Sphingomyelin Bilayers Research Articles

Preparation of Nitrogen Analogues of Ceramide and Studies of Their Aggregation in Sphingomyelin Bilayers.

Ceramides can regulate biological processes probably through the formation of laterally segregated and highly packed ceramide-rich domains in lipid bilayers. In the course of preparation of its analogues, we found that a hydrogen-bond-competent functional group in the C1 position is necessary to form ceramide-rich domains in lipid bilayers [Matsufuji; Langmuir 2018]. Hence, in the present study, we newly synthesized three ceramide analogues: CerN3, CerNH2, and CerNHAc, in which the 1-OH group of ceramide is substituted with a nitrogen functionality. CerNH2 and CerNHAc are capable of forming hydrogen bonds in their headgroups, whereas CerN3 is not. Fluorescent microscopy observation and differential scanning calorimetry analysis disclosed that these ceramide analogues formed ceramide-rich phases in sphingomyelin bilayers, although their thermal stability was slightly inferior to that of normal ceramides. Moreover, wide-angle X-ray diffraction analysis showed that the chain packing structure of ceramide-rich phases of CerNHAc and CerN3 was similar to that of normal ceramide, while the CerNH2-rich phase showed a slightly looser chain packing due to the formation of CerNH3+. Although the domain formation of CerN3 was unexpected because of the lack of hydrogen-bond capability in the headgroup, it may become a promising tool for investigating the mechanistic link between the ceramide-rich phase and the ceramide-related biological functions owing to its Raman activity and applicability to click chemistry.

Calorimetric and spectroscopic investigation of the interaction of chemotherapeutic agent carboplatin with sphingomyelin lipids

Carboplatin (CAR) is a chemo-therapeutic agent which interacts with DNA to interfer its repair, and thereby to induce apoptosis. Since CAR acts inside cell, its inevitable interaction with membrane lipids directly affects its efficacy. In the current work, we investigated the interaction profile of CAR with sphingomyelin (SM) lipids, which plays key role in different cellular functions, particularly important in cancer. By utilizing Differential Scanning Calorimetry (DSC) and Fourier transform infrared (FT-IR) spectroscopy, the effects of different concentrations of CAR (1-5-10-20-30 mol%) on phase transition temperature (Tm), enthalpy (ΔH), cooperativity (CU), lipid order, lipid fluidity and the strength of hydrogen bonding of SM lipids were monitored. The results revealed that CAR caused a decrease in Tm, ΔH, CU, lipid order, but an increase in hydrogen state of hydrophilic region of SM lipids. On the other hand, a slight decrement in fluidity solely at 30 mol% was monitored. The findings revealed that CAR incorporates into SM bilayers by binding to acyl chains and hydrophilic parts of lipids. In sum, the results may represent valuable information about the effect of CAR on biophysical properties of SM lipids, which may reflect its behavior and action in real biological systems.

The influence of ceramide and its dihydro analog on the physico-chemical properties of sphingomyelin bilayers

The influence of ceramide and its dihydro analog (Cer and DHCer, respectively; inclusively termed Cers) on sphingomyelin (SM) bilayers was examined. Fluorescent microscopy showed that SM/Cers binary bilayers undergo phase separation between Cers-rich and Cers-poor phases. Based on calorimetry, the content of Cers in the Cers-rich phase was estimated and the results show that DHCer in the DHCer-rich phase (17.5 mol%) is more condensed than Cer in the Cer-rich phase (15 mol%), likely due to a stronger intermolecular interaction of DHCer than that of Cer. Furthermore, the Cers-poor phase consists of almost pure SM. X-ray diffraction and water permeability measurements disclosed that the size of crystallites—lipid nano-clusters formed inside the Cers-rich phase—are larger for the DHCer-rich phase than those for the Cer-rich phase. Due to its stronger intermolecular interactions, DHCer could effectively suppress the inter-crystallite packing gaps to avoid the energetic disadvantage, resulting in formation of larger crystallites.

Counter-effects of Ethanol and Cholesterol on the Heterogeneous PSM-POPC Lipid Membrane: A Molecular Dynamics Simulation Study.

In a recent work, we reported that the presence of ethanol molecules leads to significant changes on the structural stability of a homogeneous sphingomyelin bilayer. We observed that at higher concentrations of ethanol (≥20 mol %), the structure of the PSM (N-palmitoyl-sphingomyelin) bilayer is almost lost. H-bond analysis revealed that ethanol molecules form H-bonds with the PSM lipids at the cost of PSM-PSM intermolecular H-bonding interactions, subsequently leading to destabilization of the homogeneous PSM bilayer. Biological membranes are heterogeneous and anisotropic in nature. Previously, it has been reported that partitioning of amphiphilic molecules across the lipid bilayer is governed by the lipid composition and cholesterol content. In order to further enhance our understanding towards the interaction of ethanol in the heterogeneous membrane environment, herein, we have investigated the influence of 5 and 20 mol % of ethanol on the structure and stability of two heterogeneous bilayers: a binary bilayer comprising PSM and POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) lipids and a ternary bilayer consisting of cholesterol along with PSM and POPC. The study reveals that the disordering and destabilizing effects of ethanol on the binary bilayer systems are enhanced as compared to that on the homogeneous PSM bilayer. However, the presence of cholesterol mitigates the influence of ethanol on the lipid bilayer because of its condensing and ordering effects on other lipids.

Mixing brain cerebrosides with brain ceramides, cholesterol and phospholipids

The properties of bilayers composed of pure brain cerebroside (bCrb) or of binary mixtures of bCrb with brain ceramide, cholesterol, egg phosphatidylcholine or brain sphingomyelin have been studied using a combination of physical techniques. Pure bCrb exhibits a rather narrow gel-fluid transition centred at ≈65 °C, with a half-width at half-height T1/2 ≈ 3 °C. bCrb mixes well with both fluid and gel phospholipids and ceramide, and it rigidifies bilayers of egg phosphatidylcholine or brain sphingomyelin when the latter are in the fluid state. Cholesterol markedly widens the bCrb gel-fluid transition, while decreasing the associated transition enthalpy, in the manner of cholesterol mixtures with saturated phosphatidylcholines, or sphingomyelins. Laurdan and DPH fluorescence indicate the formation of fluid ordered phases in the bCrb:cholesterol mixtures. Macroscopic phase separation of more and less fluid domains is observed in giant unilamellar vesicles consisting of bCrb:egg phosphatidylcholine or bCrb:sphingomyelin. Crb capacity to induce bilayer permeabilization or transbilayer (flip-flop) lipid motion is much lower than those of ceramides. The mixtures explored here contained mostly bCrb concentrations >50 mol%, mimicking the situation of cell membranes in Gaucher’s disease, or of the Crb-enriched microdomains proposed to exist in healthy cell plasma membranes.

Formation of cholesterol Bilayer Domains Precedes Formation of Cholesterol Crystals in Membranes Made of the Major Phospholipids of Human Eye Lens Fiber Cell Plasma Membranes

ABSTRACTPurpose/Aim: The goal of this study is to reveal how age-related changes in phospholipid (PL) composition in the fiber cell plasma membranes of the human eye lens affect the cholesterol (Chol) content at which Chol bilayer domains (CBDs) and Chol crystals start to form.Materials and Methods: Saturation-recovery electron paramagnetic resonance with spin-labeled cholesterol analogs and differential scanning calorimetry were used to determine the Chol contents at which CBDs and cholesterol crystals, respectively, start to form in in membranes made of the major PL constituents of the plasma membrane of the human eye lens fiber cells. To preserve compositional homogeneity throughout the membrane suspension, the lipid multilamellar dispersions investigated in this work were prepared using a rapid solvent exchange method. The cholesterol content changed from 0 to 75 mol%.Results: The saturation recovery electron paramagnetic resonance results show that CBDs start to form at 33, 50, 46, and 48 mol% Chol in the phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine, and sphingomyelin bilayers, respectively. The differential scanning calorimetry results show that Chol crystals start to form at 50, 66, 70, and 66 mol% Chol in the phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine, and sphingomyelin bilayers, respectively.Conclusions: These results, as well those of our previous studies, indicate that the formation of CBDs precedes the formation of Chol crystals in all of the studied systems, and the appearance of each depends on the type of PL forming the bilayer. These findings contribute to a better understanding of the molecular mechanisms involved in the regulation of Chol-dependent processes in eye lens fiber cell membranes.

Lamellar Phases Composed of Phospholipid, Cholesterol, and Ceramide, as Studied by 2H NMR

Sphingolipids constitute a significant fraction of cellular plasma membrane lipid content. Among sphingolipids, ceramide levels are usually very low. However, in some cell processes like apoptosis, cell membrane ceramide levels increase markedly because of the activation of enzymes like acid sphingomyelinase. This increase can change the physical state of the membrane by promoting molecular order and inducing solid-ordered (So) phase domains. This effect has been observed in a previous 2H NMR study on membranes consisting of palmitoyl sphingomyelin (PSM) and palmitoyl ceramide (PCer). Cholesterol (Chol), too, is present at high concentrations in mammalian plasma membranes and has a favorable interaction with sphingomyelin (SM), together forming domains in the liquid-ordered phase in model membranes. There are reports that Chol is able to displace ceramide (Cer) in SM bilayers and abolish the So phase domains formed by SM:Cer. This ability of Chol appears to be concentration dependent; in membranes with low Chol and high Cer contents, So phase domains rich in Cer coexist with the continuous fluid phase of the membrane. Here, we studied the effect of increasing PCer concentration in PSM:Chol bilayers, using 2H NMR. Chol:PCer mole ratios were 3:1, 3:2, and 3:3, at a fixed 7:3 phospholipid:cholesterol mol ratio. Both PSM and PCer were monitored in separate samples for changes in their physical state by introducing a perdeuterated palmitoyl chain in either molecule. Moreover, the effect of replacing PSM with DPPC was investigated to test the impact on membrane phase behavior of replacing the sphingosine with a palmitoylated glycerol backbone. We found that PCer can increase acyl chain order in both PSM:Chol and DPPC:Chol bilayers. Especially in bilayers with Chol:PCer 1:1 molar ratios, PCer induces highly stable So phase domains in both PSM and DPPC bilayers near 37°C. However, PCer has a more pronounced ordering effect on PSM compared to DPPC bilayers.

Impact of amphiphilic molecules on the structure and stability of homogeneous sphingomyelin bilayer: Insights from atomistic simulations.

Modulation of lipid membrane properties due to the permeation of amphiphiles is an important biological process pertaining to many applications in the field of pharmaceutics, toxicology, and biotechnology. Sphingolipids are both structural and functional lipids that constitute an important component of mechanically stable and chemically resistant outer leaflets of plasma membranes. Here, we present an atomistic molecular dynamics simulation study to appreciate the concentration-dependent effects of small amphiphilic molecules, such as ethanol, acetone, and dimethyl sulfoxide (DMSO), on the structure and stability of a fully hydrated homogeneous N-palmitoyl-sphingomyelin (PSM) bilayer. The study reveals an increase in the lateral expansion of the bilayer along with disordering of the hydrophobic lipid tails on increasing the concentration of ethanol. At higher concentrations of ethanol, rupturing of the bilayer is quite evident through the analysis of partial electron density profiles and lipid tail order parameters. For ethanol containing systems, permeation of water molecules in the hydrophobic part of the bilayer is allowed through local defects made due to the entry of ethanol molecules via ethanol-ethanol and ethanol-PSM hydrogen bonds. Moreover, the extent of PSM-PSM hydrogen bonding decreases with increasing ethanol concentration. On the other hand, acetone and DMSO exhibit minimal effects on the stability of the PSM bilayer at their lower concentrations, but at higher concentrations they tend to enhance the stability of the bilayer. The simulated potential of mean force (PMF) profiles for the translocation of the three solutes studied reveal that the free-energy of transfer of an ethanol molecule across the PSM lipid head region is lower than that for acetone and DMSO molecules. However, highest free-energy rise in the core hydrophobic part of the bilayer is observed for the DMSO molecule, whereas the ethanol and acetone PMF profiles show a lower barrier in the hydrophobic region of the bilayer.

Microwave measurement of giant unilamellar vesicles in aqueous solution

A microwave technique is demonstrated to measure floating giant unilamellar vesicle (GUV) membranes in a 25 μm wide and 18.8 μm high microfluidic channel. The measurement is conducted at 2.7 and 7.9 GHz, at which a split-ring resonator (SRR) operates at odd modes. A 500 nm wide and 100 μm long SRR split gap is used to scan GUVs that are slightly larger than 25 μm in diameter. The smaller fluidic channel induces flattened GUV membrane sections, which make close contact with the SRR gap surface. The used GUVs are synthesized with POPC (16:0–18:1 PC 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine), SM (16:0 Egg Sphingomyelin) and cholesterol at different molecular compositions. It is shown that SM and POPC bilayers have different dielectric permittivity values, which also change with measurement frequencies. The obtained membrane permittivity values, e.g. 73.64-j6.13 for POPC at 2.7 GHz, are more than 10 times larger than previously reported results. The discrepancy is likely due to the measurement of dielectric polarization parallel with, other than perpendicular to, the membrane surface. POPC and SM-rich GUV surface sections are also clearly identified. Further work is needed to verify the obtained large permittivity values and enable accurate analysis of membrane composition.

Preparation and Membrane Properties of Oxidized Ceramide Derivatives.

Ceramide is a bioactive lipid with important roles in several biological processes including cell proliferation and apoptosis. Although 3-ketoceramides that contain a keto group in place of the 3-OH group of ceramide occur naturally, ceramide derivatives oxidized at the primary 1-OH group have not been identified to date. To evaluate how the oxidative state of the 1-OH group affects the physical properties of membranes, we prepared novel ceramide derivatives in which the 1-OH group was oxidized to a carboxylic acid (PCerCOOH) or methylester (PCerCOOMe) and examined the rigidity of their monolayers and the formation of gel domains in palmitoyloleoylphosphatidylcholine (POPC) or sphingomyelin (SM) bilayers. As a result, PCerCOOH and PCerCOOMe exhibited membrane properties similar to those of native ceramide, although the deprotonated form of PCerCOOH, PCerCOO-, exhibited markedly lower rigidity and higher miscibility with POPC and SM. This was attributed to the electrostatic repulsion of the negative charge, which hampered the formation of the ceramide-enriched gel domain. The similarities in the properties of PCerCOOMe and ceramide revealed the potential to introduce various functional groups onto PCerCOOH via ester or amide linkages; therefore, these derivatives will also provide a new strategy for developing molecular probes, such as fluorescent ceramides, and inhibitors of ceramide-related enzymes.

Mechanical properties of milk sphingomyelin bilayer membranes in the gel phase: Effects of naturally complex heterogeneity, saturation and acyl chain length investigated on liposomes using AFM

Sphingomyelin (SM) molecules are major lipid components of plasma membranes that are involved in functional domains. Among natural SMs, that found in milk (milk-SM) exhibits important acyl chain heterogeneities in terms of length and saturation, which could affect the biophysical properties and biological functions of the milk fat globule membrane or of liposome carriers. In this study, the thermotropic and mechanical properties of milk-SM, synthetic C16:0-SM, C24:0-SM and the binary mixtures C16:0-SM/C24:0-SM (50:50% mol) and C24:0-SM/C24:1-SM (95:5% mol) bilayer membranes were investigated using differential scanning calorimetry and atomic force microscopy, respectively. Results showed that acyl chain length, heterogeneity and unsaturation affected i) the temperature of phase transition of SM bilayers, and ii) the mechanical properties of liposome (diameter<200nm) membranes in the gel phase, e.g. the Young modulus E and the bending rigidity kC. This study increases our knowledge about the key role of naturally complex lipid compositions in tailoring the physical properties of biological membranes. It could be also used in liposomes development e.g. to select the suitable lipid composition according to usage.

The improved efficacy of Sifuvirtide compared with enfuvirtide might be related to its selectivity for the rigid biomembrane, as determined through surface plasmon resonance

Most mechanistic studies on human immunodeficiency virus (HIV) peptide fusion inhibitors have focused on the interactions between fusion inhibitors and viral envelope proteins. However, the interactions of fusion inhibitors with viral membranes are also essential for the efficacy of these drugs. Here, we utilized surface plasmon resonance (SPR) technology to study the interactions between the HIV fusion inhibitor peptides sifuvirtide and enfuvirtide and biomembrane models. Sifuvirtide presented selectivity toward biomembrane models composed of saturated dipalmitoylphosphatidylcholine (DPPC) (32-fold higher compared with unsaturated 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine [POPC]) and sphingomyelin (SM) (31-fold higher compared with POPC), which are rigid compositions enriched in the HIV viral membrane. In contrast, enfuvirtide showed no significant selectively toward these rigid membrane models. Furthermore, the bindings of sifuvirtide and enfuvirtide to SM bilayers were markedly higher than those to monolayers (14-fold and 23-fold, respectively), indicating that the inner leaflet influences the binding of these drugs to SM bilayers. No obvious differences were noted in the bindings of either peptide to the other mono- and bilayer models tested, illustrating that both peptides interact with these membranes through surface-binding. The bindings of the inhibitor peptides to biomembranes were found to be driven predominantly by hydrophobic interactions rather than electrostatic interactions, as determined by comparing their affinities to those of positively charged 1-palmitoyl-2-oleoyl-sn-glycero-3-ethylphosphocholine (EPC) to zwitterionic membrane models. The improved efficiency of sifuvirtide relative to enfuvirtide might be related to its ability to adsorb on rigid lipidic areas, such as the viral envelope and lipid rafts, which results in an increased sifuvirtide concentration at the fusion site.

Calculating Membrane Area Compressibility from MD Simulations: a Novel Computational Framework

Like thin elastic sheets, lipid bilayers can bend and stretch, and are characterized by mechanical constants such as bending rigidity (Kc) and area compressibility (Ka), which define the free energy of bilayer deformation and thus have direct implications in quantifying the energetics of protein-membrane interactions. Limited experimental data for Kc and Ka of different lipid mixtures makes such calculations challenging. Existing computational methods for calculating Ka from MD simulations exhibit strong dependence on bilayer size, or require multiple simulations with constraints on the area, or tension. We present a novel computational framework that is independent of system size and requires analysis only of a single sufficiently converged simulation trajectory. The method is based on Helfrich's theory of elasticity by relating relative changes in area to relative changes in thickness. The distribution of local fluctuations in the thickness of each bilayer leaflet is used to construct a potential of mean force and recover the bilayer Ka from a quadratic fit. The rapid calculation utilizes freely available software packages. We present results for a variety of single and multicomponent bilayers of phosphatidylcholine, phosphatidylethanolamine and sphingomyelin, utilizing simulation results at different temperatures and bilayer sizes. The calculated compressibility moduli range from 170 to 320 mN/m and agree for the majority of tested cases with values reported from experiments and other computational methods. We further validate our method in a comparison with an existing polymer brush model and confirm the linear relationship between thickness and (Kc/Ka) ˆ (1/2), all calculated from the simulation trajectories.

Laurdan emission study of the cholesterol-like effect of long-chain alkylresorcinols on the structure of dipalmitoylphosphocholine and sphingomyelin membranes

Long-chain alkylresorcinols (ARs) are commonly found in plant and bacteria cells, and they exhibit a wide variety of biological effects, including antifungal, antitumor, and antiphrastic activities. The cholesterol (Chol)-like effect of ARs with hydrocarbon side-chain lengths ranging from C15 to C25 on the structure of pure and Chol-doped dipalmitoylphosphocholine (DPPC) and sphingomyelin (SM) membranes was investigated by Laurdan fluorescence spectroscopy. The Laurdan emission generalized polarization parameter was analyzed as a function of the temperature and excitation wavelength in DPPC (or SM)/Chol, DPPC (or SM)/AR, and DPPC/Chol/AR systems. It was found that AR incorporation into both DPPC and SM bilayers induces an increase in the temperature of the main lipid phase transition, similar to the effect of Chol molecule incorporation. The phase separation, lipid-chain ordering, and membrane hydration are discussed for the AR-mixed membranes and compared with DPPC (or SM)/Chol membranes.

Regulation of Sticholysin II-Induced Pore Formation by Lipid Bilayer Composition, Phase State, and Interfacial Properties.

Sticholysin II (StnII) is a pore-forming toxin that uses sphingomyelin (SM) as the recognition molecule in targeting membranes. After StnII monomers bind to SM, several toxin monomers act in concert to oligomerize into a functional pore. The regulation of StnII binding to SM, and the subsequent pore-formation process, is not fully understood. In this study, we examined how the biophysical properties of bilayers, originating from variations in the SM structure, from the presence of sterol species, or from the presence of increasingly polyunsaturated glycerophospholipids, affected StnII-induced pore formation. StnII-induced pore formation, as determined from calcein permeabilization, was fastest in the pure unsaturated SM bilayers. In 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)/saturated SM bilayers (4:1 molar ratio), pore formation became slower as the chain length of the saturated SMs increased from 14 up to 24 carbons. In the POPC/palmitoyl-SM (16:0-SM) 4:1 bilayers, SM could not support pore formation by StnII if dimyristoyl-PC was included at 1:1 stoichiometry with 16:0-SM, suggesting that free clusters of SM were required for toxin binding and/or pore formation. Cholesterol and other sterols facilitated StnII-induced pore formation markedly, but the efficiency did not appear to correlate with the sterol structure. Benzyl alcohol was more efficient than sterols in enhancing the pore-formation process, suggesting that the effect on pore formation originated from alcohol-induced alteration of the hydrogen-bonding network in the SM-containing bilayers. Finally, we observed that pore formation by StnII was enhanced in the PC/16:0-SM 4:1 bilayers, in which the PC was increasingly unsaturated. We conclude that the physical state of bilayer lipids greatly affected pore formation by StnII. Phase boundaries were not required for pore formation, although SM in a gel state attenuated pore formation.

Toxin-induced pore formation is hindered by intermolecular hydrogen bonding in sphingomyelin bilayers

Sticholysin I and II (StnI and StnII) are pore-forming toxins that use sphingomyelin (SM) for membrane binding. We examined how hydrogen bonding among membrane SMs affected the StnI- and StnII-induced pore formation process, resulting in bilayer permeabilization. We compared toxin-induced permeabilization in bilayers containing either SM or dihydro-SM (lacking the trans Δ4 double bond of the long-chain base), since their hydrogen-bonding properties are known to differ greatly. We observed that whereas both StnI and StnII formed pores in unilamellar vesicles containing palmitoyl-SM or oleoyl-SM, the toxins failed to similarly form pores in vesicles prepared from dihydro-PSM or dihydro-OSM. In supported bilayers containing OSM, StnII bound efficiently, as determined by surface plasmon resonance. However, StnII binding to supported bilayers prepared from dihydro-OSM was very low under similar experimental conditions. The association of the positively charged StnII (at pH7.0) with unilamellar vesicles prepared from OSM led to a concentration-dependent increase in vesicle charge, as determined from zeta-potential measurements. With dihydro-OSM vesicles, a similar response was not observed. Benzyl alcohol, which is a small hydrogen-bonding compound with affinity to lipid bilayer interfaces, strongly facilitated StnII-induced pore formation in dihydro-OSM bilayers, suggesting that hydrogen bonding in the interfacial region originally prevented StnII from membrane binding and pore formation. We conclude that interfacial hydrogen bonding was able to affect the membrane association of StnI- and StnII, and hence their pore forming capacity. Our results suggest that other types of protein interactions in bilayers may also be affected by hydrogen-bonding origination from SMs.

The Influence of Hydrogen Bonding on Sphingomyelin/Colipid Interactions in Bilayer Membranes

The phospholipid acyl chain composition and order, the hydrogen bonding, and properties of the phospholipid headgroup all influence cholesterol/phospholipid interactions in hydrated bilayers. In this study, we examined the influence of hydrogen bonding on sphingomyelin (SM) colipid interactions in fluid uni- and multilamellar vesicles. We have compared the properties of oleoyl or palmitoyl SM with comparable dihydro-SMs, because the hydrogen bonding properties of SM and dihydro-SM differ. The association of cholestatrienol, a fluorescent cholesterol analog, with oleoyl sphingomyelin (OSM) was significantly stronger than its association with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, in bilayers with equal acyl chain order. The association of cholestatrienol with dihydro-OSM, which lacks a trans double bond in the sphingoid base, was even stronger than the association with OSM, suggesting an important role for hydrogen bonding in stabilizing sterol/SM interactions. Furthermore, with saturated SM in the presence of 15 mol % cholesterol, cholesterol association with fluid dihydro-palmitoyl SM bilayers was stronger than seen with palmitoyl SM under similar conditions. The different hydrogen bonding properties in OSM and dihydro-OSM bilayers also influenced the segregation of palmitoyl ceramide and dipalmitoylglycerol into an ordered phase. The ordered, palmitoyl ceramide-rich phase started to form above 2 mol % in the dihydro-OSM bilayers but only above 6 mol % in the OSM bilayers. The lateral segregation of dipalmitoylglycerol was also much more pronounced in dihydro-OSM bilayers than in OSM bilayers. The results show that hydrogen bonding is important for sterol/SM and ceramide/SM interactions, as well as for the lateral segregation of a diglyceride. A possible molecular explanation for the different hydrogen bonding in SM and dihydro-SM bilayers is presented and discussed.

A weight averaged approach for predicting amide vibrational bands of a sphingomyelin bilayer.

Infrared (IR) and Raman spectra of a sphingomyelin (SM) bilayer have been calculated for the amide I, II and A modes and the double-bonded CC stretching mode by a weight averaged approach, based on an all-atom molecular dynamics (MD) simulation and a vibrational structure calculation. Representative structures and statistical weights of SM clusters connected by hydrogen bonds (HBs) are observed in MD trajectories. After constructing smaller fragments from the SM clusters, the vibrational spectra of the target modes were calculated by normal mode analysis with a correction for anharmonicity, using density functional theory. The final IR and Raman spectra of a SM bilayer were obtained as the weight averages over all SM clusters. The calculated Raman spectrum is in excellent agreement with a recent measurement, providing a clear assignment of the peak in question observed at 1643 cm(-1) to the amide I modes of a SM bilayer. The analysis of the IR spectrum has also revealed that the amide bands are sensitive to the water content inside the membrane, since their band positions are strongly modulated by the HB between SM and water molecules. The present study suggests that the amide I band serves as a marker to identify the formation of SM clusters, and opens a new way to detect lipid rafts in the biological membrane.

α-Tocopherol's Location in Membranes Is Not Affected by Their Composition.

To this day, α-tocopherol's (aToc) role in humans is not well known. In previous studies, we have tried to connect aToc's biological function with its location in a lipid bilayer. In the present study, we have determined, by means of small-angle neutron diffraction, that not only is aToc's hydroxyl group located high in the membrane but its tail also resides far from the center of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) bilayers. In addition, we located aToc's hydroxyl group above the lipid backbone in 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (POPS), and sphingomyelin bilayers, suggesting that aToc's location near the lipid-water interface may be a universal property of vitamin E. In light of these data, how aToc efficiently terminates lipid hydroperoxy radicals at the membrane center remains an open question.

CHARMM All-Atom Additive Force Field for Sphingomyelin: Elucidation of Hydrogen Bonding and of Positive Curvature

The C36 CHARMM lipid force field has been extended to include sphingolipids, via a combination of high-level quantum mechanical calculations on small molecule fragments, and validation by extensive molecular dynamics simulations on N-palmitoyl and N-stearoyl sphingomyelin. NMR data on these two molecules from several studies in bilayers and micelles played a strong role in the development and testing of the force field parameters. Most previous force fields for sphingomyelins were developed before the availability of the detailed NMR data and relied on x-ray diffraction of bilayers alone for the validation; these are shown to be too dense in the bilayer plane based on published chain order parameter data from simulations and experiments. The present simulations reveal O-H:::O-P intralipid hydrogen bonding occurs 99% of the time, and interlipid N-H:::O=C (26-29%, depending on the lipid) and N-H:::O-H (17–19%). The interlipid hydrogen bonds are long lived, showing decay times of 50 ns, and forming strings of lipids, and leading to reorientational correlation time of nearly 100 ns. The spontaneous radius of curvature for pure N-palmitoyl sphingomyelin bilayers is estimated to be 43–100 Å, depending on the assumptions made in assigning a bending constant; this unusual positive curvature for a two-tailed neutral lipid is likely associated with hydrogen bond networks involving the NH of the sphingosine group.