STRUCTURE/FUNCTION STUDIES OF E COLI F1 F0 ATPASE

线粒体F1F0ATP合成酶催化合成哺乳动物细胞所利用的绝大多数ATP，这是一个复杂的过程的高潮，称为氧化磷酸化。它是一种多亚基、膜结合的酶，已知其某些亚基的旋转运动起作用。在它的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。最近对三磷酸腺苷合成酶的结构和功能的许多见解都来自对大肠杆菌酶的研究。这种酶包含八种不同类型的亚基。在它的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合成酶的结构和功能的许多见解都来自于对大肠杆菌酶的研究。这种酶包含八种不同类型的亚基。阿尔法、贝塔、伽马、德尔塔和埃西隆形成F1，包含ATP合成的部位。亚基a、b和c形成包含质子途径的膜扇区F0。质子通过F0的运动被认为驱动了伽马和epsilon亚基相对于形成ATP催化位点的α和β亚基的旋转。在本申请中提出的研究集中在大肠杆菌ATP合成酶的两个亚基，epsilon和a亚基。将追求四个特定的目标。(A)将研究a亚基的假定功能区。跨膜跨膜将通过丙氨酸插入扫描突变来探测，保守残基将被突变以检查对功能的影响。(B)将确定a亚单位的重要结构特征。将建立a亚基跨膜跨度的近邻关系，并将通过标记程序来测试假定的“半通道”。(C)F0亚基之间的相互作用将通过半胱氨酸残基的光活性交联法进行研究。(D)将审查与功能有关的epsilon亚单位的结构问题。这包括参与与ATP合成酶中其他亚基结合的epsilon表面，以及epsilon亚单位内的灵活性对功能的作用。

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.