STRUCTURE-FUNCTION STUDIES OF E. COLI F1F0 ATPASE

大肠杆菌 F1F0 ATP酶的结构-功能研究

• 批准号: 3298112
• 负责人: STEVEN B VIK
• 金额: $ 9.4万
• 依托单位: SOUTHERN METHODIST UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-07-01 至 1991-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3298112
• 关键词: Escherichia coli; adenosine triphosphate; adenosinetriphosphatase; bacterial proteins; binding proteins; chemical structure function; chemical substitution; chloroplasts; electrochemistry; enzyme mechanism; genetic manipulation; growth media; hydrogen transport; membrane channels; membrane permeability; mitochondria; mutant; point mutation; protein biosynthesis; protein engineering; protein sequence; site directed mutagenesis

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.

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 是大肠杆菌的 F1-Fo ATP 酶。 次要的兴趣是学习 关于这个多子单元组装的各个方面， 膜结合酶。 本研究建议重点关注两个 亚单位，这似乎在两个最重要的方面发挥了作用 酶的有趣方面。 首先是α亚基， 它似乎涉及通过质子通道 膜。 该通道允许质子梯度驱动网络 ATP 合成。 第二个是 epsilon 亚基，它参与 膜结合亚基的物理连接 催化亚基，以及后者的调节。 这 任何 F1-Fo ATPase 的机制都与（如果不相同）相关 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 占膜生物学的大部分。 具体而言，本研究 旨在鉴定重要或必需的氨基酸残基 质子跨膜传导。 最近的证据 表明α亚基的羧基末端是 参与这个功能。 大肠杆菌中的单个氨基酸 蛋白质，特别是线粒体和线粒体中保守的蛋白质 叶绿体酶，会被其他氨基酸取代 通过位点特异性诱变技术（盒式 诱变）。 突变对生长特性的影响 细胞，以及分离的酶特性 将测试膜和蛋白质。 第二个具体目标 涉及 unc C（ε 亚基基因）的诱变。 含有完整基因的质粒将在体外进行 诱变，然后用于转化不能产生 ε 亚基。 将筛选转化体是否无能力 生长琥珀酸盐作为唯一的碳源。 潜在地， 将会出现几种类型的突变： F1 与膜结合但损害膜的功能 酶。 此类研究旨在将功能定位到特定的 epsilon 亚基的结构域，这些结构域稍后可以 进行定点诱变以探测更多 仔细研究这种酶的结构与功能关系。