之下，衬底 即使在流体介质中，结合和催化作用也会大大减弱。这个 转变将以低温蛋白质结晶学为特征 结合分子动力学模拟。第四个子项目 尝试应用蛋白质结晶学方法，包括 在子项目三中开发，以解开蛋白质之间的相互作用 以及细菌趋化性的构象变化。趋化作用 这项研究的重点是蛋白质，Chey是一个 在特定天冬氨酸上磷酸化的调节蛋白家族 蛋白质组氨酸激酶的酸性残留物。这个家庭的其他成员 囊中含假单胞菌藻酸盐生产调节剂 纤维化症。其余四个项目旨在确定结构和 丙氨酸消旋酶等四种酶的作用机制。

The overall objective of this proposal is to use protein crystallography to understand the structural basis for the catalytic efficiency of enzymes and how protein dynamics is coupled to protein function and thermostability. Enzymatic catalysis is central to all biochemical processes, and its defects are at the core of most inherited metabolic diseases. Detailed knowledge of how enzymes work should lead to better understanding of the biochemistry that underlies metabolic diseases and their treatment. Within this overall research program there are eight sub-projects. The specific aim of the first sub-project is to understand the protein structural features responsible for the catalytic efficiency of triosephosphate isomerase. Crystal structures of the enzyme from two sources complexed with substrates and inhibitors will be compared with structures of mutant enzymes generated by site-directed mutagenesis. Triosephosphate isomerase is a central enzyme in the metabolism of carbohydrates, and its deficiency leads to a multisystem disorder involving neurological disfunction, hemolytic anemia, and a propensity for sudden cardiac death. The second project aims to use a combination of crystallography and genetic selection to understand the relationship between protein dynamics and catalysis and thermostability. The target enzyme, 2-isopropyl malate dehydrogenase, is essential for the biosynthesis of leucine. Project 3 aims to develop methods for low-temperature crystallography of enzyme-substrate complexes, and also to map the dynamic properties of proteins as a function of temperature. There is a "glass" transition in all enzymes studied thus far that occurs about -55 degree C, leading to a marked reduction in the collective motions in the proteins. Below this transition, substrate binding and catalysis are greatly diminished, even in fluid media. The transition will be characterized by low-temperature protein crystallography combined with molecular dynamics simulations. The fourth sub-project attempts to apply protein crystallographic methods, including those developed in sub-project three, to unravel the protein-protein interactions and conformational changes in bacterial chemotaxis. The chemotactic protein that is the focus of much of this study, CheY, is a member of a family of regulatory proteins that are phosphorylated on specific aspartic acid residues by protein histidine kinases. Other members of this family include the regulator for alginate production by Pseudomonas in cystic fibrosis. The remaining four projects aim to determine the structures and mechanism of four enzymes, including alanine racemase.