Abstract
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.
Highlights
Functionalization of artificial lipid bilayers with membrane proteins is a key step in assembly of membrane model systems
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
The following few issues need to be considered for success of this experimental approach: Choice of lipid charge for proteoliposomes and target bilayers: Cationic lipids are not found in nature, while anionic lipids are abundant in biological membranes reaching, for example, ~25, 35 and 20% in inner membrane of E. coli, plasma membrane of yeast S. cerevisiae, and inner mitochondrial membranes of many species, respectively[27,28,29]
Summary
Functionalization of artificial lipid bilayers with membrane proteins is a key step in assembly of membrane model systems. Vesicle fusion is enabled and driven either by conformational rearrangements within complementary fusogenic agents (some proteins[11,12] and peptides[13] or specially modified DNA14) located in the contacting bilayers, or Coulombic interactions between lipid bilayers formed of complementarily charged cationic and anionic lipids[15,16], or cationic bilayers and negatively charged proteins[17]. The former approach requires the presence of fusogenic agents in the interacting membranes prior to fusion, is relatively slow (~30 min to reach half-maximum of fusion12,18), but can be applied to both natural and artificial membranes. A relative disadvantage of the method is that cationic lipids may exert a negative effect on functionality of membrane proteins in cationic proteoliposomes prior to fusion, especially in low ionic strength, but this effect is reversible and mitigated by a natural lipid composition of the post-fusion membrane and its return to the normal ionic strength medium
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