Controlled Co-reconstitution of Multiple Membrane Proteins in Lipid Bilayer Nanodiscs Using DNA as a Scaffold
Controlled Co-reconstitution of Multiple Membrane Proteins in Lipid Bilayer Nanodiscs Using DNA as a Scaffold
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DOI：
10.1021/acschembio.5b00627
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发表时间：
2015-11-01
影响因子：
4
通讯作者：
Wagner, Gerhard
中科院分区：
文献类型：
--
作者：
Raschle, Thomas;Lin, Chenxiang;Wagner, Gerhard
纳米盘构成了一种用于溶解脂质双层中的膜蛋白的工具，从而提供了接近天然的膜环境。许多膜蛋白与其他膜蛋白相互作用;然而，在单个纳米盘中的多个膜蛋白的共重构是受包括蛋白质聚集的若干因素不利影响的随机过程。在这里，我们提出了一种方法，用于在单个纳米盘中控制多个膜蛋白的共重构。指定的寡核苷酸与单个膜蛋白的暂时连接使得能够通过互补寡核苷酸序列的碱基配对形成稳定的去污剂溶解的膜蛋白复合物，从而促进膜蛋白复合物插入具有限定的化学计量和组成的纳米盘中。作为原理的证明，使用荧光标记的电压门控阴离子通道（VDAC）作为模型系统重构含有异源二聚体和异源三聚体膜蛋白复合物的纳米盘。
Nanodiscs constitute a tool for the solubilization of membrane proteins in a lipid bilayer, thus offering a near native membrane environment. Many membrane proteins interact with other membrane proteins; however, the co-reconstitution, of multiple membrane proteins in a single nanodisc is a random process that is adversely affected by several factors, including protein aggregation. Here, we present an approach for the controlled co-reconstitution of multiple membrane proteins in a single nanodisc. The temporary attachment of designated oligonucleotides to individual membrane proteins enables the formation of stable; detergent-solubilized membrane protein complexes by base-pairing of complementary oligonucleotide sequences, thus facilitating the insertion of the membrane protein complex into nanodiscs with defined stoichiometry and composition. As a proof of principle nanodiscs containing a heterodimeric and heterotrimeric membrane protein complex were reconstituted using a fluorescently labeled voltage-gated anion channel (VDAC) as a model system.