STRUCTURE & DYNAMICS OF METALLO BETA LACTAMASE

结构

• 批准号: 6410436
• 负责人: HELEN JANE DYSON
• 金额: $ 14.53万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-01-01 至 2001-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6410436
• 关键词: Bacteroides; X ray crystallography; active sites; affinity chromatography; bacterial proteins; beta lactamase; conformation; drug resistance; enzyme inhibitors; enzyme mechanism; enzyme structure; ligands; metalloenzyme; molecular dynamics; nuclear magnetic resonance spectroscopy; protein purification; site directed mutagenesis

Project 2 is concerned with the elucidation of the structure and dynamics of a metallo-beta-lactamase from a clinically important anaerobic organism, Bacteroides fragilis. The goals of the project are two-fold, to obtain information on a molecule that is a key component of antibiotic resistance in the organism, and to utilize this system as one of the paradigms to investigate coupling between polypeptide chain dynamics and enzymatic function. Since NMR is the premier method for obtaining site- specific information on polypeptide dynamics in solution, the focus of the project is a extensive NMR study of the B. fragilis metallo-beta- lactamase, the wild type enzyme free and in complex with an inhibitor and mutant proteins specifically designed on the basis of the kinetic, structural and dynamic information available on the wild-type protein. Complete resonance assignments will be made for the active form of the enzyme, which contains two zinc ions. Preliminary data are excellent, and indicate that such assignments will be possible using a combination of Calpha and CO-based strategies. Complete resonance assignments will also be made for a complex of the enzyme with a substrate-analog, and a solution structure determination will be made for this complex. Polypeptide dynamics will be investigated by NMR for both the free enzyme an the complex, using 15N, 13C and 2H relaxation measurements. The study will provide the basis for mutagenesis of the metallo-beta-lactamase (Project 3) and for the elucidation of the reaction pathway (Project 4). The fourth specific aim concerns the study of local structure in the active site of the enzyme, using cadmium-substituted metallo-beta- lactamase. The identity and bonding of the ligands and the solvent exposure of the metal site can be obtained from these measurements, together with an estimate of the dissociation constants of the metal ions. The final specific aim will follow from the interactions between the components of the Program Project. Resonance assignments and relaxation measurements will be made for mutants designed on the basis of accumulated information form wild-type dynamics (Project 2), kinetic studies (Project 3) and calculations (Project 4). Structures will be determined for selected mutants either in solution by NMR or by X-ray crystallography by a collaborator, Dr. Osnat Herzberg. Information obtained from the synergistic interaction between the components of the Program Project should provide important insights into the role of dynamics in enzyme action.

项目2涉及阐明结构和 来自临床上重要的厌氧的金属β-内酰胺酶的动力学 生物体，菌丝的脆弱。该项目的目标是两个方面的 获取有关抗生素关键成分的分子的信息 有机体的抗性，并利用该系统作为其中之一 研究多肽链动力学和 酶促功能。由于NMR是获得站点的主要方法 有关溶液中多肽动力学的特定信息， 项目是一项广泛的NMR研究，对脆弱的金属链球链球菌 - β- 乳糖酶，无野生酶无酶，并与抑制剂和复杂 突变蛋白专为基础设计而设计 有关野生型蛋白的结构和动态信息。 将针对主动形式进行完整的共振分配 酶，其中包含两个锌离子。初步数据非常好，并且 表明将这种作业结合在一起 Calpha和基于CO的策略。完整的共振分配也将 用底物 - 分析制成酶的复合物，A 该复合物将确定解决方案结构。 NMR将研究两种游离酶的多肽动力学 一个复合物，使用15N，13C和2H弛豫测量。研究 将为金属β-内酰胺酶的诱变提供基础 （项目3）和阐明反应途径（项目4）。 第四个特定目的涉及研究本地结构 酶的活性位点，使用镉取代的金属β- 乳糖酶。配体和溶剂的身份和键合 可以从这些测量值中获得金属位点的暴露， 以及金属离子解离常数的估计。 最终的具体目的将来自 程序项目的组成部分。共振分配和放松 将对根据累积设计的突变体进行测量 信息形成野生型动力学（项目2），动力学研究（项目 3）和计算（项目4）。将确定结构 通过NMR或通过X射线晶体学在溶液中选择突变体 合作者Osnat Herzberg博士。从 程序项目组件之间的协同互动 应该为动态在酶中的作用提供重要的见解 行动。