PROTEIN BINDING SITE STRUCTURE BY SOLID-STATE NMR

通过固态 NMR 分析蛋白质结合位点结构

• 批准号: 2727331
• 负责人: MATTHEW P. ESPE
• 金额: $ 10.95万
• 依托单位: UNIVERSITY OF AKRON
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-09-30 至 2003-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2727331
• 关键词: Escherichia coli; X ray crystallography; active sites; ammonia lyase; binding sites; bioimaging /biomedical imaging; enzyme structure; glutamates; lyophilization; nuclear magnetic resonance spectroscopy; protein binding; protein protein interaction; protein sequence; structural biology

DESCRIPTION (Adapted from abstract): The goal of this work is to understand the allosteric effects at the molecular level on the activity of the enzyme, L-aspartase, which catalyzes the reversible deamination of L-aspartic acid to form fumarate. L-aspartase is a member of a family of enzymes that have fumarate as a substrate. The enzyme contains an allosteric activator site, which binds the substrate, L-aspartic acid, as well as a series of analogs. The enzyme has the interesting property that the presence of an activator is only required at basic pH. At low pH the activator site appears decoupled from catalytic operation. The location and structure of the activator site is unknown and the mechanism by which an activator affects the enzyme activity is not understood. The specific aims of the work are: to identify the activator binding site by determining the amino acids that interact with the activator, determine the orientation of the activator in the binding site, and measure the distance between the activator and the active sites. The primary technique to be used is magic-angle-spinning solid-state NMR. Inter-nuclear distances will be measured using recently developed dipolar recoupling techniques of REDOR, DRAMA, and SEDRA. Distances will be measured between at the activator binding site between the enzyme amino acids and the carbon, nitrogen, or phosphorous of several activators. In addition to measuring distance, the NMR results will also detail the type of amino acids that form the activator binding site. By using this information, along with the activator-protein distances, a model for the binding site will be constructed. By measuring the distances between a bound substrate analog and a bound activator, the inter-site distance will be determined. With knowledge of the location and structure of the activator binding site the structural changes that occur in the enzyme upon activator binding will be investigated and the role of the activator understood.

描述(改编自摘要)：这项工作的目标是理解 分子水平上的变构效应对酶活性的影响， L-天冬氨酸酶，催化L-天冬氨酸的可逆脱氨反应 形成富马酸。L-天冬氨酸酶是一种酶家族的成员 富马酸作为底物。该酶含有变构激活剂部位， 它与底物L-天冬氨酸以及一系列类似物结合。这个 酶具有一个有趣的性质，即活化剂的存在只是 在碱性pH值时需要。在低pH时，激活剂位置似乎与 催化操作。激活部位的位置和结构如下 未知，激活剂影响酶活性的机制是 我不明白。这项工作的具体目标是：确定激活者 通过确定与激活剂相互作用的氨基酸， 确定激活剂在结合部位的取向，并测量 激活剂和活性部位之间的距离。实现以下目标的主要技术 使用的是魔角旋转固态核磁共振。核间距离将 使用最近开发的Redor的偶极重新耦合技术进行测量， 戏剧，还有塞德拉。将在激活剂结合处测量距离 位于酶的氨基酸和碳、氮或磷之间的位置 几个激活剂。除了测量距离外，核磁共振结果还将 还详细说明了构成激活剂结合部位的氨基酸类型。通过 利用这一信息以及激活剂-蛋白质距离，建立了一个模型 将构建结合位点。通过测量两个对象之间的距离 结合底物模拟物和结合激活剂，位点间距离将为 下定决心。了解激活剂的位置和结构 结合部位激活剂上的酶发生的结构变化 将对结合进行调查，并了解激活剂的作用。