Showing papers by "Michio Murata published in 2022"

Molecular substructure of the liquid-ordered phase formed by sphingomyelin and cholesterol: sphingomyelin clusters forming nano-subdomains are a characteristic feature

Abstract: As a model of lipid rafts, the liquid-ordered (Lo) phase formed by sphingomyelin (SM) and cholesterol (Cho) in bilayer membranes has long attracted the attention of biophysics researchers. New approaches and methodologies have led to a better understanding of the molecular basis of the Lo domain structure. This review summarizes studies on model membrane systems consisting of SM/unsaturated phospholipid/Cho implying that the Lo phase contains SM-based nanodomains (or nano-subdomains). Some of the Lo phase properties may be attributed to these nanodomains. Several studies suggest that the nanodomains contain clustered SM molecules packed densely to form gel-phase-like subdomains of single-digit nanometer size at physiological temperatures. Cho and unsaturated lipids located in the Lo phase are likely to be concentrated at the boundaries between the subdomains. These subdomains are not readily detected in the Lo phase formed by saturated phosphatidylcholine (PC) molecules, suggesting that they are strongly stabilized by homophilic interactions specific to SM, e.g., between SM amide groups. This model for the Lo phase is supported by experiments using dihydro-SM, which is thought to have stronger homophilic interactions than SM, as well as by studies using the enantiomer of SM having opposite stereochemistry to SM at the 2 and 3 positions and by some molecular dynamics (MD) simulations of lipid bilayers containing Lo-lipids. Nanosized gel subdomains seem to play an important role in controlling membrane organization and function in biological membranes.

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.

Behavior of Triterpenoid Saponin Ginsenoside Rh2 in Ordered and Disordered Phases in Model Membranes Consisting of Sphingomyelin, Phosphatidylcholine, and Cholesterol.

Abstract: The ginsenoside Rh2 (Rh2) is a saponin of medicinal ginseng, and it has attracted much attention for its pharmacological activities. In this study, we investigated the interaction of Rh2 with biological membranes using model membranes. We examined the effects of various lipids on the membrane-disrupting activity of Rh2 and found that cholesterol and sphingomyelin (SM) had no significant effect. Furthermore, the effects of Rh2 on acyl chain packing (DPH anisotropy) and water molecule permeability (GP340 values) did not differ significantly between bilayers containing SM and saturated phosphatidylcholine. These results suggest that the formation of the liquid-ordered (Lo) phase affects the behavior of Rh2 in the membrane rather than a specific interaction of Rh2 with a particular lipid. We investigated the effects of Rh2 on the Lo and liquid-disordered (Ld) phases using surface tension measurements and fluorescence experiments. In the surface tension-area isotherms, we compared the monolayers of the Ld and Lo lipid compositions and found that Rh2 is abundantly bound to both monolayers, with the amount being greater in the Ld phase than in the Lo phase. In addition, the hydration state of the bilayers, mainly consisting of the Lo or Ld phase, showed that Rh2 tends to bind to the surface of the bilayer in both phases. At higher concentrations, Rh2 tends to bind more abundantly to the relatively shallow interior of the Ld phase than the Lo phase. The phase-dependent membrane behavior of Rh2 is probably due to the phase-selective affinity and binding mode of Rh2.

Lipid chain-driven interaction of a lipidated Src-family kinase Lyn with the bilayer membrane.

Abstract: N-Myristoylation is a process of ubiquitous protein modification, which promotes the interaction of lipidated proteins on cell surfaces, in conjunction with reversible S-palmitoylation. We report the cooperative lipid-lipid interaction of two acyl chains of proteins, which increases the protein-membrane interaction and facilitates selective targeting of membranes containing anionic lipids. Lyn is a member of the Src family kinases distributed on the membrane surface by N-myristoyl and neighbouring S-palmitoyl chain anchors at the unique N-terminus domain. We prepared N-terminal short segments of lipidated Lyn to investigate the behaviour of each acyl chain in the lipid composition-dependent membrane interaction by solid-state nuclear magnetic resonance (NMR) analysis. Solid-state 31P-NMR studies revealed that S-palmitoylation of N-myristoylated Lyn peptides increased the interaction between peptides and phospholipid head groups, particularly with the anionic phosphatidylserine-containing bilayers. The solid-state 2H-NMR of Lyn peptides with a perdeutero N-myristoyl chain indicated an increase (0.6-0.8 Å) in the extent of the N-myristoyl chain in the presence of nearby S-palmitoyl chains, probably through the interaction via the acyl chains. The cooperative hydrocarbon chain interaction of the two acyl chains of Lyn increased membrane binding by extending the hydrocarbon chains deeper into the membrane interior, thereby promoting the peptide-membrane surface interaction between the cationic peptide side chains and the anionic lipid head groups. This lipid-driven mechanism by S-palmitoylation promotes the partition of the lipidated proteins to the cytoplasmic surface of the cell membranes and may be involved in recruiting Lyn at the signalling domains rich in anionic lipids.

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

Abstract: 2 H solid-state NMR is a method for examining the mobility and orientation of molecules in the field of biophysics. In studies on lipid bilayer membranes, 2 H NMR is often adopted to detect a phase transition from the gel to the liquid-crystal phase, which is observed as a change in spectral shape, and to evaluate the ordering of lipid alkyl chains using quadrupole coupling values. Since the mobility of membrane lipids is highly temperature dependent, precise temperature control is a prerequisite for evaluating the physical properties of membranes. Generally, NMR instruments monitor the temperature of the VT gas. The temperature inside the sample tube and the VT gas match only when the heat generated by the rf pulse emitted from the coil or magic angle spinning is significantly lower than the cooling capacity of the VT gas. In other words, the sample temperature inside the tube depends on the measurement method. Therefore, in this study, we took advantage of temperature-dependent changes in the chemical shift of a paramagnetic metal-ligand complex. We designed and synthesized a deuterated ligand molecule and evaluated its temperature dependence as a thermometer for 2 H solid-state NMR spectroscopy. We chose Tb, Dy, Ho, and Er as the paramagnetic central metals. We then measured the 2 H NMR spectrum of each metal complex and confirmed the 2 H chemical shift to be temperature dependent. Furthermore, using the thermometer molecule with Er, we succeeded in accurately evaluating the segmental melting of an alkyl chain in lipid bilayers with 0.1°C accuracy.