Molecular mechanism of the ATP synthase proton channel

三磷酸腺苷合成酶是绝大多数生物体内能量代谢的关键酶。它将以跨膜离子梯度形式存储的电化学能量转换为三磷酸腺苷(ATP)分子中高能键的纯化学形式，三磷酸腺苷(ATP)分子作为细胞的通用能量流通单位。三磷酸腺苷合成酶作为一个分子涡轮，通过蛋白质的膜结构域驱动质子的跨膜流动。质子梯度能转化为转子运动动能的分子机制还不是很清楚。虽然转子的高分辨率结构已经解决，但定子的结构在很大程度上仍然未知。这项工作的长期目标是阐明通过三磷酸腺苷合成酶的膜通道进行质子转移的机制。我们计划结合高分辨率核磁共振光谱(核磁共振)、X射线结晶学、生化方法和分子建模来分析质子通道的结构。我们将使用设计用于模拟质子通道的基本结构元素的工程蛋白质，通过X射线结晶学来解决结构问题。这种方法可能为解决ATP合成酶完整膜结构域的结构提供了另一种选择，到目前为止，这种方法还没有成功。同时，我们将继续用多维核磁共振技术研究‘a’亚基的全球折叠。核磁共振研究提供的增量结构信息与大量已有的生化数据结合在一起，将指导通过受限分子动力学对质子通道进行分子建模，直到高分辨率X射线结构变得可用。核磁共振是研究膜蛋白分子动力学以及瞬时蛋白质-脂质和蛋白质-蛋白质相互作用的唯一有效工具。它还可以提供有价值的结构信息，而这正是蛋白质结晶学所不能做到的。A亚基的研究将推动高分辨率核磁共振技术在膜蛋白中的应用。

ATP synthase is a key enzyme of the energy metabolism in the vast majority of living organisms. It converts electrochemical energy stored in the form of transmembrane ion gradients into a pure chemical form of a high-energy bond in the adenosine triphosphate (ATP) molecule, which serves as a universal energy currency unit of the cell. ATP synthase functions as a molecular turbine driven by the transmembrane flow of protons through the membrane domain of the protein. The molecular mechanism of transformation of the energy of proton gradient into the kinetic energy of rotor movement is not well understood. While the high resolution structure of the rotor has been solved, the structure of the stator remains largely unknown. The long-term goal of this work is to elucidate the mechanism of proton transfer through the membrane channel of ATP synthase. We plan to analyze the structure of the proton channel using a combination of high-resolution nuclear magnetic resonance spectroscopy (NMR), X-ray crystallography, biochemical methods, and molecular modeling. We will use engineered proteins designed to model the essential structural elements of the proton channel to solve the structure by X-ray crystallography. This approach may offer an alternative to the attempts to solve the structure of the complete membrane domain of the ATP synthase, which so far have been unsuccessful. In parallel, we will continue investigation of the global fold of subunit 'a' by multidimensional NMR. The incremental structural information provided by NMR studies taken together with the large body of already available biochemical data will guide molecular modeling of the proton channel by restrained molecular dynamics until the high-resolution X-ray structure becomes available. NMR is a uniquely powerful tool for investigation of molecular dynamics of membrane proteins, as well as transient protein-lipid and protein-protein interactions. It can also provide valuable structural information, where protein crystallography fails. Investigation of subunit 'a' will advance application of high-resolution NMR to membrane proteins.