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
Functionalization of larger vesicles by this approach is more challenging, as vesicle size gets reduced after detergent removal, and it is not possible for giant unilamellar vesicles (GUV, >1 μm), as they are destabilized by detergents
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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