Detection and quantification of asymmetric lipid vesicle fusion using deuterium NMR.
It is demonstrated that deuterium nuclear magnetic resonance (2H NMR) spectroscopy can be used to detect and to quantify fusion between anionic giant unilamellar vesicles (GUVs) and cationic small unilamellar vesicles (SUVs). The sensitivity to fusion relies on the conformational response of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) to changes in membrane surface electrostatic charge due to lipid mixing upon fusion. This conformational change is reported in the 2H NMR spectrum as a change in the quadrupolar splitting from choline-deuterated POPC. GUVs were composed of varying molar ratios of the anionic lipid 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG), plus cholesterol (CHOL), plus POPC. SUVs were composed of the cationic lipid 1,2-dioleoyloxy-3-(dimethylammonio)-propane (DODAP), plus POPC with or without 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE). Using a quantitative model that relates the 2H NMR quadrupolar splitting to the mole fractions of cationic, anionic, and neutral lipids in the vesicle membrane, it was possible to deduce the extent of fusion between the two oppositely-charged vesicle populations directly from the quadrupolar splitting. SUVs composed of DODAP + POPC + POPE (40/40/20) fused 100% with GUVs composed of POPC + CHOL + POPG (60/30/10). Removing POPE from the SUVs reduced the extent of fusion, as did reducing the POPG content of the GUVs.
Franzin
C M
CM
Department of Chemistry, Erindale College, University of Toronto, Mississauga, Ontario, Canada.
Macdonald
P M
PM
D000838:Anions; D002412:Cations; D003903:Deuterium; D008051:Lipid Bilayers / Q000737:chemistry*; D008081:Liposomes / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008561:Membrane Fusion / Q000187:drug effects; D010714:Phosphatidylethanolamines / Q000494:pharmacology; D013050:Spectrometry, Fluorescence
D000838:Anions; D002412:Cations; D008051:Lipid Bilayers; D008081:Liposomes; D010714:Phosphatidylethanolamines; D003903:Deuterium