STRUCTURE-FUNCTION STUDIES OF E. COLI F1F0 ATPASE

本研究的主要长期目标是了解 细胞（即细菌、线粒体和 叶绿体）合成ATP。 本研究选择的系统 是E.杆菌 次要的兴趣是学习 关于这个多亚单位组装的各个方面， 膜结合酶 本研究建议重点关注两个 亚单位，这似乎是工具，在两个最 酶的有趣方面。 首先是α亚基， 它似乎参与了一个质子通道， 膜的 这个通道允许质子梯度驱动净 ATP合成。 第二个是α亚基，它参与了 膜结合亚基和膜结合亚基之间的物理连接， 催化亚基，并在后者的调节。 的 任何F1-Fo ATP酶的机制都与（如果不相同） 与）线粒体酶，因此，对人类 条件，例如心脏病。 此外，该质子通道是 可能与各种离子泵和通道有关， 占膜生物学的很大一部分。 具体来说，这项研究 目的是鉴定对蛋白质的合成重要或必需的氨基酸残基， 质子穿过膜的传导。 最近的证据 表明α亚基的羧基末端是 参与这项功能。 E.杆菌 蛋白质，特别是那些在线粒体和 叶绿体酶，将被其他氨基酸取代 通过位点特异性诱变技术（盒 诱变）。 突变对生长特性的影响 细胞，以及分离的酶性质 膜和蛋白质将被测试。 第二个具体目标 涉及突变的unc C，基因的α亚基。 含有整个基因的质粒将在体外进行 诱变，然后用于转化不能产生 α亚基。 将对转化体进行筛选， 以琥珀酸作为唯一碳源。 很有可能， 几种类型的突变将出现：那些减少 F1与膜的结合，但损害膜的功能。 酵素 这些研究旨在将功能定位到特定的 这些结构域可以在以后被 进行定点突变以探测更多 仔细研究这种酶的结构与功能关系。

The primary long-term objective of this study is to understand the mechanism by which cells (i.e. bacteria, mitochondria and chloroplasts) synthesize ATP. The system of choice for this study is the F1-Fo ATPase of E. coli. Of secondary interest is to learn about the various aspects of assembly of this multi-subunit, membrane-bound enzyme. This study proposes to focus on two subunits, which seem to be instrumental in two of the most interesting aspects of the enzyme. First is the alpha subunit, which seems to be involved in a proton channel through the membrane. This channel allows a proton gradient to drive net ATP synthesis. Second is the epsilon subunit, which is involved in the physical linkage of the membrane-bound subunits and the catalytic subunits, and in the regulation of the latter. The mechanism of any F1-Fo ATPase is relevant to (if not identical with) the mitochondrial enzyme, and hence, to the human condition, e.g. heart disease. Furthermore, this proton channel is probably related to the various ion pumps and channels which account for much of membrane biology. Specifically, this study aims to identify amino acid residues important or essential for the conduction of protons across the membrane. Recent evidence suggests that the carboxy-terminus of the alpha subunit is involved in this function. Individual amino acids in the E. coli protein, in particular those conserved among mitochondrial and chloroplast enzymes, will be replaced by other amino acids through a site-specific mutagenesis technique (cassette mutagenesis). The effect of mutations on growth properties of the cells, as well as the enzymatic properties of isolated membranes and proteins will be tested. The second specific aim involves mutagenesis of unc C, the gene for the epsilon subunit. A plasmid containing the entire gene will be subjected to in vitro mutagenesis and then used to transform cells unable to produce an epsilon subunit. Transformants will be screened for the inability to grow an succinate as the sole carbon source. Potentially, several types of mutations will arise: Those which diminish binding of F1 to the membrane but impair the functioning of the enzyme. Such studies intend to locate functions to specific domains of the epsilon subunit, and these domains can later be subjected to site-specific mutagenesis in order to probe more carefully structure-function relationships in this enzyme.