Function and dynamics of rotor-stator interactions in the proton-translocating Fo motor of ATP synthase

项目总结 F1Fo ATP合成酶是一种基本的能量转换复合体，存在于所有已知的细胞中。这个综合体使用一种 离子的电化学梯度，以驱动导致腺苷合成的旋转机制 三磷酸(ATP)，生命中普遍存在的化学能源货币。在大肠杆菌中，质子驱动的火焰马达， 嵌入细胞膜，由十聚体c亚基转子环组成，与a亚基接触。 定子。质子通过两个偏置的水溶液半通道通过FO。质子通过亚基a从 C-环的周质(P侧)。C环旋转后，质子以半通道进入细胞质(N侧) 在交流界面处形成。N侧通道上的一簇簇残基与质子有关 转位和可能的门控机制，但这些残基的作用尚不清楚。这个项目寻求 让本科生研究人员参与生化和生物物理相结合的方法来 描述转子和定子之间的相互作用，并确定它们在质子传输中的作用(S)。目标1将 系统检测Ac基因N侧几个关键残基的突变和化学修饰 与体外生化分析电池的接口，以确定每个残基在质子驱动中的作用 三磷酸腺苷合成和三磷酸腺苷驱动的质子泵AIM 2将使用定点定向自旋标记和电子 顺磁共振(EPR)谱探测环区的结构和结构动力学 与C-环相互作用的亚基A。自旋标签移动性、可访问性和接近性的模式将揭示 A-c界面上相互作用的细胞质环在ATP过程中是否发生构象变化 合成或水解。结构数据和功能数据一起将有助于理解 质子在这一重要生物分子机器中传输的分子机制及其对未来药物的启示 发展。

PROJECT SUMMARY F1Fo ATP synthase is a fundamental energy conversion complex found in all known cells. This complex uses an electrochemical gradient of ions to drive a rotary mechanism that results in the synthesis of adenosine triphosphate (ATP), the universal chemical energy currency of life. In E. coli, the proton-driven Fo motor, embedded in the cell membrane, consists of a decameric subunit c rotor ring in contact with subunit a of the stator. Protons move through Fo via two offset aqueous half channels. Protons enter through subunit a from the periplasm (P-side) to the c-ring. After c-ring rotation, protons exit to the cytoplasm (N-side) by a half channel formed at the a-c interface. Clusters of residues along the N-side channel have been implicated in proton translocation and a possible gating mechanism, but the roles of these residues are unclear. This project seeks to engage undergraduate student researchers in a combined biochemical and biophysical approach to characterize interactions between the rotor and stator and establish their role(s) in proton transport. Aim 1 will systematically examine mutations and chemical modifications of several key residues on the N-side of the a-c interface with a battery of in vitro biochemical assays to determine the role of each residue in proton-driven ATP synthesis and ATP-driven proton pumping. Aim 2 will use site-directed spin labeling and electron paramagnetic resonance (EPR) spectroscopy to probe the structure and structural dynamics of the loop regions of subunit a that interact with the c-ring. Patterns of spin label mobility, accessibility, and proximity will reveal whether interacting cytoplasmic loops at the a-c interface undergo conformational changes during ATP synthesis or hydrolysis. Together, the structural and functional data will contribute to understanding the molecular mechanism of proton transport in this important biomolecular machine and inform future drug development.