Sample preparation and measurements of 2H NMR

Sample preparation and ²H NMR measurements were conducted in a manner similar to that in our previous work (37). In this study, the bilayer compositions prepared were 7.0 μmol of d₃₁-POPC, 0.78 μmol of PCer, and 0.78 μmol of either Chol or CholPC; 31.3 μmol of POPC, 3.48 μmol of d₂-PCer, and 3.48 μmol of either Chol or Chol-PC; and POPC and 3-d₁-Chol (9:1, 34.8 μmol/3.87 μmol) with or without PCer (3.87 μmol). These mixtures were dissolved in MeOH-CHCl₃, and the solvents were evaporated, after which they were kept in vacuo overnight. MLVs were prepared by hydrating the dried lipid films with ∼1 mL of deuterium-depleted water at 65°C followed by vigorous vortexing. Each suspension was freeze-thawed 10 times, then lyophilized, rehydrated with deuterium-depleted water until the films were 50% hydrated (w/w), and freeze-thawed another 10 times. Each sample was transferred into a 5 mm glass tube (Wilmad, Vineland, NJ), which was sealed with epoxy glue.

All the ²H NMR spectra were recorded on a 300 MHz CMX300 spectrometer (Chemagnetics, Agilent, Palo Alto, CA) fitted with a 5 mm ²H static probe (Otsuka Electronics, Osaka, Japan) using a quadrupolar echo sequence. The 90° pulse width was 2.5 μs, interpulse delay was 30 μs, and the repetition rate was 0.6 s. The sweep width was 250 MHz and the number of scans was around 100,000. ²H data analysis was performed as described previously (37, 38).