Freely suspended liposomes are widely used as model membranes for studying lipid–lipid and protein–lipid interactions. Liposomes prepared by conventional methods have chemically identical bilayer leaflets. By contrast, living cells actively maintain different lipid compositions in the two leaflets of the plasma membrane, resulting in asymmetric membrane properties that are critical for normal cell function. Here, we present a protocol for the preparation of unilamellar asymmetric phospholipid vesicles that better mimic biological membranes. Asymmetry is generated by methyl-β-cyclodextrin-catalyzed exchange of the outer leaflet lipids between vesicle pools of differing lipid composition. Lipid destined for the outer leaflet of the asymmetric vesicles is provided by heavy-donor multilamellar vesicles containing a dense sucrose core. Donor lipid is exchanged into extruded unilamellar acceptor vesicles that lack the sucrose core, facilitating the post-exchange separation of the donor and acceptor pools by centrifugation because of differences in vesicle size and density. We present two complementary assays allowing quantification of each leaflet’s lipid composition: the overall lipid composition is determined by gas chromatography–mass spectrometry, whereas the lipid distribution between the two leaflets is determined by NMR, using the lanthanide shift reagent Pr3+. The preparation protocol and the chromatographic assay can be applied to any type of phospholipid bilayer, whereas the NMR assay is specific to lipids with choline-containing headgroups, such as phosphatidylcholine and sphingomyelin. In ~12 h, the protocol can produce a large yield of asymmetric vesicles (up to 20 mg) suitable for a wide range of biophysical studies.
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The authors acknowledge support from University of Windsor startup funds (to D.M.); Natural Sciences and Engineering Research Council of Canada (NSERC) funding ref. no. 2018-04841 (to D.M.); Austrian Science Fund (FWF) project P27083 (to G.P.); U.S. National Science Foundation (NSF) grant DMR 1709035 (to E.L.); NSF grant MCB-1817929 (to F.A.H.); and the Laboratory Directed Research and Development Program of Oak Ridge National Laboratory (to F.A.H., J.K., and R.F.S.), managed by UT-Battelle for the U.S. Department of Energy under contract no. DE-AC05 00OR22725.
The authors declare no competing interests.
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Key references using this protocol
1. Eicher, B. et al. Biophys. J. 114, 146–157 (2018): https://doi.org/10.1016/j.bpj.2017.11.009
2. Eicher, B. et al. J. Appl. Crystallogr. 50, 419–429 (2017): https://doi.org/10.1107/S1600576717000656
3. Marquardt, D. et al. Langmuir 33, 3731–3741 (2017): https://doi.org/10.1021/acs.langmuir.6b04485
4. Heberle, F. A. et al. Langmuir 32, 5195–5200 (2016): https://doi.org/10.1021/acs.langmuir.5b04562
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Doktorova, M., Heberle, F.A., Eicher, B. et al. Preparation of asymmetric phospholipid vesicles for use as cell membrane models. Nat Protoc 13, 2086–2101 (2018). https://doi.org/10.1038/s41596-018-0033-6
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