Galkin Mikhail A, Russell Aidan N, Vik Steven B, Berry Richard M, Ishmukhametov Robert R
First Pavlov State Medical University.
Clarendon Laboratory, Department of Physics, Oxford University.
J Vis Exp. 2018 Apr 5(134):56909. doi: 10.3791/56909.
Detergents are indispensable for delivery of membrane proteins into 30-100 nm small unilamellar vesicles, while more complex, larger model lipid bilayers are less compatible with detergents. Here we describe a strategy for bypassing this fundamental limitation using fusogenic oppositely charged liposomes bearing a membrane protein of interest. Fusion between such vesicles occurs within 5 min in a low ionic strength buffer. Positively charged fusogenic liposomes can be used as simple shuttle vectors for detergent-free delivery of membrane proteins into biomimetic target lipid bilayers, which are negatively charged. We also show how to reconstitute membrane proteins into fusogenic proteoliposomes with a fast 30-min protocol. Combining these two approaches, we demonstrate a fast assembly of an electron transport chain consisting of two membrane proteins from E. coli, a primary proton pump bo3-oxidase and F1Fo ATP synthase, in membranes of vesicles of various sizes, ranging from 0.1 to >10 microns, as well as ATP production by this chain.
去污剂对于将膜蛋白递送至30 - 100纳米的小单层囊泡中是不可或缺的，而更复杂、更大的模型脂质双层与去污剂的兼容性较差。在此，我们描述了一种策略，即使用携带感兴趣膜蛋白的促融合带相反电荷脂质体来绕过这一基本限制。此类囊泡之间的融合在低离子强度缓冲液中5分钟内即可发生。带正电荷的促融合脂质体可用作简单的穿梭载体，用于在无去污剂的情况下将膜蛋白递送至带负电荷的仿生靶脂质双层中。我们还展示了如何通过一个快速的30分钟方案将膜蛋白重构到促融合蛋白脂质体中。结合这两种方法，我们证明了由来自大肠杆菌的两种膜蛋白（一种初级质子泵bo3 - 氧化酶和F1Fo ATP合酶）组成的电子传递链能够在大小从0.1微米到大于10微米不等的各种囊泡膜中快速组装，以及该链产生ATP的过程。
