F1-ATPase Chemical-Mechanical Coupling Mechanisms

F1-ATP酶化学机械耦合机制

• 批准号: 7049224
• 负责人: WAYNE D FRASCH
• 金额: $ 27.46万
• 依托单位: ARIZONA STATE UNIVERSITY-TEMPE CAMPUS
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-08-01 至 2009-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7049224
• 关键词: Chlamydomonas; active sites; adenosine triphosphate; conformation; electron spin resonance spectroscopy; enzyme activity; enzyme mechanism; fluorescence microscopy; gene mutation; hydrogen transporting ATP synthase; interferometry; liposomes; magnesium ion; molecular dynamics; oxidative phosphorylation; phosphates; protein biosynthesis; site directed mutagenesis; tyrosine

DESCRIPTION (provided by applicant): The F1Fo ATP synthase is a multisubunit, membrane-bound enzyme that catalyzes the synthesis of the majority of cellular ATP. Mutations in several subunits in the human mitochondrial enzyme are of important clinical relevance. The enzyme works as a molecular motor that drives the rotary motion of some of the subunits. The enzymes from mitochondria, chloroplasts and the plasma membranes of bacteria are closely similar. In E. coli, F1 is composed of an (alpha-beta)3 ring that surrounds the gamma subunit, and also contains delta and epsilon subunits. The membrane embedded Fo is composed of a c10 subunit ring to which is attached the a subunit and two b subunits. During ATP synthesis, the movement of protons through Fo drives the rotation of the c10 subunit ring to which the gamma and epsilon subunits are attached that forces sequential conformational changes in the (alpha-beta)3 ring and results in the synthesis of ATP from each of the three (alpha-beta)3 heterodimers. The hydrolysis of ATP can also drive the rotation of the gamma subunit in the opposite direction. When F1 molecules are attached to a microscope cover slip, and a probe that can be observed under a microscope is attached to the gamma subunit, single molecules can be observed to rotate upon addition of ATP. Our long term objective is to elucidate the mechanism of ATPase-driven rotation of the gamma subunit. We propose the induction mechanism as a working hypothesis for rotation that will be critically examined. This hypothesis posits that Coulombic potential originating from residues that form hydrogen bonds and salt bridges between the (alpha-beta)3 ring and the gamma subunit contribute to the generation of gamma subunit torque. We will test this hypothesis by accomplishing the following aims. (1) The contribution of beta Catch Loop-gamma Subunit interactions to gamma subunit rotation will be examined by measuring the effects of mutations that eliminate these interactions on the rate of gamma subunit rotation measured using an innovative single molecule assay that we developed for this purpose. (2) The contribution of other known (alpha-beta)3 ring-gamma subunit interactions to gamma subunit rotation will be determined that include contacts at the gamma subunit N and C termini. (3) The contribution to rotation of (alpha-beta)3 ring residues that do not interact directly with the gamma subunit will be examined including those that communicate between the gamma subunit and the catalytic sites. (4) The contribution to gamma subunit rotation of (alpha-beta)3 ring-gamma subunit interactions that have not been identified in known crystal structures of F1 but are identified by tracking the rotational path of charged and polar gamma subunit residues around the inside of the (alpha-beta)3 ring.

描述(申请人提供)：F1Fo ATP合成酶是一种多亚基、膜结合的酶，催化大多数细胞ATP的合成。人类线粒体酶几个亚基的突变具有重要的临床意义。这种酶就像一个分子马达，驱动一些亚基的旋转运动。细菌的线粒体、叶绿体和质膜中的酶非常相似。在大肠杆菌中，F1由一个围绕伽马亚基的(α-β)3环组成，还包含Delta和epsilon亚基。包埋Fo的膜由连接有a亚基和两个b亚基的C10亚基环组成。在ATP合成过程中，质子通过Fo的运动驱动连接有伽马和epsilon亚基的C10亚单位环的旋转，从而迫使(α-β)3环发生连续的构象变化，并导致从三个(α-β)3异二聚体中的每一个合成ATP。ATP的水解也可以驱动伽马亚基朝着相反的方向旋转。当F1分子连接到显微镜盖片上，并将在显微镜下可以观察到的探针连接到伽马亚基上时，可以观察到单分子在添加ATP后旋转。我们的长期目标是阐明ATPase驱动的伽马亚基旋转的机制。我们提出了诱导机制作为旋转的工作假说，这将得到严格的检验。这一假设假设，来自(α-β)3环和伽马亚单位之间形成氢键和盐桥的残基的库仑势有助于伽马亚单位扭矩的产生。我们将通过实现以下目标来检验这一假设。(1)将通过测量消除这些相互作用的突变对伽马亚单位旋转速度的影响来检查βCatch Loop-伽马亚单位相互作用对伽马亚单位旋转的贡献，该突变是用我们为此开发的创新型单分子分析方法测量的。(2)将确定其他已知的(α-β)3环-伽马亚单位相互作用对伽马亚单位旋转的贡献，其中包括在伽马亚单位N和C末端的接触。(3)将考察不直接与伽马亚基相互作用的(α-β)3环残基对旋转的贡献，包括那些在伽马亚基和催化中心之间通信的残基。(4)(α-β)3环-伽马亚单位相互作用对伽马亚单位旋转的贡献，这些相互作用在已知的F1晶体结构中尚未被识别，但通过跟踪带电和极性伽马亚单位残基围绕(α-β)3环内部的旋转路径而被识别。