STRUCTURE/FUNCTION STUDIES OF E COLI F1F0 ATPASE

本研究的长期目标是通过以下方法了解其机制： 这些细胞合成ATP， 有机体 ATP合成发生在大多数细菌的质膜上， 真核生物的线粒体和叶绿体中也有。 选择系统 用于本研究的是来自大肠杆菌的F1 F0 ATP合酶。 的 该系统的优点是易于构建和分析， 突变体，和相对简单的亚基组成的酶。 的 已知ATP的净合成与质子的运动相耦合 但这个过程的细节仍有待确定。 F1 F0 ATP合酶家族的所有成员的机制将是： 与已被充分研究的哺乳动物线粒体密切相关， 因此，我们的研究结果将与 人体和细菌代谢。 特别是，许多线粒体 肌病与不能产生足够的ATP有关。 本研究 将重点放在如下两个 这种酶的结构和功能。 F1F0 ATP 合成酶是一种膜结合酶，由8种不同类型组成 亚基（α、β、γ、δ、α、a、B、c）的亚基 化学计量，3：3：1：1：1：2：10。 在结构上，该酶由以下组成： 2个可分离的部分; F1，由α、β、γ组成的ATP水解酶， δ和δ亚基，以及F0，一种由 a，B，和c亚基。 F1必须连接到F0（在膜中）以获得ATP 功能上，F1是ATP合成和水解的位点， 其中活性位点区域由α、β和γ亚基组成。 Fo是质子穿过膜的运动位点， 由a和c亚单位协调。 功能的第三个方面是 这两个过程的联系，这可能涉及其余的 亚基：δ、β和B。 首先，我们建议继续我们的 Fo.质子转位的研究。 这些研究将包括（一） A亚基的定点突变，以测试最近开发的 质子转移模型，（II）a亚基与B的共价键 和c亚基的基因融合，以测试F0亚基的可能性， 旋转，和（III）跨膜区段的结构分析，和 连接它们的环，在α亚基中，通过引入 独特的活性半胱氨酸残基。 这些半胱氨酸然后可以通过测试 对于二硫化物形成，当成对引入时，或者对于标记， 表面或膜可溶性试剂。 第二，我们建议研究 质子转运与ATP合成的偶联， γ和γ亚基与c亚基的相互作用。 我们有 最近绘制了γ-射线与γ-射线相互作用时的结合表面， 和C亚基的突变。 未来的研究将 继续绘制生物多样性的功能区域图，并开始分析 γ亚基的残基，其通过以下方式与γ和c亚基相互作用： 诱变

The long term objective of this study is to understand the mechanism by which cells synthesize ATP, the basic unit of chemical energy in all organisms. ATP synthesis occurs at the plasma membrane of most bacteria, and for eukaryotes, in mitochondria and chloroplasts. The system of choice for this study is the F1F0 ATP synthase from Escherichia coli. The advantages of this system are the ea se of construction and analysis of mutants, and athe relatively simple subunit composition of the enzyme. The net synthesis of ATP is known to be coupled to the movement of protons across a membrane, but the details of this process remain to be determined. The mechanism of all members of the F1F0 ATP synthase family will be closely related to that of the well-studied, mammalian mitochondrial enzymes, and therefore, the results of our studies will be relevant both to human and bacterial metabolism. In particular, many mitochondrial myopathies are related to the inability to make sufficient ATP. This study will focus on two aspects of the structure and function of this enzyme. The F1F0 ATP synthase is a membrane-bound enzyme that is composed of 8 different types of subunits (alpha, beta, gamma, delta, epsilon, a, b, c) in the following stoichiometry, 3:3:1:1:1:1:2:10. Structurally, the enzyme is composed of 2 separable parts; F1, an ATP hydrolase composed of alpha, beta, gamma, delta, and epsilon subunits, and F0, an integral membrane complex composed of a, b, and c subunits. F1 must be attached to F0 (in a membrane) for ATP synthesis, Functionally, F1 is the site of ATP synthesis and hydrolysis, with the active site region composed of alpha, beta, and gamma subunits. Fo is the site of proton movement through the membrane, which is coordinated by a and c subunits. The third aspect of function is the linking of these 2 processes, which probably involves the remaining subunits: delta, epsilon, and b.. First,we propose to continue our studies of proton translocation by Fo. These studies will include (I) site-specific mutagenesis of the a subunit to test a recently developed model of proton translocation, (II) covalent linkage of the a subunit to b and c subunits via gene fusions, to test the possibility of F0 subunit rotation, and (III) structural analysis of the transmembrane segments, and of the loops connecting them, in the alpha subunit, by introduction of uniquely reactive cysteine residues. These cysteines can then by tested for disulfide formation, when introduced in pairs, or for labeling by surface or membrane-soluble reagents. Second, we propose to study the coupling of proton translocation to ATP synthesis by focusing on interactions of the gamma and epsilon subunits with the c subunit. We have recently mapped the binding surface of epsilon as it interacts with gamma and c subunit, by alanine-scanning mutagenesis. Future studies will continue to map functional regions of epsilon, and initiate analysis of residues of the gamma subunit that interact with epsilon and c subunits by mutagenesis.