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: 8248706
• 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/8248706
• 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; public health relevance; 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、视网膜色素变性、肌肉无力和家族性双侧纹状体坏死可由编码FoF1 ATP合成酶亚基的基因受损引起。FoF1 ATP合成酶有两个相对的旋转分子马达，由一个共同的轴连接。整体式膜Fo马达利用质子动力（PMF）驱动车轴旋转进行f1依赖性ATP合成。在体内，FoF1维持[ATP]/[ADP][Pi]的比例远离平衡，使高[ATP]为细胞过程提供能量来源。Fo电机使用布朗棘轮来对F1电机施加的负载进行顺时针旋转。我们最近观察到，当atp酶驱动的旋转被粘度诱导的负载减慢时，FoF1的Fo亚基a和c之间存在一种以前未知的相互作用。这种相互作用的一个显著特征是，它形成了一个将旋转限制在360度的系绳。cD44N/cR50突变体消除了系链的形成，并导致氧化磷酸化依赖性大肠杆菌生长的丧失，表明系链是体内ATP合成的重要运动成分。提出了一种机械假说，其中系绳使Fo电机在ATP合成过程中对F1电机施加的负载进行顺时针棘轮旋转。本文提出的工作重点是验证FoF1合成ATP时的Fo运动机制假说。与F1相比，人们对Fo的机制知之甚少，部分原因是由于膜相关的技术问题，使得对Fo进行单分子研究非常困难。在显微镜载玻片上，一种新型的含有定向FoF1 ATP合成酶分子的支撑平面脂质双层组装将在前所未有的信噪比下，以5秒的时间分辨率测量ATP合成过程中的单分子旋转。该项目的具体目标将：(1)确定ATP合成过程中Fo电机产生的转速和扭矩作为PMF的函数；(2)通过增加ATP/ADP，确定ATP合成过程中系链的形成和持续时间，作为粘度对Fo施加负荷的函数。Pi，通过相对于F1载荷减小Fo驱动力，并通过突变体分析；(3)确定ATP合成过程中9ms的催化停留时间，并确定其他9ms的停留时间是否为“等待底物”；(4)通过突变体分析验证旋转与ATP合成耦合的擒纵机制假说。我们对亚基a和亚基c残基cD44和cR50之间先前未知的系链的发现，为研究Fo马达驱动旋转催化ATP合成的机制提供了一个新的窗口。通过使用我们的创新方法来组装支持的平面脂质双层，结合我们的新型纳米棒分析，这里提出的实验将提供有关Fo分子马达机制的几个基本方面的重要新见解，以及它与F1马达接口以催化ATP合成的方法。