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: 8448316
• 负责人: WAYNE D FRASCH
• 金额: $ 29.43万
• 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/8448316
• 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.

描述（由申请人提供）：共济失调、Leigh综合征、色素性视网膜炎、肌无力和家族性双侧纹状体坏死可由编码FoF 1 ATP合酶亚单位的基因损伤引起。FoF 1 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。完整的膜Fo马达使用质子动力（PMF）驱动轴旋转，用于F1依赖的ATP合成。在体内，FoF 1使[ATP]/[ADP][Pi]的比例远离平衡，使高[ATP]能够为细胞过程提供能量来源。Fo电机使用布朗棘轮来偏置顺时针旋转以抵抗F1电机施加的负载。 最近，我们观察到一个以前未知的Fo亚基之间的相互作用的FoF 1的A和C时，ATP酶驱动的旋转是由粘度引起的负载减慢。这种相互作用的一个显著特征是，它形成了一条将旋转限制在360度的系绳。cD 44 N/cR 50突变体消除了系链形成，并导致氧化磷酸化依赖性E.大肠杆菌生长，表明系链是一个重要的Fo马达组件的ATP合成在体内。提出了一种机制假说，其中系绳使Fo电机棘轮顺时针旋转对F1电机施加的负载在ATP合成。这里提出的工作的重点是测试这一Fo运动机制的假设时，FoF 1合成ATP。与F1相比，Fo机制知之甚少，部分原因是膜相关的技术问题使得对Fo进行单分子研究非常困难。 一种新的组装支持平面脂质双层含有定向FoF 1 ATP合酶分子在显微镜载玻片上，现在将使单分子旋转测量ATP合成过程中的时间分辨率为5秒，在前所未有的信噪比。该项目的具体目标将：（1）确定在ATP合成期间由Fo马达产生的旋转速度和扭矩作为PMF的函数;（2）通过增加ATP/ADP.Pi、通过相对于F1负载减小Fo驱动力以及通过突变体分析，确定在ATP合成期间作为经由粘度施加在Fo上的负载的函数的系链形成和持续时间;（3）识别ATP合成期间的9 ms催化停留，并确定其他9 ms停留是否是“底物等待”;以及（4）通过突变体分析来测试用于耦合旋转与ATP合成的擒纵机构假设。 我们发现的亚基a和亚基c残基cD 44和cR 50之间的以前未知的系链提供了一个新的窗口，以检查的机制，通过该机制Fo电机功率旋转催化ATP的合成。通过使用我们的创新方法支持的平面脂质双层的组装结合我们的新的纳米棒测定，这里提出的实验将提供重要的新的见解有关的几个基本方面的Fo分子马达的机制，以及通过它的接口与F1马达催化ATP的合成的手段。