Department of Biology, Moscow Lomonosov State University, 119234, Moscow, Russia.
Department of Physics, University of Osnabrück, 49069, Osnabrück, Germany.
Chem Phys Lipids. 2019 Mar;219:50-57. doi: 10.1016/j.chemphyslip.2019.01.013. Epub 2019 Jan 31.
Styrene-maleic acid (SMA) copolymers are used to extract lipid-encased membrane proteins from lipid bilayers in a detergent-free manner, yielding SMA lipid particles (SMALPs). SMALPs can serve as stable water-soluble nanocontainers for structural and functional studies of membrane proteins. Here, we used SMA copolymers to study full-length pore-forming α-subunits hKCNH5 and hKCNQ1 of human neuronal and cardiac voltage-gated potassium (Kv) channels, as well as the fusion construct comprising of an α-subunit hKCNQ1 and its regulatory transmembrane KCNE1 β-subunit (hKCNE1-hKCNQ1) with added affinity tags, expressed in mammalian COS-1 cells. All these recombinant proteins were shown to be functionally active. Treatment with the SMA copolymer, followed by purification on the affinity column, enabled extraction of all three channels. A DLS experiment demonstrated that negative stain electron microscopy and single particle image analysis revealed a four-fold symmetry within channel-containing SMALPs, which indicates that purified hKCNH5 and hKCNQ1 channels, as well as the hKCNE1-hKCNQ1 fusion construct, retained their structural integrity as tetramers.
苯乙烯-马来酸（SMA）共聚物用于以无去污剂的方式从脂质双层中提取包裹脂质的膜蛋白，得到 SMA 脂质颗粒（SMALPs）。SMALPs 可作为膜蛋白结构和功能研究的稳定水溶性纳米容器。在这里，我们使用 SMA 共聚物研究了全长的人神经元和心脏电压门控钾（Kv）通道的孔形成α-亚基 hKCNH5 和 hKCNQ1，以及包含 α-亚基 hKCNQ1 和其调节跨膜 KCNE1β-亚基（hKCNE1-hKCNQ1）的融合构建体，并添加了亲和标签，在哺乳动物 COS-1 细胞中表达。所有这些重组蛋白均显示出功能活性。用 SMA 共聚物处理，然后在亲和柱上纯化，可提取所有三种通道。DLS 实验表明，负染色电子显微镜和单颗粒图像分析显示通道包含的 SMALPs 具有四重对称性，这表明纯化的 hKCNH5 和 hKCNQ1 通道以及 hKCNE1-hKCNQ1 融合构建体作为四聚体保留了其结构完整性。
