STRUCTURE-FUNCTION STUDIES OF E. COLI F1F0 ATPASE

这项研究的主要长期目标是了解 细胞(即细菌、线粒体和 叶绿体)合成三磷酸腺苷。这项研究的选择系统 是大肠杆菌的F1-Fo-ATPase。次要的兴趣是学习 关于这种多亚基组装的各个方面， 膜结合酶。这项研究建议重点关注两个方面 亚基，这似乎在两个最重要的 酶的有趣方面。首先是阿尔法亚单位， 它似乎参与了一个质子通道，通过 薄膜。这个通道允许质子梯度驱动网 三磷酸腺苷合成。第二个是epsilon亚基，它参与了 膜结合亚单位与细胞周期的物理联系 催化亚基，以及对后者的调节。这个 任何F1-Fo ATPase的作用机制都与(如果不是相同的)相关 线粒体酶，因此，对人类来说 病情，例如心脏病。此外，这个质子通道是 可能与各种离子泵和通道有关 占膜生物学的很大一部分。具体地说，这项研究 目的是确定氨基酸残基对人体健康的重要性 质子在膜上的传导。最近的证据 表明阿尔法亚基的羧基末端是 参与这项工作的。大肠埃希菌中的个别氨基酸 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 通过定点突变技术(盒式磁带 诱变)。突变对乳酸菌生长特性的影响 分离的细胞及其酶学性质 将对膜和蛋白质进行测试。第二个具体目标 涉及到UNC C的突变，这是epsilon亚单位的基因。 含有整个基因的质粒会在体外进行 突变，然后用来转化不能产生 Epsilon亚基。将对变形者进行筛查，看他们是否无法 将琥珀酸根作为唯一的碳源。有可能， 将会出现几种类型的突变：那些会减少的突变 F1与膜结合，但会损害细胞的功能 酵素。这类研究旨在定位特定的功能 Epsilon亚单位的结构域，这些结构域稍后可以 为了探测到更多 这种酶的结构-功能关系很仔细。

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.