STRUCTURE AND DYNAMICS OF METAL CONTAINING PROTEINS

含金属蛋白质的结构和动力学

• 批准号: 2182418
• 负责人: Thomas Charles Pochapsky
• 金额: $ 15.79万
• 依托单位: BRANDEIS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1990
• 资助国家: 美国
• 起止时间: 1990-04-01 至 1999-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2182418
• 关键词: Pseudomonas; active sites; bacterial proteins; chemical binding; crystallization; cytochrome P450; electron transport; ferredoxin; intermolecular interaction; molecular dynamics; molecular site; nuclear magnetic resonance spectroscopy; protein structure; site directed mutagenesis

This project represents a continuing effort to determine the role of protein structure in protein-protein interaction and electron transfer in the P-450cam system from Pseudomonas putida. The P-450cam system consists of three soluble proteins, cytochrome P-450 cam, putidaredoxin (Pdx), a 2Fe-2S ferredoxin, and the flavin-containing NADH-dependent putidaredoxin reductase (PdR). The cytochrome catalyzed the 5-exo-hydroxylation of camphor by molecular oxygen, a reaction which requires two electrons, which are supplied to the cytochrome sequentially by Pdx, which is also required as an effector substrate turnover. The electrons are supplied to Pdx by PdR by oxidation of PdR-bound NADH. The camphor hydroxylase system is a good model for human P-450 enzymes involved in steroid hormone biosynthesis and processing of xenobiotics and carcinogens, which can result in carcinogen activation. The P. putida system has been studied in detail because it is easy to purify, all the components are soluble in their active forms, and the genes have been cloned for all components. This project aims to refine the NMR-derived solution structure of Pdx by obtaining more NOE and dihedral angle restraints using multidimensional 13C, 15N-edited NMR methods. The current structure was determined using 1H NMR methods, from which a limited number of unambiguous NOE restraints were obtained. The paramagnetism of the 2Fe- 2S cluster is another impediment to structure refinement, and selective labeling schemes and diamagnetic metal center substitutions are proposed to overcome this problem. Several bound waters are suspected in the structure, and are expected on the surface of Pdx, and pulsed-field- gradient NMR methods will be employed to identify these. Redox-dependent structural changes have been identified in Pdx, and the dynamics of two accessible oxidation states will be examined by amide proton exchange measurements. Based on the known structures of Pdx and p-450cam, models for the diprotein complex will be proposed and tested using heteronuclear- edited NMR methods. Crystallization trials for pdx, PdR and their complexes with each other and with P-450cam are in progress for determination of crystal structures. Finally, the mechanism of metal center incorporation into Pdx will be investigated by a variety of physico-chemical methods.

该项目代表了确定角色的持续努力 蛋白质 - 蛋白质相互作用和电子转移的蛋白质结构 Pseudomonas Putida的P-450CAM系统。 P-450CAM系统组成 三种可溶性蛋白，细胞色素P-450 CAM，putidaredoxin（PDX），A 2FE-2S铁氧还蛋白和含黄素的NADH依赖性putidaredoxin 还原酶（PDR）。 细胞色素催化了5-二羟基化的 通过分子氧樟脑，一种需要两个电子的反应，该反应 由PDX顺序依次提供给细胞色素，这也需要 作为效应子基板的营业额。 电子通过 PDR通过氧化PDR结合的NADH。 樟脑羟化酶系统是人类P-450酶的良好模型 参与类固醇激素的生物合成和异种生物的加工和 致癌物，这可能导致致癌物激活。 P. putida 系统已被详细研究，因为它易于净化，所有 组件以其活性形式可溶，基因已经 克隆所有组件。 该项目旨在完善NMR衍生的 PDX的溶液结构通过获得更多NOE和二面角 使用多维13C，15N编辑的NMR方法的约束。 电流 使用1H NMR方法确定结构，从中数量有限 获得了明确的NOE约束。 2fe-的promagnetism- 2S簇是结构改进和选择性的另一个障碍 提出了标签方案和DiamaMagnetic Metal Center取代 克服这个问题。 怀疑有几个被绑定的水 结构，预期在PDX表面，脉冲场 - 将采用梯度NMR方法来识别这些方法。 氧化还原依赖性 在PDX中已经确定了结构变化，并且两个动力学 可访问的氧化态将通过酰胺质子交换检查 测量。 基于PDX和P-450CAM的已知结构，模型 对于二蛋白蛋白复合物，将使用异核 - 编辑的NMR方法。 PDX，PDR及其的结晶试验 彼此之间的复合物和P-450CAM正在进行中 确定晶体结构。 最后，金属的机制 中心融合到PDX将通过各种 物理化学方法。