Folding and stabilizing membrane proteins in amphipol A8-35
Folding and stabilizing membrane proteins in amphipol A8-35
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DOI：
10.1016/j.ymeth.2018.04.012
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发表时间：
2018-09-01
期刊：
影响因子：
4.8
通讯作者：
Zoonens, Manuela
中科院分区：
文献类型：
--
作者：
Le Bon, Christel;Marconnet, Anais;Zoonens, Manuela
膜蛋白（MP）是重要的药理学靶点，因为它们参与许多重要的细胞过程，其功能障碍可导致多种疾病。详细了解MP的结构及其活性的分子机制对于设计新的治疗剂是必不可少的。然而，在体外研究MP是具有挑战性的，因为它通常意味着它们在功能形式下的过表达，然后从膜中提取和纯化。将过表达的MP靶向膜通常是有毒的，并且表达产量往往是有限的。一种替代方法是在细胞的胞质溶胶中形成包涵体（IB），然后在可以考虑进行结构和功能分析之前，MP需要从包涵体折叠成其天然构象。折叠针对IB的MP是一项艰巨的任务。被称为“amphipols”（APols）的专门设计的两亲聚合物（其最初是为了与洗涤剂相比改善MP在水溶液中的稳定性而开发的）可用于折叠α-螺旋和β-桶MP。APols代表了一种有趣的新型两亲介质，其中可以实现高折叠产率。在这篇综述中，APol A8-35和他们形成的复合物与MP的性质进行了总结。概述了迄今为止报道的使用A8-35折叠MP的最重要的研究。最后，从实用的角度出发，对折叠和陷印方法进行了详细的说明。
Membrane proteins (MPs) are important pharmacological targets because of their involvement in many essential cellular processes whose dysfunction can lead to a large variety of diseases. A detailed knowledge of the structure of MPs and the molecular mechanisms of their activity is essential to the design of new therapeutic agents. However, studying MPs in vitro is challenging, because it generally implies their overexpression under a functional form, followed by their extraction from membranes and purification. Targeting an overexpressed MP to a membrane is often toxic and expression yields tend to be limited. One alternative is the formation of inclusion bodies (IBs) in the cytosol of the cell, from which MPs need then to be folded to their native conformation before structural and functional analysis can be contemplated. Folding MPs targeted to IBs is a difficult task. Specially designed amphipathic polymers called 'amphipols' (APols), which have been initially developed with the view of improving the stability of MPs in aqueous solutions compared to detergents, can be used to fold both alpha-helical and beta-barrel MPs. APols represent an interesting novel amphipathic medium, in which high folding yields can be achieved. In this review, the properties of APol A8-35 and of the complexes they form with MPs are summarized. An overview of the most important studies reported so far using A8-35 to fold MPs is presented. Finally, from a practical point of view, a detailed description of the folding and trapping methods is given.