Bavarian NMR Center at the Department of Chemistry and Institute for Advanced Study, Technical University of Munich, Lichtenbergstrasse 4, 85747, Garching, Germany.
Institute of Structural Biology, Helmholtz Zentrum München, Ingolstädter Landstrasse 1, 85764, Neuherberg, Germany.
Chemistry. 2018 Apr 11;24(21):5493-5499. doi: 10.1002/chem.201800812. Epub 2018 Mar 23.
Structural studies on membrane-anchored proteins containing a transmembrane (TM) helix have been hampered by difficulties in producing these proteins in a natively folded form. Detergents that are required to solubilize the hydrophobic TM helix usually destabilize the soluble domain. Thus, TM helices are removed for structural studies, which neglects the pivotal role of a membrane on protein function. This work presents a versatile strategy for the production of this protein class attached to phospholipid nanodiscs. By inserting the TM-helix into nanodiscs and a subsequent SortaseA-mediated ligation of the soluble domain, membrane-anchored BclxL could be obtained in a folded conformation. This strategy is suitable for high-resolution structure determination as well as for probing membrane location by NMR. This method will be applicable to a wide range of membrane-anchored proteins and will be useful to decipher their functional role in a native membrane environment.
含有跨膜（TM）螺旋的膜锚定蛋白的结构研究受到以下困难的阻碍：难以以天然折叠形式生产这些蛋白。溶解疏水性 TM 螺旋所需的去污剂通常会使可溶性结构域不稳定。因此，TM 螺旋被去除用于结构研究，这忽略了膜在蛋白质功能中的关键作用。这项工作提出了一种将此类蛋白连接到磷脂纳米盘的通用策略。通过将 TM 螺旋插入纳米盘中，然后通过 SortaseA 介导的可溶性结构域连接，可获得折叠构象的膜锚定 BclxL。该策略适用于高分辨率结构测定以及通过 NMR 探测膜定位。该方法将适用于广泛的膜锚定蛋白，并有助于在天然膜环境中破译它们的功能作用。
