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

ATP合酶质子易位Fo马达中转子-定子相互作用的功能和动力学

• 批准号: 9812498
• 负责人: Phillip Ryan Steed
• 金额: $ 32.67万
• 依托单位: UNIVERSITY OF NORTH CAROLINA ASHEVILLE
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-08-01 至 2023-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9812498
• 关键词: Address; Adenosine Triphosphate; Adopted; Affect; Automobile Driving; Biochemical; Biological Assay; Biological Models; Cell membrane; Cells; Chemicals; Collaborations; Complex; Coupled; Coupling; Cryoelectron Microscopy; Cytoplasm; Data; Drug Targeting; Electron Spin Resonance Spectroscopy; Escherichia coli; Future; Heterogeneity; Hydrolysis; In Vitro; Investigation; Ion Pumps; Ions; Knowledge; Libraries; Life; Light; Maps; Mechanics; Membrane; Modeling; Modification; Molecular; Molecular Conformation; Molecular Machines; Motor; Mutagenesis; Mutation; Oxidative Phosphorylation; Pathway interactions; Pattern; Permeability; Positioning Attribute; Process; Proton Pump; Protons; Pump; Research; Research Personnel; Resolution; Role; Rotation; Side; Site; Site-Directed Mutagenesis; Spin Labels; Structure; Students; Testing; Torque; Universities; Variant; aqueous; biophysical techniques; chemical property; crosslink; drug development; experimental study; interdisciplinary approach; mutant; periplasm; tuberculosis drugs; undergraduate student

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.

项目摘要 F1 Fo ATP合成酶是一种存在于所有已知细胞中的基本能量转换复合物。该综合体使用一个 离子的电化学梯度驱动旋转机制，导致腺苷的合成 三磷酸盐（ATP），生命的通用化学能量货币。在大肠大肠杆菌，质子驱动的Fo马达， 包埋在细胞膜中，由十聚体亚基c转子环与亚基a接触组成。 定子质子通过两个偏移的水半通道移动通过Fo。质子通过a亚基从 周质（P侧）到C环。C环旋转后，质子通过半通道离开细胞质（N侧 在a-c界面处形成。沿着N-侧通道沿着的残基簇与质子交换有关。 易位和可能的门控机制，但这些残基的作用尚不清楚。项目的目标是 让本科生研究人员参与生物化学和生物物理学相结合的方法， 表征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。目标1将 系统地检查了a-c的N侧上几个关键残基的突变和化学修饰， 与一组体外生物化学测定接口，以确定每个残基在质子驱动的 ATP合成和ATP驱动的质子泵。AIM 2将使用定点自旋标记和电子 顺磁共振（EPR）光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。自旋标签的移动性、可及性和邻近性的模式将揭示 在ATP作用期间，a-c界面上相互作用的胞质环是否经历构象变化 合成或水解。总之，结构和功能数据将有助于理解 质子在这一重要生物分子机器中传输的分子机制，并为未来的药物提供信息 发展