Krasnoselska Ganna O, Meier Thomas
Department of Structural Biology, Max Planck Institute of Biophysics, Frankfurt am Main, Germany.
Department of Life Sciences, Imperial College London, London, UK.
Methods Mol Biol. 2025;2881:65-86. doi: 10.1007/978-1-0716-4280-1_3.
F-type Adenosine triphosphate (ATP) synthase is a membrane-bound macromolecular complex, which is responsible for the synthesis of ATP, the universal energy source in living cells. This enzyme uses the proton- or sodium-motive force to power ATP synthesis by a unique rotary mechanism and can also operate in reverse, ATP hydrolysis, to generate ion gradients across membranes. The FF-ATP synthases from bacteria consist of eight different structural subunits, forming a complex of ~550 kDa in size. In the bacterium Ilyobacter tartaricus, the ATP synthase has the stoichiometry αβγδεabc. This chapter describes a wet-lab working protocol for the purification of several tens of milligrams of pure, heterologously (E. coli-) produced I. tartaricus Na-driven FF-ATP synthase and its subsequent efficient reconstitution into proteoliposomes. The methods are useful for a broad range of subsequent biochemical and biotechnological applications.
F型三磷酸腺苷（ATP）合酶是一种膜结合大分子复合物，负责合成ATP，即活细胞中的通用能量来源。该酶利用质子动力或钠动力，通过独特的旋转机制为ATP合成提供能量，并且还可以反向运行，即ATP水解，以在膜上产生离子梯度。细菌的F₀F₁-ATP合酶由八个不同的结构亚基组成，形成一个大小约为550 kDa的复合物。在嗜酒伊氏杆菌中，ATP合酶的化学计量为αβγδεabc。本章描述了一种湿实验室工作方案，用于纯化几十毫克纯的、异源（大肠杆菌）产生的嗜酒伊氏杆菌钠驱动的F₀F₁-ATP合酶，并随后将其高效重组到蛋白脂质体中。这些方法对于广泛的后续生化和生物技术应用非常有用。
