Structure-Function Studies of E. coli F1Fo-ATPase

大肠杆菌 F1Fo-ATP 酶的结构功能研究

• 批准号: 7253386
• 负责人: STEVEN B VIK
• 金额: $ 23.01万
• 依托单位: SOUTHERN METHODIST UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-07-01 至 2009-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7253386
• 关键词: ATP Synthesis Pathway; ATP phosphohydrolase; Abbreviations; Acridines; Address; Alanine; Area; Bacteria; Binding; Binding Sites; Biological Assay; Catalytic Domain; Chloroplasts; Coupling; Cysteine; Disulfides; Engineering; Enzymes; Epitopes; Escherichia coli; F1F0-ATP synthase; Genes; Helix (Snails); Hemagglutinin; Hydrolysis; Laboratories; Mammalian Cell; Mechanics; Mediating; Membrane; Mitochondria; Mono-S; Motion; Movement; Mus; Mutagenesis; Mutate; Mutation; N-(4-azido-2,3,5,6-tetrafluorobenzyl)-3-maleimidopropionamide; Oxidative Phosphorylation; Pathway interactions; Process; Proteins; Proteolysis; Proton-Motive Force; Protons; Reagent; Relative (related person); Research; Research Personnel; Role; Rotation; Site; Spin Labels; Structure; Surface; Thinking; biocytin; crosslink; ear helix; insight; nickel nitrilotriacetic acid; polymyxin B nonapeptide; programs; propionamide; transmission process

DESCRIPTION (provided by applicant): The mitochondrial F1-F0 ATP synthase catalyzes synthesis of the vast majority of ATP that is utilized by mammalian cells, in the culmination of an intricate process known as oxidative phosphorylation. It is a multisubunit, membrane-bound enzyme that is known to function with rotary motion of some of its subunits. Mutations found in several of its subunits are manifested clinically. Close relatives of the mitochondrial enzyme are found in chloroplasts and in some bacteria. Many of the recent insights into the structure and function of the ATP synthase have come from studies of the E. coli enzyme. This version of the enzyme contains eight different types of subunits. Alpha, beta, gamma, delta, and epsilon form FI, containing the sites of ATP synthesis. Subunits a, b and c form the membrane sector F0, containing the proton pathway. The movement of protons through F0 is thought to drive the rotation of gamma and epsilon subunits, relative to the alpha and beta subunits, which form the ATP catalytic sites. The studies proposed in this application focus on two of the subunits from the E. coli ATP synthase, epsilon and subunit a. The long term objectives of this project are to elucidate mechanisms of proton translocation and conformational coupling in the F1F0 ATP synthase. The focus of these studies is on the pathways of the protons that drive conformational and rotational movements of subunits in the ATP synthase, and on the conformational changes and binding sites of proteins involved in the transmission of mechanical energy to the sites of ATP synthesis. Four specific aims will be pursued. (A) Structure and dynamics of subunit a will be examined using cysteine-substitution mutagenesis, followed by disulfide formation and spin-labeling (B) Functional issues in subunit a will be addressed by mutagenesis, followed by assays of ATP synthesis. (C) Subunit interactions among F0 subunits will be investigated by engineering disulfide cross-linking. (D) Structural issues in the epsilon subunit that relate to its role in function during ATP synthesis and hydrolysis will be examined.

描述（由申请人提供）：线粒体F1-F0 ATP合成酶催化绝大多数ATP的合成，这些ATP被哺乳动物细胞利用，这是一个被称为氧化磷酸化的复杂过程的高潮。它是一种多亚基的膜结合酶，已知其一些亚基的旋转运动起作用。在其几个亚基中发现的突变在临床上表现出来。线粒体酶的近亲存在于叶绿体和一些细菌中。最近对ATP合酶的结构和功能的许多见解都来自对大肠杆菌酶的研究。这种酶含有八种不同类型的亚基。-, -， -和-形成FI，包含ATP合成的位点。亚基a， b和c形成膜扇区F0，包含质子途径。质子通过F0的运动被认为驱动了相对于形成ATP催化位点的α和β亚基的γ和ε亚基的旋转。本申请中提出的研究重点是大肠杆菌ATP合成酶的两个亚基，epsilon和亚基a。本项目的长期目标是阐明F1F0 ATP合成酶中质子易位和构象偶联的机制。这些研究的重点是质子驱动ATP合成酶亚基的构象和旋转运动的途径，以及参与将机械能传递到ATP合成位点的蛋白质的构象变化和结合位点。将追求四个具体目标。(A)亚基A的结构和动力学将使用半胱氨酸取代诱变进行检查，然后是二硫化物形成和自旋标记(B)亚基A的功能问题将通过诱变来解决，然后是ATP合成的分析。(C) F0个亚基之间的相互作用将通过工程二硫交联进行研究。(D) epsilon亚基的结构问题与其在ATP合成和水解过程中的功能作用有关。