Sample preparation and measurements of 2H NMR

Sample preparation and ²H NMR measurements were conducted in a manner similar to that described in our previous work (30). The bilayers were composed of POPC with 10 mol % stearoyl ceramide, which had site-specific deuteration at 12,12 in the long-chain base or at 10′,10′ of the stearoyl chain. The lipid 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, followed by lyophilization, rehydration with deuterium-depleted water until 50% hydration was achieved (w/w), and freeze-thawed 10 times again. Each sample was transferred into a 5 mm glass tube (Wilmad, Vineland, NJ) that was sealed with epoxy glue. All of 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, the interpulse delay was 30 μs, and the repetition rate was 0.6 s. The sweep width was 250 kHz and the number of scans was ∼100,000.