Depth-Dependent Segmental Melting of the Sphingomyelin Alkyl Chain in Lipid Bilayers

Abstract

The chain melting of lipid bilayers has often been investigated in detail using calorimetric methods, such as differential scanning calorimetry (DSC), and the resultant main transition temperature is regarded as one of the most important parameters in model membrane experiments. However, it is not always clear whether the hydrocarbon chains of lipids are gradually melting along the depth of the lipid bilayer or whether they all melt concurrently in a very narrow temperature range, as implied by DSC. In this study, we focused on stearoyl-D-sphingomyelin (SSM) as an example of raft-forming lipids. We synthesized deuterium-labeled SSMs at the 4′, 10′, and 16′ positions, and their depth-dependent melting was measured using solid-state deuterium NMR by changing the temperature by 1.0 °C, and comparing with that observed from a saturated lipid, palmitoylstearoylphosphatidylcholine (PSPC). The results showed that SSM exhibited a characteristic depth-dependent melting, which was not observed for PSPC. The strong intermolecular hydrogen bonds between the sphingomyelin amide moiety probably caused the chain melting to start from the chain terminus through the middle part and end in the upper part. This depth-dependent melting implies that the small gel-like domains of SSM remain at temperatures slightly above the main transition temperature. These sphingomyelin features may be responsible for the biological properties of SM-based lipid rafts.

LnDOTA‐d8, a versatile chemical‐shift thermometer for 2H solid‐state NMR

“…In our previous study, we examined the segment‐specific melting of the SSM acyl chain by examining thermal changes in the chemical shifts of deuterated methanol, [ 35 ] which showed similar results to the present results (see Figure 5). However, there is a small difference of about 1°C in the segmental melting point of the C16′ position from the present study.…”

“…We recently reported the membrane depth dependence of acyl chain melting in lipid bilayers using site‐selective labeled lipids and solid‐state NMR spectra. [ 35 ] We found that sphingomyelin gradually melts from the terminus of the alkyl chains in the bilayers, and the segment near the amide group then melts at a temperature that is higher by about 1.2°C than that determined by differential scanning calorimetry. However, because the level of inaccuracy in temperature measurements was generally large in NMR measurements, we needed to verify these results with higher accuracy using the 2 H chemical‐shift thermometer, ErDOTA‐ d 8 .…”

“…Based on these findings, it is clear that segmental melting in the SSM bilayer occurs from the terminal to the upper segment along the acyl chain, thus confirming our previous results. [ 35 ]…”

“…On the other hand, the internal rotation is suppressed at the C4′ and C10′ positions, so the Pake doublet does not appear until the disappearance of the gel‐like nanodomains, and the spectral shapes are less dependent on the pulse sequence, and so similar results to previous reports are expected for those positions. [ 35 ]…”

“…[ 33 ] Three kinds of deuterium‐labeled stearoyl‐sphingomyelin (SSM), d 2 ‐SSM, at the 4′, 10′, and 16′ positions of the acyl group were chemically synthesized following our previous paper (purity > 95%). [ 34,35 ] All other chemicals were purchased from standard vendors. 1 H, 2 H, and 13 C solution NMR spectra were recorded with an ECS400 NMR instrument (JEOL, Japan) equipped with a conventional 5 mm tunable probe.…”