STRUCTURE & DYNAMICS OF METALLO BETA LACTAMASE

结构

• 批准号: 6301782
• 负责人: HELEN JANE DYSON
• 金额: $ 14.77万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-01-01 至 2000-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6301782
• 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涉及结构的阐明， 临床上重要的厌氧细菌中金属β-内酰胺酶的动力学 脆弱拟杆菌Bacteroides fragilis该项目的目标是双重的， 获取抗生素关键成分分子的信息 生物体中的抗性，并利用该系统作为 研究多肽链动力学和 酶的功能由于核磁共振是获得位置的首要方法- 关于溶液中多肽动力学的具体信息， 项目是对B进行广泛的核磁共振研究。脆性金属β- 内酰胺酶，野生型酶游离的和与抑制剂复合的， 基于动力学特别设计的突变蛋白， 野生型蛋白质的结构和动力学信息。 完整的共振分配将作出积极的形式， 酶，含有两个锌离子。初步数据非常好， 表明这种分配将可能使用以下组合 Calpha和基于CO的策略。完整的共振分配也将 酶与底物类似物的复合物，以及 将对该络合物进行溶液结构测定。 多肽动力学将通过NMR研究两种游离酶 用~（15）N、~（13）C和~ 2 H弛豫测量了配合物的结构。研究 将为金属-β-内酰胺酶的诱变提供基础 （项目3）和用于阐明反应途径（项目4）。 第四个具体目标是研究 酶的活性位点，使用镉取代的金属-β- 内酰胺酶配体和溶剂的身份和键合 金属位点的暴露可以从这些测量中获得， 以及金属离子的离解常数的估计值。 最后的具体目标将来自于 计划项目的组成部分。共振分配和弛豫 将对基于累积的 野生型动态（项目2）、动力学研究（项目3） 3)计算（项目4）。结构将确定为 通过NMR或X射线晶体学在溶液中选择突变体， 一位合作者，奥斯纳特·赫兹伯格博士所用信息均来自 计划项目各组成部分之间的协同作用 应该提供重要的洞察力的作用，动力学在酶 行动上