Fo Motor Mechanisms that Power FoF1 ATP Synthesis

为 FoF1 ATP 合成提供动力的 Fo 电机机制

• 批准号: 8086570
• 负责人: WAYNE D FRASCH
• 金额: $ 29.45万
• 依托单位: ARIZONA STATE UNIVERSITY-TEMPE CAMPUS
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-04-01 至 2015-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8086570
• 关键词: ATP Synthesis Pathway; ATP phosphohydrolase; Affect; Appearance; Ataxia; Bilateral; Binding; Biological Assay; Cardiomyopathies; Catalysis; Catalytic Domain; Cell physiology; Corpus striatum structure; Coupling; Dependence; Employee Strikes; Energy-Generating Resources; Equilibrium; Escherichia coli; Genes; Gold; Growth; Independent Living; Leigh Disease; Lipid Bilayers; Measurement; Measures; Membrane; Microscope; Molecular Motors; Motor; Muscle Weakness; Mutation; Necrosis; Noise; Oxidative Phosphorylation; Parkinson Disease; Patients; Proton-Motive Force; Relative (related person); Resolution; Retinitis Pigmentosa; Rotation; Signal Transduction; Site; Slide; Sodium Chloride; Source; Testing; Time; Torque; Viscosity; Work; aqueous; driving force; in vivo; innovation; insight; mutant; nanorod; novel; research study; single molecule

DESCRIPTION (provided by applicant): Ataxia, Leigh syndrome, retinitis pigmentosa, muscle weakness and familial bilateral striatal necrosis can result from damage to the genes that encode FoF1 ATP synthase subunits. The FoF1 ATP synthase has two opposed rotary molecular motors connected by a common axle. The integral membrane Fo motor uses proton- motive force (PMF) to drive axle rotation for F1-dependent ATP synthesis. In vivo, FoF1 maintains the [ATP]/[ADP][Pi] ratio far from equilibrium, enabling high [ATP] to provide an energy source for cellular processes. The Fo motor uses a Brownian ratchet to bias clockwise rotation against an F1 motor-imposed load. We recently observed a previously unknown interaction between Fo subunits a and c of FoF1 when ATPase-driven rotation is slowed by a viscosity-induced load. A striking feature of this interaction is that it forms a tether that limits rotation to 360. The cD44N/cR50 mutant eliminates tether formation and causes loss of oxidative phosphorylation-dependent E. coli growth, indicating that the tether is an important Fo motor component for ATP synthesis in vivo. A mechanistic hypothesis is proposed where the tether enables the Fo motor to ratchet clockwise rotation against an F1 motor-imposed load during ATP synthesis. The focus of the work proposed here is to test this Fo motor mechanism hypothesis when FoF1 synthesizes ATP. The Fo mechanism is poorly understood compared to that of F1, in part, because of membrane-associated technical problems that make it very difficult to carry out single-molecule studies on Fo. A novel assembly of supported planar lipid bilayers containing oriented FoF1 ATP synthase molecules on a microscope slide will now be made to enable single molecule rotation measurements during ATP synthesis with a time resolution to 5 s at unprecedented signal-to-noise. The specific aims of the project will: (1) determine rotational velocity and torque generated by the Fo motor during ATP synthesis as a function of PMF; (2) determine tether formation and duration during ATP synthesis as a function of a load on Fo imposed via viscosity, by increasing ATP/ADP.Pi, by decreasing Fo driving force relative to the F1 load, and by mutant analysis; (3) identify the 9 ms catalytic dwell during ATP synthesis, and determine if the other 9ms dwell is "substrate-waiting"; and (4) test the escapement mechanism hypothesis for coupling rotation with ATP synthesis through mutant analysis. Our discovery of the previously unknown tether between subunit a and subunit-c residues cD44 and cR50 provides a new window with which to examine the mechanism by which the Fo motor powers rotation to catalyze ATP synthesis. Through the use of our innovative approach for the assembly of supported planar lipid bilayers in combination with our novel nanorod assay, the experiments proposed here will provide important new insight concerning several fundamental aspects of the mechanism of the Fo molecular motor, and the means by which it interfaces with the F1 motor to catalyze the synthesis of ATP. PUBLIC HEALTH RELEVANCE: Ataxia, Leigh syndrome, retinitis pigmentosa, muscle weakness and familial bilateral striatal necrosis can result from damage to the genes that encode subunits of the FoF1 ATP synthase. Some patients with cardiomyopathies or Parkinson's disease also have increased damage to these genes. All independent life forms use the FoF1 as the main source of cellular ATP.

描述（由申请人提供）：共济失调，Leigh综合征，色素性视网膜炎，肌肉无力和家族性双边纹状体坏死可能是由于对编码FOF1 ATP合酶亚基的基因的损害造成的。 FOF1 ATP合酶具有两个相对的旋转分子电机，该电动机由公共轴连接。积分膜FO电动机使用质子运动力（PMF）驱动轴旋转，以依赖F1依赖性ATP合成。在体内，FOF1维持[ATP]/[ADP] [PI]比率远离平衡，使高[ATP]能够为细胞过程提供能源。 FO电动机使用布朗棘轮将偏置旋转与F1电动机施加的负载偏置。 最近，当粘度诱导的载荷减慢了ATPase驱动的旋转时，我们观察到FOF1的FO亚基A和C之间的先前未知的相互作用。这种相互作用的一个惊人特征是它形成了一个将旋转限制至360的束缚。CD44N/CR50突变体消除了束缚的形成并导致氧化磷酸化依赖性大肠杆菌生长的丧失，这表明该链球是在体内ATP合成的重要FO FO MOTOR成分。提出了一个机械假设，即束缚使FO电动机在ATP合成过程中以F1电动机载荷的顺时针旋转。此处提出的工作的重点是当Fof1合成ATP时测试该FO运动机制假设。与F1相比，FO机制的理解很少，部分原因是由于膜相关的技术问题，因此很难进行有关FO的单分子研究。 现在，将制作一个新颖的平面脂质双层组合，这些脂质双层在显微镜载玻片上含有方向的FOF1 ATP合酶分子，以实现ATP合成期间的单分子旋转测量值，并在未预言的信号到噪声的情况下，将时间分辨率分辨率为5 s。项目的具体目的将：（1）确定ATP合成过程中FO电动机在PMF中产生的旋转速度和扭矩； （2）通过粘度，通过降低相对于F1载荷的FO驱动力和突变体分析来确定通过粘度增加的FO，通过粘度增加的FO载荷的函数，确定束缚的形成和持续时间； （3）确定在ATP合成过程中确定9 ms催化停留物，并确定其他9ms停留是否是“底物等待”； （4）测试通过突变分析与ATP合成偶联旋转旋转的逃逸机制假设。 我们发现了亚基A和亚基-C残基CD44和CR50之间先前未知的系绳，提供了一个新窗口，可以使用该窗口检查FO运动能力旋转以催化ATP合成的机制。通过使用我们的创新方法将支持的平面脂质双层组合在一起，并结合我们的新型纳米棒测定法，此处提出的实验将提供有关FO分子运动机制的几个基本方面的重要新见解，并与F1运动与F1 Motor互动以催化Atphessiss of Atpeanseiss of Atpeans。 公共卫生相关性：共济失调，Leigh综合征，色素性视网膜炎，肌肉无力和家族性双侧纹状体坏死可能是由于对编码FOF1 ATP ATP合酶亚基的损害而造成的。一些患有心肌病或帕金森氏病的患者对这些基因的损害也增加了。所有独立的生命形式都使用FOF1作为细胞ATP的主要来源。