Structure-Function Studies of E. coli F1Fo-ATPase

描述(申请人提供)：线粒体F1-F0ATP合成酶催化合成哺乳动物细胞所利用的绝大多数ATP，这是一个称为氧化磷酸化的复杂过程的顶峰。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。阿尔法、贝塔、伽马、德尔塔和埃西隆形式的FI，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。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。本项目的长期目标是阐明F1F0ATP合成酶中质子转运和构象偶联的机制。这些研究的重点集中在驱动ATP合成酶亚基的构象和旋转运动的质子的途径，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部位。将实现四个具体目标。(A)a亚基的结构和动力学将通过半胱氨酸取代突变、随后的二硫键形成和自旋标记来研究。(B)a亚基的功能问题将通过突变来解决，随后将进行ATP合成的分析。(C)将通过工程二硫键交联法研究F0亚基之间的亚基相互作用。(D)将研究与其在ATP合成和水解过程中的功能作用有关的epsilon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.