Investigating phase behaviour and nano-domain characteristics of sphingomyelin/cholesterol membrane using 2H NMR

Sphingomyelin (SM) and cholesterol are major components of the mammalian cell plasma membrane. It is suggested that assemblies of sphingolipids and cholesterol lead to the formation of domains known as “rafts”. In the SM/Cholesterol binary system cholesterol induces formation of a “liquid-ordered” (lo) phase that can coexist with either the “liquid-disordered” (ld) phase or the “solid-ordered” (so) phase. Similar lo-phase domain formation is observed in DPPC/Cholesterol and DPPC/ergosterol systems. Furthermore, cholesterol interacts more strongly with sphingomyelin than DPPC. Deuterium nuclear magnetic resonance spectroscopy (NMR) is used to study the dependence of the phase and nanodomain structure of sphingomyelin/cholesterol membranes on sterol content and temperature. NMR spectra of N-palmitoyl (D31)-D-erythro-sphingosylphosphorylcholine (PSM) were taken for temperatures from 25 to 70oC and cholesterol concentrations 0 - 40%. Analogous experiments were done using 1-palmitoyl,2-palmitoyl(D31)-sn-glycero-3-phosphocholine (DPPC)/cholesterol membranes in order to carefully compare the data obtained using palmitoyl chains which have similar “kinked” conformations. The constructed phase diagrams exhibit both so + lo and ld + lo phase coexistence regions, however macroscopic (micron-sized) coexistence of ld + lo does not occur. Instead, we observed line-broadening in the ld + lo region which was characterized by examining the cholesterol dependence of the quadrupolar splittings and linewidths of the peaks in the depaked spectra, at a given temperature. These results were analyzed by assuming fast exchange of lipids between ld and lo nanodomains which were found to be generally less than 10 nm across. By analyzing the depaked spectrum, information from all orientations of the lipid long axis relative to the magnetic field is pooled. This may obscure the results used to characterize domain size. Selectively deuterated sphingomyelin, labeled at C9 of the N-linked palmitoyl chain, was used to measure the orientation dependence of T2 relaxation time and to more directly calculate domain size. A phenomenological theory based on the hypothesis that fluctuations in local curvature and bilayer thickness are responsible for the orientation dependence of T2 provides an excellent fit to the data. The ld + lo domain sizes calculated using the C9 data agree well with those calculated from the depaked spectrum of PSM.