Linking functions of the FOF1 ATP Synthase

FOF1 ATP 合酶的连接功能

• 批准号: 6625644
• 负责人: ROBERT K. NAKAMOTO
• 金额: $ 29.42万
• 依托单位: UNIVERSITY OF VIRGINIA
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-05-01 至 2006-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6625644
• 关键词: Escherichia coli; bioenergetics; biomechanics; cysteine; electron spin resonance spectroscopy; enzyme activity; enzyme mechanism; enzyme structure; hydrogen transporting ATP synthase; mutant; nanotechnology; protein sequence

DESCRIPTION(provided by applicant): The long term goal of this project is to understand how the catalytic reaction of the hydrolysis or synthesis of adenosine triphosphate is coupled to the transport of protons across the membrane in the multi-subunit FOF 1 ATP synthase. Recent observations have established that the catalytic and transport functions utilize rotational mechanisms and energy is transferred between them, in part, by the torque generated on the rotor subunits. Characterization of several mutant enzymes with substitutions in the interfaces between rotor and stator indicate that the efficiency of energy transfer between transport and catalysis involves transmission of conformational information. To understand how the conformational effects modulate the catalytic and transport mechanisms, we will carry out two Specific Aims. Specific Aim 1: the site-directed spin labeling strategy of EPR spectroscopy and determination of solvent accessibility of cysteine substitutions by reactivity with hydrophilic sulfhydryl reagents will be used to generate a structure-function map of the gamma and epsilon rotor subunits. Physical properties of the rotor and how it interacts with the stator will be elucidated. Specific Aim 2: we will dissect the coordination between the catalytic transition state and the rotational behavior of the catalytic motor. The effects of amino acid replacements on ATP-driven gamma subunit rotation, which is observed directly on a nano-fabricated experimental platform, will be correlated to the partial reaction steps of the catalytic mechanism, which are determined chemically by pre-steady state quench-flow and fluorescence stopped-flow kinetic measurements. Mutant enzymes will be analyzed that have amino acid replacements that perturb rotor-stator interactions and have effects on the catalytic transition state and rotation. These studies seek to understand the molecular mechanisms of a true molecular rotary motor, and in particular, the mechanisms of coupling efficiency that generate high torque forces which makes it the most powerful nanoelectromechanical motor.

描述（由申请人提供）：该项目的长期目标是 了解水解或合成的催化反应如何 三磷酸腺苷与质子的运输偶联 多支亚基FOF 1 ATP合酶中的膜。最近的观察结果 确定催化和运输功能利用旋转 机理和能量在它们之间部分通过扭矩传递 在转子亚基上生成。几种突变酶的表征 在转子和定子之间的接口中取代，表明 运输和催化之间的能量转移效率涉及 构象信息的传输。了解如何 构象作用调节催化和运输机制，我们将 执行两个具体目标。特定目标1：定向的自旋标记 EPR光谱策略和确定溶剂可及性 通过与亲水性硫赖尔试剂反应性的半胱氨酸取代 用于生成伽马和epsilon转子的结构功能图 亚基。转子的物理特性及其如何与定子相互作用 将阐明。特定目的2：我们将剖析 催化过渡状态和催化的旋转行为 发动机。氨基酸替代对ATP驱动的伽马亚基的影响 旋转，直接在纳米制造的实验上观察到 平台将与催化的部分反应步骤相关 机制，通过稳态的状态淬火流量化学确定 荧光停止流动动力学测量。将分析突变酶 具有氨基酸的替代品，可扰动转子 - 定子相互作用和 对催化过渡状态和旋转产生影响。这些研究寻求 了解真实分子旋转电动机的分子机制，并在 特别是产生高扭矩的耦合效率的机制 使其成为最强大的纳米机电电动机的力。