CALIXAR, 60 avenue Rockefeller 69008, Lyon, France; CHEM2STAB, laboratoire commun, 301 rue Baruch de Spinoza, 84916 Avignon cedex 9, France.
Institut des Biomolécules Max Mousseron (UMR 5247 UM-CNRS-ENSCM), Avignon University, Equipe Chimie Bioorganique et Systèmes amphiphiles, 301 rue Baruch de Spinoza, 84916 Avignon cedex 9, France; CHEM2STAB, laboratoire commun, 301 rue Baruch de Spinoza, 84916 Avignon cedex 9, France.
Methods. 2020 Aug 1;180:19-26. doi: 10.1016/j.ymeth.2020.02.005. Epub 2020 Feb 13.
Membrane proteins (MPs) are important drug discovery targets for a wide range of diseases. Conventional detergents such as n-Dodecyl β-D-maltoside have been used largely and efficiently to solubilize MPs with varying degrees of success concerning MPs functionality and stability. Fluorinated surfactants (FSs) have shown a great potential for the stabilization of various MPs. However, so far only a limited number of reports have demonstrated the ability of FSs to solubilize MPs from biological membranes. We report herein the use of a fluorinated lactobionamide-based detergent named FLAC6 for functional and structural stabilization of membrane proteins. We first demonstrated that FLAC6 efficiently solubilized three membrane proteins i.e. the native adenosine receptor AR, a G protein-coupled receptor, and two native transporters AcrB and BmrA. The resulting affinity purified MPs were highly pure, homogenous and aggregates free. Furthermore, the functionality of each MP was well maintained. Finally, striking overstabilization features were observed. Indeed, the Tm of native AR, AcrB and BmrA could be improved by 7, ~9 and ~ 23 °C, respectively when FLAC6 was used instead of the reference detergent. This work illustrates that FLAC6 is an efficient tool to maintain structural and functional integrities of different MPs belonging to different classes, providing a new avenue for functional stabilization of highly druggable and challenging membrane proteins involved in unmet medical needs.
膜蛋白（MPs）是广泛疾病的重要药物发现靶点。传统的去污剂，如 n-十二烷基 β-D-麦芽糖苷，已被广泛有效地用于溶解 MP，在 MP 功能和稳定性方面取得了不同程度的成功。氟化表面活性剂（FSs）已显示出稳定各种 MPs 的巨大潜力。然而，到目前为止，只有有限的报道表明 FSs 能够从生物膜中溶解 MPs。本文报告了一种名为 FLAC6 的基于氟代乳糖酰胺的去污剂用于膜蛋白的功能和结构稳定。我们首先证明 FLAC6 可以有效地溶解三种膜蛋白，即天然的腺苷受体 AR、一种 G 蛋白偶联受体以及两种天然的转运体 AcrB 和 BmrA。所得亲和纯化的 MPs 高度纯净、均一且无聚集体。此外，每个 MP 的功能都得到了很好的维持。最后，观察到了惊人的过稳定特征。事实上，当使用 FLAC6 代替参考去污剂时，天然 AR、AcrB 和 BmrA 的 Tm 可以分别提高 7、9 和23°C。这项工作表明，FLAC6 是一种有效的工具，可以维持不同类别 MPs 的结构和功能完整性，为解决未满足医疗需求的高度可成药和具有挑战性的膜蛋白的功能稳定提供了新途径。
