CRYSTALLOGRAPHIC STUDIES OF PROTEIN STRUCTURE/FUNCTION

这项提案的总体目标是利用蛋白质晶体学， 了解酶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结合。 酶催化是所有生物化学过程的核心，其 缺陷是大多数遗传性代谢疾病的核心。 详细 酶如何工作的知识应导致更好地了解 代谢性疾病及其治疗的基础生物化学。 内 这一总体研究计划有八个子项目。 具体 第一个子项目的目的是了解蛋白质结构 负责磷酸丙糖催化效率的特征 异构酶 来自两种来源的酶的晶体结构复合 将与突变体的结构进行比较 通过定点诱变产生的酶。 磷酸丙糖异构酶 是碳水化合物代谢的中心酶，其缺乏 导致多系统紊乱包括神经功能障碍， 溶血性贫血和心源性猝死倾向 第二 该项目旨在将晶体学和遗传选择相结合， 了解蛋白质动力学和催化作用之间的关系， 热稳定性 目标酶，2-异丙基苹果酸脱氢酶， 对亮氨酸的生物合成至关重要。 项目3旨在开发 酶-底物复合物的低温晶体学方法， 也可以绘制蛋白质的动态特性， 温度 在所研究的所有酶中都有一个“玻璃化”转变 到目前为止，发生约-55摄氏度，导致显着减少， 蛋白质的集体运动。 在这种过渡下，衬底 即使在流体介质中，粘合和催化作用也大大减弱。 的 转变将通过低温蛋白质晶体学来表征 结合分子动力学模拟。 第四个分项目 尝试应用蛋白质晶体学方法，包括那些 在子项目三中开发，以解开蛋白质-蛋白质相互作用 和细菌趋化性的构象变化。 趋化 蛋白质是这项研究的重点，CheY，是一个成员， 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基。 这一家族的其它成员 包括胞囊假单胞菌产生藻酸盐的调节剂， 纤维化 其余四个项目旨在确定结构和 包括丙氨酸消旋酶在内的四种酶的作用机制。

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.