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Transhydrogenase: Structure, Dynamics, and Mechanism

转氢酶：结构、动力学和机制

基本信息

批准号：
9251822
负责人：
Qinghai Zhang
金额：
$ 36.58万
依托单位：
SCRIPPS RESEARCH INSTITUTE, THE
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-06-01 至 2019-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9251822
关键词：
Aging Alzheimer&apos s Disease Amyotrophic Lateral Sclerosis Binding Biochemical Biochemical Genetics Biological Assay Biological Process C-terminal Catalysis Charge Complex Coupled Couples Coupling Crystallization Data Data Set Detergents Dimerization Electron Microscopy Enzymes Huntington Disease Hyperglycemia Individual Inner mitochondrial membrane Insulin Resistance Knowledge Lead Link Lipids Malignant Neoplasms Maps Measurement Membrane Methods Microscopy Mitochondria Mitochondrial Diseases Molecular Molecular Conformation Morphology Motion Movement Multienzyme Complexes Mutagenesis Mutation NAD(P)+ transhydrogenase NADH NADP Neurodegenerative Disorders Niacinamide Non-Insulin-Dependent Diabetes Mellitus Nucleotides Oxidation-Reduction Oxidative Stress Parkinson Disease Periodicity Phase Positioning Attribute Production Proton Pump Proton-Motive Force Protons Reactive Oxygen Species Reducing Agents Resolution Resources Respiration Roentgen Rays Role Signal Transduction Site-Directed Mutagenesis Structure Structure of beta Cell of islet System Thermus thermophilus Time design and construction dimer electron density electron diffraction experimental study gene product improved insulin secretion mitochondrial dysfunction mutant particle public health relevance reconstruction vector

项目摘要

DESCRIPTION (provided by applicant): The energy-linked inner mitochondrial membrane enzyme nicotinamide nucleotide transhydrogenase (TH) couples the proton motive force (pmf) generated by respiration to formation of NADPH. In the absence of NADPH reactive oxygen species (ROS) lead to mitochondrial dysfunction which is strongly correlated with neurodegenerative diseases. In pancreatic beta cells, oxidative stress arising from TH mutations is correlated with type 2 diabetes due to loss of the redox signaling that controls insulin secretion. To understand the relationship between ROS, mitochondrial dysfunction and disease, it is necessary to understand the mechanism by which TH couples the pmf with formation of NADPH. Extensive biochemical and genetic data are available, but to define a mechanism crystal structures of the entire enzyme and its constituent subunits are required. Further, knowledge of the dispositions of the subunits during hydride transfer and proton pumping are required. A viable strategy employs TH from Thermus thermophilus for crystallization of the enzyme and its individual components. Diffraction quality crystals of the membrane intercalated domains have been obtained in the lipidic cubic phase (LCP); structures of the membrane domains in complex with the soluble, NADP(H) binding domain (domain III) will also be obtained via construct design and co- crystallization. High resolution structures of the soluble NAD(H) subunit alone and in complex with domain III have been solved. Large prismatic crystals of intact TH in multiple morphologies are available for diffraction experiments. The component crystal structures will be positioned within the TH electron density envelop using lower resolution X-ray data and EM methods (single particle averaging and electron diffraction), revealing for the first time a complete structure of TH. SAXS experiments in the presence of NAD(H) and NADP(H), together with the crystal structures, will identify motions of domain III within the complex. Structure guided site- directed mutagenesis and biochemical assays (cyclic and reverse hydride transfer, vectorial proton pumping) will define residue functions. Knowledge of structure, conformational states, and function will enable a mechanism for coupling proton translocation and NADPH formation to be deduced.

描述（由申请方提供）：能量相关线粒体内膜酶烟酰胺核苷酸转氢酶（TH）将呼吸产生的质子动力（pmf）与NADPH的形成偶联。在NADPH缺乏的情况下，活性氧（ROS）导致线粒体功能障碍，这与神经退行性疾病密切相关。在胰腺β细胞中，由TH突变引起的氧化应激与2型糖尿病相关，这是由于控制胰岛素分泌的氧化还原信号的丢失。为了了解ROS、线粒体功能障碍和疾病之间的关系，有必要了解TH将pmf与NADPH形成偶联的机制。广泛的生物化学和遗传学数据是可用的，但要定义整个酶及其组成亚基的机制，需要晶体结构。此外，氢化物转移和质子泵期间的亚单位的处置的知识是必需的。一种可行的策略是采用来自嗜热栖热菌的TH来结晶酶及其单个组分。已经在介晶立方相（LCP）中获得了膜嵌入结构域的衍射质量晶体;还将通过构建体设计和共结晶获得与可溶性NADP（H）结合结构域（结构域III）复合的膜结构域的结构。已经解决了单独的可溶性NAD（H）亚基和与结构域III复合的可溶性NAD（H）亚基的高分辨率结构。完整的TH在多种形态的大棱柱晶体可用于衍射实验。组分晶体结构将使用较低分辨率的X射线数据和EM方法（单粒子平均和电子衍射）定位在TH电子密度包络内，首次揭示TH的完整结构。在NAD（H）和NADP（H）存在下的SAXS实验以及晶体结构将鉴定复合物内结构域III的运动。结构引导的定点诱变和生物化学测定（循环和反向氢化物转移、矢量质子泵）将定义残基功能。结构，构象状态和功能的知识将使耦合质子易位和NADPH形成的机制被推导出来。