STRUCTURE & DYNAMICS OF METALLO BETA LACTAMASE

项目2涉及对结构的阐明和 临床重要厌氧菌金属β-内酰胺酶的动态研究 有机体，脆弱类杆菌。该项目的目标有两个，即 获取作为抗生素关键成分的分子的信息 在有机体中的抗药性，并利用这个系统作为 多肽链动力学和多肽链动力学偶联的研究范式 酶功能。由于核磁共振是获得地点的首要方法- 关于溶液中多肽动力学的特定信息，重点是 项目是对脆弱杆菌金属-贝塔的广泛的核磁共振研究- 内酰胺酶，不含野生型酶，并与一种抑制剂和 突变蛋白质是在动力学的基础上设计的， 关于野生型蛋白质的结构和动态信息。 完整的共鸣作业将为主动形式的 含有两个锌离子的酶。初步数据很好，而且 指示可以使用以下组合进行此类赋值 Calpha和基于CO的策略。完整的共振作业也将 被制成酶与底物类似物的络合物，以及 将对该络合物进行溶液结构的确定。 核磁共振将研究这两种游离酶的多肽动力学 用~(15)N、~(13)C和~(2H)H弛豫测量。这项研究 为金属β-内酰胺酶的诱变奠定了基础 (项目3)和阐明反应途径(项目4)。 第四个具体目标涉及到对当地结构的研究 酶的活性部位，用镉取代的金属-β- 内酰胺酶。配体和溶剂的同一性和成键 金属部位的曝光可以从这些测量中获得， 并估算了金属离子的离解常数。 最终的具体目标将来自于 计划项目的组成部分。共鸣赋值与松弛 将根据累积的变种设计进行测量 来自野生型动力学的信息(项目2)、动力学研究(项目 3)和计算(项目4)。将为以下项目确定结构 通过核磁共振或X射线结晶学方法在溶液中选择突变体 合作者奥斯纳特·赫兹伯格博士。从以下网站获得的信息 计划项目各组成部分之间的协同作用 应该为酶的动力学作用提供重要的见解 行动。

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.