STRUCTURE/FUNCTION STUDIES OF E COLI F1F0 ATPASE

这项研究的长期目标是通过以下方式了解这一机制 哪些细胞合成三磷酸腺苷，这是所有细胞中化学能的基本单位 有机体。ATP合成发生在大多数细菌的质膜上， 对于真核生物，在线粒体和叶绿体中。选择的系统 本研究的目的是从大肠杆菌中提取F1F0三磷酸腺苷合成酶。这个 该系统的优点是易于构建和分析 突变株，以及酶的相对简单的亚基组成。这个 已知三磷酸腺苷的净合成与质子的运动相耦合。 跨膜，但这一过程的细节仍有待确定。 F1F0 ATP合成酶家族的所有成员的机制将是 与研究得很好的哺乳动物线粒体密切相关 因此，我们的研究结果将与 人类和细菌的代谢。特别是，许多线粒体 肌病与无法制造足够的三磷酸腺苷有关。本研究 将重点放在两个 这种酶的结构和功能的各个方面。F1F0的ATP 合酶是一种膜结合酶，由8种不同类型组成 亚基(阿尔法、贝塔、伽马、德尔塔、依西隆、a、b、c) 化学计量比，3：3：1：1：1：1：2：10。从结构上讲，该酶由 2个可分离的部分；F1，一种由α，β，γ， Delta和epsilon亚基，以及F0，组成的完整的膜复合体 包括a、b和c亚基。对于ATP，F1必须连接到F0(在膜中) 合成，从功能上讲，F1是ATP合成和水解的部位， 活性部位区域由α、β和伽马亚基组成。 FO是质子通过膜运动的位置，它是 由A和C亚基协调。函数的第三个方面是 这两个进程的链接，这可能涉及剩余的 亚单位：Delta、epsilon和b.首先，我们建议继续我们的 Fo.质子迁移的研究。这些研究将包括：(I) 用a亚基定点突变检测新近发展起来的 质子转移模型，(Ii)a亚基与b亚基的共价键 和c亚基，通过基因融合，测试F0亚基的可能性 旋转，以及(Iii)跨膜片段的结构分析，以及 在阿尔法亚单位中，通过引入 独一无二的活性半胱氨酸残基。然后，这些半胱氨酸可以通过测试 用于二硫化物的形成，当成对引入时，或用于标记 表面或膜可溶试剂。第二，我们建议研究 质子转运与ATP合成的偶联 γ和epsilon亚基与c亚基的相互作用。我们有 最近绘制了epsilon与伽马相互作用时的结合表面 和c亚基，丙氨酸扫描诱变。未来的研究将 继续绘制epsilon的功能区图，并启动对 通过与epsilon和c亚基相互作用的伽马亚基残基 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.