Structure, Function and Dynamics of Transhydrogenase

转氢酶的结构、功能和动力学

• 批准号: 7088826
• 负责人: Charles David Stout
• 金额: $ 43.62万
• 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: 2000-07-01 至 2008-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7088826
• 关键词: Escherichia coli; NAD(P) transhydrogenase; Rhodospirillum; X ray crystallography; bioenergetics; cofactor; conformation; crystallization; enzyme mechanism; enzyme structure; hydrogen transport; mutant; nuclear magnetic resonance spectroscopy; site directed mutagenesis

DESCRIPTION (provided by applicant): This project is focused on the structure, function, and conformation of an integral membrane proton pump, transhydrogenase. Transhydrogenase (TH) is an essential enzyme of the respiratory system in mitochondria and bacteria. Proton translocation through membrane-intercalated domain II is coupled to hydride transfer between NAD(H) and NADP(H) bound to extramembranous domains I and III. In the forward direction, the proton motive force shifts the equilibrium toward NADPH formation; in the reverse direction, transhydrogenation from NADPH to NAD results in creation of a proton gradient. The structure of the intact enzyme is unknown, and the mechanism for coupling binding energy and conformational change with proton translocation is unsolved. Structural and functional experiments will develop a picture of this complex molecular machine and its catalytic mechanism. Three Aims address key aspects of TH function: the sites of proton uptake and release in domain Ill, the mechanism of hydride transfer between domains I and III, and the structure and conformation of the intact enzyme. A fourth Aim addresses the mechanism by applying mutagenesis and functional assays to evaluate the coupling of proton transfer in domain II with conformational and binding events in domain III. The experiments entail protein expression and purification, mutagenesis, enzymatic activity assays, preparation of monodisperse, detergent-solubilized TH, negative stain electron microscopy, crystallization in lipid bilayers, single-particle and 2D crystal electron cryo-microscopy, crystallization of detergent-solubilized TH, NMR of expressed domain III, and x-ray crystallography of expressed domains I and III, individually and in cocrystals.

描述（由申请人提供）：该项目重点研究整体膜质子泵转氢酶的结构、功能和构象。转氢酶（TH）是线粒体和细菌呼吸系统的必需酶。通过膜插入结​​构域 II 的质子易位与结合到膜外结构域 I 和 III 的 NAD(H) 和 NADP(H) 之间的氢化物转移偶联。在向前的方向上，质子动力将平衡向 NADPH 的形成方向移动；反之，从 NADPH 到 NAD 的转氢作用会产生质子梯度。完整酶的结构尚不清楚，结合能和构象变化与质子易位的耦合机制也尚未解决。结构和功能实验将描绘出这种复杂的分子机器及其催化机制。三个目标解决了 TH 功能的关键方面：结构域 III 中的质子摄取和释放位点、结构域 I 和 III 之间的氢化物转移机制以及完整酶的结构和构象。第四个目标是通过应用诱变和功能测定来评估结构域 II 中的质子转移与结构域 III 中的构象和结合​​事件的耦合来解决该机制。实验包括蛋白质表达和纯化、诱变、酶活性测定、单分散、去污剂溶解的 TH 的制备、负染色电子显微镜、脂质双层结晶、单颗粒和 2D 晶体电子冷冻显微镜、去污剂溶解的 TH 的结晶、表达结构域 III 的 NMR 以及表达结构域 I 和 III 的 X 射线晶体学（单独的和共晶中的）。