Molecular mechanism of the ATP synthase proton channel

ATP合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储存的电化学能量转化为三磷酸腺苷（ATP）分子中高能键的纯化学形式，ATP是细胞的通用能量货币单位。ATP合酶的功能就像一个分子涡轮机，由质子通过蛋白质的膜结构域的跨膜流动驱动。质子梯度能转化为转子运动动能的分子机理尚不清楚。虽然转子的高分辨率结构已经解决，定子的结构仍然很大程度上是未知的。本工作的长期目标是阐明质子通过ATP合酶膜通道转移的机制。我们计划结合高分辨率核磁共振波谱（NMR）、x射线晶体学、生化方法和分子模型来分析质子通道的结构。我们将使用工程蛋白设计来模拟质子通道的基本结构元素，通过x射线晶体学来解决结构问题。这种方法可能为解决ATP合酶的完整膜结构域的尝试提供了一种替代方法，迄今为止尚未成功。同时，我们将继续利用多维核磁共振对亚基“a”的整体褶皱进行研究。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一起，将通过受限制的分子动力学指导质子通道的分子建模，直到高分辨率x射线结构可用。核磁共振是研究膜蛋白分子动力学以及瞬时蛋白-脂质和蛋白-蛋白相互作用的独特有力工具。它还可以提供蛋白质晶体学无法提供的有价值的结构信息。亚基“a”的研究将推动高分辨率核磁共振在膜蛋白研究中的应用。

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.