Comprehensive Molecular Motion Capture for Sphingomyelin by Site-Specific Deuterium Labeling

Abstract

Lipid rafts have attracted much attention because of their significant functional roles in membrane-associated processes. It is thought that sphingomyelin and cholesterol are essential for forming lipid rafts; however, their motion characteristics are not fully understood despite numerous studies. Here we show accurate local motions encompassing an entire sphingomyelin molecule, which were captured by measuring quadrupole splittings for 19 kinds of site-specifically deuterated sphingomyelins (that is, molecular motion capture of sphingomyelin). The quadrupole splitting profiles, which are distinct from those reported from perdeuterated sphingomyelins or simulation studies, reveal that cholesterol enhances the order in the middle parts of the alkyl chains more efficaciously than at the shallow positions. Comparison with dimyristoylphosphocholine bilayers suggests that cholesterol is deeper in sphingomyelin bilayers, which likely explains the so-called umbrella effect. The experiments also demonstrate that (i) the C2'-C3' bond predominantly takes the gauche conformation, (ii) the net ordering effect of cholesterol in sphingomyelin bilayers is not larger than that in phosphatidylcholine bilayers, (iii) cholesterol has no specific preference for the acyl or sphingosine chain, (iv) the acyl and sphingosine chains seem mismatched by about two methylene lengths, and (v) the motion of the upper regions of sphingomyelin chains is less temperature dependent than that of lower regions probably due to intermolecular hydrogen bond formation among SM molecules. These insights into the atomic-level dynamics of sphingomyelin provide critical clues to understanding the mechanism of raft formation.

Sphingomyelin distribution in lipid rafts of artificial monolayer membranes visualized by Raman microscopy

“…The outer and inner Pake doublets correspond to the L o and L d domains, respectively (Fig. 7C) (28). The diyne-SM mixture gave a pair of Pake doublets at Δν = 48.1 kHz, which is close to the outer Pake obtained from the SM mixture (Δν = 51.9 kHz) (Fig.…”

“…S9 and S10); d 2 -diyne-SM was synthesized similarly to diyne-SM, and d 2 -SM was prepared (details in Figs. S11 and S12), as reported (28). Other chemicals were purchased from Wako Pure Chemical Industries, Ltd.…”

Deuterium NMR of Raft Model Membranes Reveals Domain-Specific Order Profiles and Compositional Distribution

“…We recently synthesized site-specifically deuterated N-stearoylsphingomyelin (SSM, Fig. 1 a) and measured their quadrupole splitting in single-component SSM and binary SSM-Chol membrane systems, which revealed accurate segmental motions of methylene groups encompassing an entire SSM molecule (29,30). In this report, we further applied the site-specifically deuterated SSMs to raft-exhibiting ternary mixtures (SSM/DOPC/Chol) and acquired the quadrupole coupling profiles of SSM from L o and L d domains separately and simultaneously.…”

Novel Raman-tagged sphingomyelin that closely mimics original raft-forming behavior

“…Herein, site-selectively 2 H-labeled compounds were used to avoid overlapping of quadrupole doublet signals found in perdeuterated acyl chains, 24 which can alter the physical properties of lipid bilayers as well. 25 Our previous study indicated that chol enhances the order of the C10 position more effectively than other positions in the acyl chains of SM, 15 implying that the 2 H NMR spectrum should show clearly separated signals for the L o and L d phases. However, the clear phase separation of 5 is hardly observed in liposome preparation.…”