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(即细菌、线粒体和叶绿体)合成ATP， 生物体中最重要的代谢物。所选择的系统 本研究是从大肠杆菌中分离的F1 FO-ATP合成酶。这张网 已知，三磷酸腺苷的合成与质子的运动相耦合。 一层膜。任何F1 F0-ATPase的机制都被认为是密切相关的 与研究得很好的哺乳动物线粒体酶有关，以及 因此，这样的研究将与人类状况相关。在……里面 特别是，心脏病的许多方面很可能与 制造ATP和利用跨膜质子梯度的能力。这 研究将集中在该酶的两个亚基上，这两个亚基涉及两个 其功能中最有趣的方面。首先是阿尔法 亚基，这是通过酶的质子通道的一部分。这 通道允许质子梯度驱动净ATP合成。第二个是 Epsilon亚基，这是物理连接之间所必需的 膜结合亚基(F0)和催化亚基(F1)，以及 作为一种内在的抑制因子。这项研究试图将 这两个亚基的结构与其功能有关。在这种情况下 Epsilon亚单位，将进行定点突变，以便 确定氨基酸残基对抑制作用有重要结合作用。会是 有趣的是，如果两者的所有突变都是相似的缺陷 结合和抑制。在阿尔法亚单位的情况下，几个氨基 酸残基已被确认与质子有关 有动静。定点突变将继续按顺序进行 以确定其他重要的氨基酸残基。其他方法 将有助于巩固从诱变中获得的信息。 地形信息将通过引入独特的半胱氨酸来收集 不同位置的残留物，以及被标记的能力的测试 从膜的一侧。最后，会产生抗体。 通过对lambda表达系统的筛选，对原生F0进行了筛选。这些可以 中的局部更改和全局更改 不同突变体之间的结构。

The long term objective of this study is to understand the mechanism by which cells (i.e., bacteria, mitochondria and chloroplasts) synthesize ATP, the most important metabolite in any organism. The system of choice for this study is the F1 FO-ATP synthase from Escherichia coli. The net synthesis of ATP is known to be coupled to the movement of protons across a membrane. The mechanism of any F1 F0-ATPase is thought to be closely related to that of the well-studied, mammalian mitochondrial enzymes, and therefore, such studies will be relevant to the human condition. In particular, many aspects of heart disease are likely to be related to the ability to make ATP and to utilize a transmembrane proton gradient. This study will focus on two subunits of the enzyme, which are involved in two of the most interesting aspects of its function. First is the alpha subunit, which makes a part of the proton channel through the enzyme. This channel allows a proton gradient to drive net ATP synthesis. Second is the epsilon subunit, which is necessary for the physical linkage between the membrane-bound subunits (F0) and the catalytic subunits (F1), and which functions as an intrinsic inhibitor. This study seeks to relate the structure of these two subunits to their function. In the case of the epsilon subunit, site-directed mutagenesis will be carried out in order to identify amino acid residues important binding for inhibition. It will be interesting to learn if all mutations are similarly defective in both binding and inhibition. In the case of the alpha subunit, several amino acid residues have already been identified as being involved in proton movement. Site-directed mutagenesis will continue to be applied in order to identify other amino acid residues that are important. Other approaches will serve to consolidate the information gained from mutagenesis. Topographical information will be gathered by introducing unique cysteine residues at various locations, and testing for the ability to be labelled from one side of the membrane. Finally, antibodies will be produced against native F0, by screening the lambda expression system. These can then be used to distinguish between local and global alterations in structure among the various mutants.