STRUCTURE AND DYNAMICS OF METAL CONTAINING PROTEINS

该项目代表了一项持续的努力，以确定 蛋白质相互作用中的蛋白质结构和电子传递 恶臭假单胞菌P-450 cam系统。 P-450 cam系统包括 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）。 该细胞色素催化的5-外羟基化 樟脑与分子氧反应，该反应需要两个电子， 通过Pdx顺序地提供给细胞色素，这也是需要的 作为效应器衬底翻转。 电子通过以下方式提供给Pdx： PdR通过氧化PdR结合的NADH。 樟脑羟化酶系统是研究人P-450酶的良好模型 参与类固醇激素的生物合成和外源性物质的加工， 致癌物，这可能导致致癌物活化。 恶臭假单胞菌 系统已经详细研究，因为它很容易纯化，所有的 成分是可溶于其活性形式，基因已被 克隆所有组件。 该项目旨在完善NMR衍生的 通过获得更多NOE和二面角Pdx溶液结构 限制使用多维13 C，15 N-编辑的NMR方法。 当前 使用1H NMR方法确定结构，从中得到有限数量的 的明确NOE约束。 2Fe-2Fe的顺磁性 2S聚类是结构细化的另一个障碍， 提出了标记方案和抗磁性金属中心取代， 克服这个问题。 几个绑定沃茨被怀疑在 结构，并预计在表面上的Pdx，和脉冲场- 将采用梯度NMR方法来鉴定这些化合物。 氧化还原依赖性 在Pdx中已经确定了结构变化，并且两个 可用的氧化态将通过酰胺质子交换进行检查 测量. 基于Pdx和p-450 cam的已知结构， 对于双蛋白复合物，将提出并使用杂合物进行测试， NMR方法。 用于pdx、PdR及其组合的结晶试验 彼此之间以及与P-450 cam的复合物正在进行中， 晶体结构的测定。 最后，金属的机制 中心并入Pdx将通过多种方法进行研究。 物理化学方法

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.