STRUCTURE/FUNCTION STUDIES OF E COLI F1 F0 ATPASE

线粒体F1F0 ATP合成酶催化了哺乳动物细胞所使用的绝大多数ATP的合成，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的质膜中。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。在其几个亚基中发现的突变在临床上表现出来。线粒体酶的近亲在其几个亚基中被发现，并在临床上表现出来。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F1，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。将追求四个具体目标。(A)亚基A的假定功能区将被检查。利用丙氨酸插入扫描诱变技术探测跨膜间隙，并对保守残基进行突变，以检验其对功能的影响。(B)亚单元a的重要结构特征将被识别。亚基a跨膜跨度的近邻关系将被建立，假定的“半通道”将通过标记程序进行测试。(C) F0个亚基之间的相互作用将通过Cys残基的光活性交联来研究。(D)将审查与功能有关的epsilon亚单位的结构问题。这包括参与ATP合酶中与其他亚基结合的epsilon表面，以及epsilon亚基内的灵活性在功能中的作用。

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.