Fo Motor Mechanisms that Power FoF1 ATP Synthesis

为 FoF1 ATP 合成提供动力的 Fo 电机机制

基本信息

批准号：
8640195
负责人：
WAYNE D FRASCH
金额：
$ 30.5万
依托单位：
ARIZONA STATE UNIVERSITY-TEMPE CAMPUS
依托单位国家：
美国
项目类别：
财政年份：
2011
资助国家：
美国
起止时间：
2011-04-01 至 2016-03-31
项目状态：
已结题

项目摘要

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）：编码 FoF1 ATP 合酶亚基的基因受损可能导致共济失调、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。 FoF1 ATP 合酶有两个通过公共轴连接的相对旋转分子马达。集成膜 Fo 电机使用质子动力 (PMF) 驱动轴旋转，以实现依赖于 F1 的 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量来源。 Fo 电机使用布朗棘轮来偏置顺时针旋转以对抗 F1 电机施加的负载。我们最近观察到当 ATP 酶驱动的旋转因粘度引起的负载减慢时，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着特征是，它形成了一条将旋转限制为 360 度的系链。cD44N/cR50 突变体消除了系链形成，并导致氧化磷酸化依赖性大肠杆菌生长丧失，表明系链是 Fo 运动的重要组成部分。 ATP 体内合成。提出了一种机械假设，其中系绳使 Fo 电机能够在 ATP 合成过程中针对 F1 电机施加的负载进行顺时针旋转。这里提出的工作重点是测试 FoF1 合成 ATP 时的 Fo 运动机制假设。与 F1 相比，人们对 Fo 机制了解甚少，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。现在将在显微镜载玻片上制作一种新型支撑平面脂质双层组件，其中包含定向 FoF1 ATP 合酶分子，以便能够在 ATP 合成过程中进行单分子旋转测量，时间分辨率达到 5 秒，信噪比前所未有。该项目的具体目标是：（1）确定 Fo 电机在 ATP 合成过程中产生的转速和扭矩作为 PMF 的函数； (2) 通过增加 ATP/ADP.Pi、通过相对于 F1 负载减少 Fo 驱动力以及通过突变体分析，确定 ATP 合成过程中系链的形成和持续时间作为通过粘度施加在 Fo 上的负载的函数； (3) 识别 ATP 合成过程中的 9 ms 催化停留，并确定其他 9 ms 停留是否为“底物等待”； (4)通过突变分析检验旋转与ATP合成耦合的擒纵机构假说。我们发现了亚基 a 和亚基 c 残基 cD44 和 cR50 之间先前未知的连接链，这为研究 Fo 马达驱动旋转以催化 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撑平面脂质双层，并结合我们的新型纳米棒测定，这里提出的实验将为 Fo 分子马达机制的几个基本方面提供重要的新见解，以及它与 F1 马达连接，催化 ATP 的合成。