STRUCTURE/FUNCTION STUDIES OF E COLI F1 F0 ATPASE

大肠杆菌 F1 F0 ATP酶的结构/功能研究

• 批准号: 6329697
• 负责人: STEVEN B VIK
• 金额: $ 19.16万
• 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 2003-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6329697
• 关键词: Escherichia coli; adenosine triphosphate; bacterial proteins; binding sites; conformation; crosslink; cysteine; disulfide bond; enzyme mechanism; enzyme structure; hydrogen transport; hydrogen transporting ATP synthase; microorganism metabolism; oxidative phosphorylation; protein biosynthesis; protein structure function; site directed mutagenesis

The mitochondrial F1F0 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 multi- subunit, 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 the plasma membranes of 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. Mutations found in several of its subunits are manifested clinically. Close relatives of the mitochondrial enzyme are found in several of its subunits are manifested clinically. Close relatives of the mitochondrial enzymes are found in chloroplasts and in the plasma membranes of some bacteria. Many of the recent insight 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 F1, 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. Four specific aims will be pursued. (A) Putative functional regions of subunit a will be examined. Transmembrane spans will be probed by alanine insertion scanning mutagenesis and conserved residues will be mutated for examining of effects on function. (B) Important structural features of subunit a will be identified. Near- neighbor relationships of transmembrane spans in subunit a will be established, and the putative "half-channels" will be tested by labeling procedures. (C) Subunit interactions among F0 subunits will be investigated by photoactive crosslinking from Cys residues. (D) Structural issues in the epsilon subunit that relate to function will be examined. This includes the surface of epsilon involved in binding to other subunits in the ATP synthase, and the role of flexibility within the epsilon subunit for function.

线粒体F1F0 ATP合酶催化哺乳动物细胞利用的绝大多数ATP的合成，在称为氧化磷酸化的复杂过程的顶点。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的质膜中。最近对ATP合酶的结构和功能的许多认识来自于对E。大肠杆菌酶。这种酶含有八种不同类型的亚基。 在其几个亚基中发现的突变在临床上表现出来。线粒体酶的近亲被发现在其几个亚基的临床表现。线粒体酶的近亲存在于叶绿体和一些细菌的质膜中。最近对ATP合酶的结构和功能的许多认识来自于对大肠杆菌的研究。大肠杆菌酶。这种酶含有八种不同类型的亚基。α、β、γ、δ和β型F1，含有ATP合成位点。亚基a、B和c形成包含质子路径的膜扇区F0。质子通过F0的运动被认为驱动γ和γ亚基相对于α和β亚基的旋转，α和β亚基形成ATP催化位点。本申请中提出的研究集中在E. coliATP合成酶、ATP酶和ATP酶a亚基。将实现四个具体目标。(A)亚基a的推定功能区域将被检查。将通过丙氨酸插入扫描诱变探测跨膜跨度，并将突变保守残基以检查对功能的影响。(B)将鉴定亚基a的重要结构特征。将建立亚基a中跨膜跨度的近邻关系，并通过标记程序测试推定的“半通道”。(C)F0亚基之间的亚基相互作用将通过Cys残基的光活性交联进行研究。(D)将检查与功能相关的结构性问题。这包括ATP合酶中与其他亚基结合的ATP酶表面，以及ATP酶亚基中的柔性功能。