School of Biomedical Sciences, Faculty of Biological Sciences & Astbury Centre for Structural and Molecular Biology, University of Leeds, Leeds, LS2 9JT, UK.
Université de Paris, Laboratoire de Biologie Physico-Chimique des Protéines Membranaires, CNRS, UMR 7099, F-75005, Paris, France.
Commun Biol. 2021 Nov 25;4(1):1337. doi: 10.1038/s42003-021-02834-3.
Membrane proteins are essential for cellular growth, signalling and homeostasis, making up a large proportion of therapeutic targets. However, the necessity for a solubilising agent to extract them from the membrane creates challenges in their structural and functional study. Although amphipols have been very effective for single-particle electron cryo-microscopy (cryoEM) and mass spectrometry, they rely on initial detergent extraction before exchange into the amphipol environment. Therefore, circumventing this pre-requirement would be a big advantage. Here we use an alternative type of amphipol: a cycloalkane-modified amphiphile polymer (CyclAPol) to extract Escherichia coli AcrB directly from the membrane and demonstrate that the protein can be isolated in a one-step purification with the resultant cryoEM structure achieving 3.2 Å resolution. Together this work shows that cycloalkane amphipols provide a powerful approach for the study of membrane proteins, allowing native extraction and high-resolution structure determination by cryoEM.
膜蛋白对于细胞生长、信号传递和内稳态至关重要，它们构成了很大比例的治疗靶点。然而，为了从膜中提取它们，需要使用增溶剂，这给它们的结构和功能研究带来了挑战。尽管两性离子聚合物在单颗粒电子冷冻电镜（cryoEM）和质谱分析中非常有效，但它们依赖于最初用去污剂提取，然后再交换到两性离子聚合物环境中。因此，规避这个前提条件将是一个很大的优势。在这里，我们使用了一种替代类型的两性离子聚合物：环烷烃修饰的两亲聚合物（CyclAPol），直接从膜中提取大肠杆菌 AcrB，并证明该蛋白可以通过一步纯化进行分离，得到的 cryoEM 结构分辨率达到 3.2Å。这项工作共同表明，环烷烃两性离子聚合物为膜蛋白的研究提供了一种强大的方法，允许通过 cryoEM 进行天然提取和高分辨率结构测定。
