Molecular structure of H^+ transporting ATPase and its rotation mechanisms in the catalysis

在活细胞内，各种离子（包括H^+和Na^+）的浓度受到严格的调节。这些调节对于保持细胞的内部环境和细胞的生长至关重要，而细胞的生长与癌症等几种疾病有关。各种生物膜中的离子转运蛋白在这一调控中起着非常重要的作用。此外，其中一些在ATP合成等能量转导中起重要作用。在本研究中，我们利用基因工程和生物化学技术研究了其中两种蛋白质ATP合酶的结构、功能和调控。大肠杆菌ATP合成酶有8个独立的亚基，对亚基基因进行了克隆和测序。跨生物膜的电化学质子梯度和H +通过膜结合酶的流动是催化ATP合成所必需的。然而，这种复杂的亚基结构与酶的催化机制之间的关系尚不清楚。要实现子单元的功能，必须了解子单元的拓扑结构。在这里，我们开发了一种结合遗传学和生物化学的新方法。我们已经证明了一种新的遗传方法，酵母双杂交系统，可以适用于揭示酶内的亚基相互作用。通过这个过程，我们已经证明了- - -和- -的紧密相互作用。这些互动是进一步的。亚基复合物的体外重构证明了这一点。通过引入相互作用的各种功能缺陷突变，分析了相互作用的功能重要性。其中最重要的发现之一是b- δ相互作用，它有助于膜积分和外周部分的相互作用。

Concentrations of various ions including H^+ and Na^+ are strictly regulated within living cells. These regulations are important to keep internal environment of cells and also their growth , which are related to several diseases like cancer, when they are deregulated. Ion transporting proteins in various biological membranes have very important roles in such regulations. Further, some of them have essential roles in energy transduction such as ATP synthesis. In the present study, we have studied structure, function, and regulation for two of these proteins, ATP synthase with techniques of genetic engineering and biochemistry.ATP synthase of Escherichia coli has 8 independent subunits and the genes of subunits were cloned and sequenced. Electrochemical proton gradient across biological membranes and H^+ flow through this membrane bound enzyme is essential for catalysis of ATP synthesis. However, relationship bewteen such complex subunit structure and the catalytic mechanisms of the enzyme is not well understood. Topological arrangement of the subunits should be understood to realize the function of subunits. Here, we developed a new approach with a combination of genetics and biochemistry. We have shown that a new genetic approach, the yeast two-hybrid system, could be applicable to reveal subunit interactions within the enzyme. We have shown tight interactions of alpha-beta gamma-epsilon and b-delta by this procedure. These interactions were further. evidenced by in vitro reconstitution of the subunit complex. Functional importance of the interactions was analyzed by introducing various functionally defective mutations for the interactions. One of the most important findings was b-delta interaction which contributed for the interaction of membrane integral and peripheral portions.