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(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合成酶亚单位的基因受损引起。FoF1-ATP合成酶有两个相对的旋转分子马达，通过一个共同的轴连接。整体式薄膜燃料发动机使用质子动力(PMF)来驱动车轴旋转，以实现F1依赖的ATP合成。在体内，FoF1保持远离平衡的[ATP]/[ADP][PI]比率，使高[ATP]为细胞过程提供能量来源。Fo马达使用布朗棘轮来偏置顺时针旋转，以对抗F1马达施加的负载。我们最近观察到当ATPase驱动的旋转被粘度诱导的负荷减慢时，FoF1的Fo亚基a和c之间存在先前未知的相互作用。这种相互作用的一个显著特点是，它形成了一条系绳，将自转限制在360度。CD44N/cR50突变体消除了系链的形成，并导致氧化磷酸化依赖的大肠杆菌生长丧失，表明系链是体内合成ATP的重要Fo马达成分。提出了一个机械假说，在ATP合成过程中，系绳使Fo马达能够对抗F1马达施加的负载进行顺时针旋转。本文提出的工作重点是在FoF1合成ATP时验证这一Fo运动机制假说。与F1相比，人们对Fo的作用机制知之甚少，部分原因是膜相关的技术问题使Fo的单分子研究变得非常困难。一种新型的含有定向FOF1三磷酸腺苷合成酶分子的支撑平面脂质双层将在显微镜载玻片上组装，以前所未有的信噪比实现三磷酸腺苷合成过程中单分子旋转测量，时间分辨率为5 S。该项目的具体目标将：(1)确定作为PMF的函数的Fo马达在ATP合成过程中产生的转速和扭矩；(2)通过增加ATP/ADP.PI、通过降低Fo相对于F1负载的驱动力以及通过突变分析来确定ATP合成过程中Fo的系绳形成和持续时间；(3)确定ATP合成过程中的9ms催化停留时间，并确定其他9ms停留时间是否为“底物等待”；以及(4)通过突变分析测试将旋转与ATP合成相结合的逃逸机制假说。我们在a亚基和c亚基残基CD44和cR50之间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。通过使用我们用于组装支持的平面脂质双层的创新方法与我们的新型纳米棒实验相结合，这里提出的实验将提供关于Fo分子马达机制的几个基本方面的重要的新见解，以及它与F1马达接口以催化合成ATP的方法。 公共卫生相关性：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合成酶亚单位的基因受损引起。一些患有心肌病或帕金森氏症的患者也增加了这些基因的损伤。所有独立的生命形式都使用FoF1作为细胞内ATP的主要来源。