Structure-Function Studies of E. coli F1Fo-ATPase

描述（由申请人提供）：线粒体F1-F0 ATP合酶催化哺乳动物细胞利用的绝大多数ATP的合成，这是一个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。α、β、γ、δ和β型FI，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。本研究的长期目标是阐明F1 F0 ATP合酶中质子转运和构象偶联的机制。这些研究的重点是质子的路径，驱动ATP合酶中的亚基的构象和旋转运动，并在构象变化和蛋白质的结合位点参与机械能的传输到ATP合成的网站。将实现四个具体目标。(A)亚基a的结构和动力学将使用半胱氨酸取代诱变，然后二硫化物形成和自旋标记（B）来检查。亚基a中的功能问题将通过诱变，然后ATP合成测定来解决。(C)将通过工程化二硫键交联研究F0亚基之间的亚基相互作用。(D)将研究与ATP合成和水解过程中其功能作用有关的α亚基的结构问题。

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.