Reconstitution of purified membrane protein dimers in lipid nanodiscs with defined stoichiometry and orientation using a split GFP tether.
Reconstitution of purified membrane protein dimers in lipid nanodiscs with defined stoichiometry and orientation using a split GFP tether.
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DOI：
10.1016/j.jbc.2022.101628
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发表时间：
2022-04
期刊：
影响因子：
--
通讯作者：
Weis WI
中科院分区：
文献类型：
--
作者：
Bruguera ES;Mahoney JP;Weis WI
许多膜蛋白作为二聚体或更大的寡聚体起作用，包括转运蛋白、通道、某些信号受体和粘附分子。在某些情况下，单个蛋白质之间的相互作用可能很弱和/或依赖于特定的脂质，使得用于生物化学和结构研究的去污剂溶解破坏功能性寡聚化。溶解的膜蛋白低聚物可以被捕获在脂质纳米盘中，但这是一个低效的过程，可以产生化学计量和拓扑异构的制剂。在这里，我们描述了一种技术，以获得纯化的均匀的膜蛋白二聚体的纳米盘使用分裂GFP（sGFP）系链。互补的sGFP标签关联到系链的共表达的二聚体和控制纳米盘内的化学计量和方向，如通过定量Western印迹和负染色EM评估的。与其他方法相比，sGFP系链具有几个优点：它在溶液中和SDS-PAGE中高度稳定，这有助于通过荧光筛选二聚体表达和纯化，并且还提供了与GFP纳米抗体缀合的树脂一起使用的二聚体特异性纯化手柄。我们使用这种方法来纯化纳米盘中的卷曲蛋白-4同源二聚体和卷曲蛋白-4/低密度脂蛋白受体相关蛋白6异源二聚体。这些实施例证明了这种方法的实用性和灵活性，这使得膜蛋白对的后续机制分子和结构研究成为可能。
Many membrane proteins function as dimers or larger oligomers, including transporters, channels, certain signaling receptors, and adhesion molecules. In some cases, the interactions between individual proteins may be weak and/or dependent on specific lipids, such that detergent solubilization used for biochemical and structural studies disrupts functional oligomerization. Solubilized membrane protein oligomers can be captured in lipid nanodiscs, but this is an inefficient process that can produce stoichiometrically and topologically heterogeneous preparations. Here, we describe a technique to obtain purified homogeneous membrane protein dimers in nanodiscs using a split GFP (sGFP) tether. Complementary sGFP tags associate to tether the coexpressed dimers and control both stoichiometry and orientation within the nanodiscs, as assessed by quantitative Western blotting and negative-stain EM. The sGFP tether confers several advantages over other methods: it is highly stable in solution and in SDS-PAGE, which facilitates screening of dimer expression and purification by fluorescence, and also provides a dimer-specific purification handle for use with GFP nanobody–conjugated resin. We used this method to purify a Frizzled-4 homodimer and a Frizzled-4/low-density lipoprotein receptor–related protein 6 heterodimer in nanodiscs. These examples demonstrate the utility and flexibility of this method, which enables subsequent mechanistic molecular and structural studies of membrane protein pairs.
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影响因子：
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