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: 3298108
• 负责人: STEVEN B VIK
• 金额: $ 14.41万
• 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 1994-05-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3298108
• 关键词: Escherichia coli; adenosinetriphosphatase; bacterial proteins; binding proteins; chloroplasts; electrochemistry; enzyme linked immunosorbent assay; enzyme mechanism; fluorescent dye /probe; genetic manipulation; hydrogen transport; immunochemistry; membrane channels; membrane permeability; mitochondria; mutant; point mutation; protein biosynthesis; protein engineering; protein sequence; protein structure function; radiotracer; site directed mutagenesis

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.

本研究的长期目标是通过以下方法了解其机制： 哪些细胞（即，细菌、线粒体和叶绿体）合成ATP， 任何生物体中最重要的代谢物 选择的系统 本研究是大肠杆菌F1代FO-ATP合酶的研究。 净 已知ATP的合成与质子的移动相耦合， 一种膜。 任何F1 F0-ATP酶的机制被认为是密切相关的。 与研究充分的哺乳动物线粒体酶有关， 因此，这类研究将与人类状况相关。 在 特别是，心脏病的许多方面可能与心脏病有关。 制造ATP和利用跨膜质子梯度的能力。 这 这项研究将集中在酶的两个亚基上，这两个亚基涉及两个 最有趣的功能。 首先是阿尔法 亚基，它构成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。 二是 这是必需的物理连接之间的 膜结合亚基（F0）和催化亚基（F1），并且 作为内在抑制剂发挥作用。 本研究试图将 这两个亚基的结构与它们的功能。 的情况 为了使突变体中的每个亚基都能表达，将进行定点诱变， 鉴定对抑制作用重要结合的氨基酸残基。 将 有趣的是，如果所有的突变都是类似的缺陷， 结合和抑制。 在α亚基的情况下，几个氨基 酸残基已经被确定为参与质子 运动 将继续应用定点突变， 来鉴定其他重要的氨基酸残基。 其他方法 将有助于巩固从诱变获得的信息。 通过引入独特的半胱氨酸， 不同位置的残留物，并测试被标记的能力 从膜的一侧。 最后，抗体会产生 针对天然F0，通过筛选λ表达系统。 这些可以 然后用于区分局部和全局变化， 各种突变体之间的结构。