Multi-dimensional 1H– 13C HETCOR and FSLG-HETCOR NMR study of sphingomyelin bilayers containing cholesterol in the gel and liquid crystalline states

Abstract

13C cross polarization magic angle spinning (CP-MAS) and 1H MAS NMR spectra were collected on egg sphingomyelin (SM) bilayers containing cholesterol above and below the liquid crystalline phase transition temperature ( T m). Two-dimensional (2D) dipolar heteronuclear correlation (HETCOR) spectra were obtained on SM bilayers in the liquid crystalline ( L α ) state for the first time and display improved resolution and chemical shift dispersion compared to the individual 1H and 13C spectra and significantly aid in spectral assignment. In the gel ( L β ) state, the 1H dimension suffers from line broadening due to the 1H– 1H homonuclear dipolar coupling that is not completely averaged by the combination of lipid mobility and MAS. This line broadening is significantly suppressed by implementing frequency switched Lee–Goldburg (FSLG) homonuclear 1H decoupling during the evolution period. In the liquid crystalline ( L α ) phase, no improvement in line width is observed when FSLG is employed. All of the observed resonances are assignable to cholesterol and SM environments. This study demonstrates the ability to obtain 2D heteronuclear correlation experiments in the gel state for biomembranes, expands on previous SM assignments, and presents a comprehensive 1H/ 13C NMR assignment of SM bilayers containing cholesterol. Comparisons are made to a previous report on cholesterol chemical shifts in dimyristoylphosphatidylcholine (DMPC) bilayers. A number of similarities and some differences are observed and discussed.