Raftlike Mixtures of Sphingomyelin and Cholesterol Investigated by Solid-State ²H NMR Spectroscopy

Abstract

Sphingomyelin is a lipid that is abundant in the nervous systems of mammals, where it is associated with putative microdomains in cellular membranes and undergoes alterations due to aging or neurodegeneration. We investigated the effect of varying the concentration of cholesterol in binary and ternary mixtures with N-palmitoylsphingomyelin (PSM) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) using deuterium nuclear magnetic resonance ( 2 H NMR) spectroscopy in both macroscopically aligned and unoriented multilamellar dispersions. In our experiments, we used PSM and POPC perdeuterated on the N-acyl and sn-1 acyl chains, respectively. By measuring solidstate 2 H NMR spectra of the two lipids separately in mixtures with the same compositions as a function of cholesterol mole fraction and temperature, we obtained clear evidence for the coexistence of two liquid-crystalline domains in distinct regions of the phase diagram. According to our analysis of the first moments M 1 and the observed 2 H NMR spectra, one of the domains appears to be a liquidordered phase. We applied a mean-torque potential model as an additional tool to calculate the average hydrocarbon thickness, the area per lipid, and structural parameters such as chain extension and thermal expansion coefficient in order to further define the two coexisting phases. Our data imply that phase separation takes place in raftlike ternary PSM/POPC/cholesterol mixtures over a broad temperature range but vanishes at cholesterol concentrations equal to or greater than a mole fraction of 0.33. Cholesterol interacts preferentially with sphingomyelin only at smaller mole fractions, above which a homogeneous liquid-ordered phase is present. The reasons for these phase separation phenomena seem to be differences in the effects of cholesterol on the configurational order of the palmitoyl chains in PSM-d 31 and POPC-d 31 and a difference in the affinity of cholesterol for sphingomyelin observed at low temperatures. Hydrophobic matching explains the occurrence of raftlike domains in cellular membranes at intermediate cholesterol concentrations but not saturating amounts of cholesterol.

The Lipid Modifications of Ras that Sense Membrane Environments and Induce Local Enrichment

“…Figure 4 d shows a plot of the order parameters of the phospholipids in the mixture and of N-Ras. Consistent with the differences of l o and l d domains, [20][21][22] the order parameters of PSM are higher than those of POPC. Nevertheless, the lengths of the chains are relatively similar (15.1 for POPC and 16.1 for PSM).…”

“…[19] The raft (liquid-ordered, l o ) and liquid-disordered (l d ) domains of the membrane are characterized by different thicknesses. [20][21][22] Mostly long-chain saturated sphingomyelin (SM) and cholesterol are segregated into l o domains, whereas l d domains contain mostly unsaturated lipids, which are more disordered and therefore shorter. Recently, it was shown that the N-Ras proteins exhibited diffusion and subsequent clustering in the l o /l d phase boundaries.…”

Influence of the sterol aliphatic side chain on membrane properties: a molecular dynamics study

“…For these sterols, a preferential interaction with the saturated sn-1 chain of POPC (over the unsaturated sn-2 chain) was detected. Importantly, this preferential association with saturated lipid chains, previously suggested as a possible driving force for the formation of cholesterol-induced membrane domains such as lipid rafts, 5,8,83,84 is attenuated for both shorter-and longerchained sterols.…”

Area per Lipid and Cholesterol Interactions in Membranes from Separated Local-Field 13 C NMR Spectroscopy

“…Lipid-protein interactions (1) and the associated functions of biomembranes (2)(3)(4)(5)(6) are known to be significantly influenced by the composition (2,(7)(8)(9)(10)(11) and structure of the lipid bilayer (6,(11)(12)(13)(14)(15)(16). Recently, the importance of lipids in cellular membranes and tissues has made lipidomics (17) an emerging field in biomedical research.…”