Skip to main content
NCBI home page
Biophysical Journal logoLink to Biophysical Journal
. 2016 Jan 19;110(2):431–440. doi: 10.1016/j.bpj.2015.11.3515

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

Tomokazu Yasuda 1, Md Abdullah Al Sazzad 1, Niklas Z Jäntti 1, Olli T Pentikäinen 2, J Peter Slotte 1,∗
PMCID: PMC4724628  PMID: 26789766

Abstract

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.

Introduction

Sphingomyelin (SM) is a common sphingolipid in the plasma membranes of most eukaryotic cells (1). Its general structure (N-acyl-sphingosine-1-phosphocholine) has been known for a long time (2), and its enantiomeric configuration of 2NH and 3OH (2S,3R) was solved in the 1960s (3). These two polar functions are the main hydrogen bond donors in nonglycosylated sphingolipids. In SM, the hydrogen bond accepting atoms are the phosphate oxygens, the carbonyl ester oxygen, the oxygen of the 3OH, and the nitrogen of the 2NH functional groups (4). The interfacial, glycerol-based structures of other common glycerophospholipids do not usually have proton-donating polar functions.

The role of hydrogen bonding in the stability and properties of membranes was suggested in the 1970s (5, 6, 7). Early 1H and 31P NMR spectroscopy studies, comparing SM and phosphatidylcholine (PC) bilayers, suggested that differences observed in proton line widths, spin lattice relaxation times, and chemical shifts were due to both inter- and intramolecular hydrogen bonding in the SM bilayers (8). Further Fourier transform infrared spectroscopy studies suggested that the phosphate group of SM was involved in intermolecular hydrogen bonding (9). A later Fourier transform infrared spectroscopy study demonstrated strong intermolecular hydrogen bonding involving the 3OH of the long-chain base of SM (10). Much more recent 1H and 31P NMR studies (11), as well as computational simulations (12, 13), indicated that the 3OH functional group was hydrogen bonding with phosphate oxygen, and that long-lived hydrogen bonds formed between the 2NH functional group of SM and hydrogen bond accepting groups of neighboring lipid species (14).

Early studies did not observe a direct role for hydrogen bonding in the stabilization of the cholesterol/SM interaction (15, 16). However, a recent 2H NMR study showed that hydrogen bonding between SMs in bilayers affected the depth profile of cholesterol when compared to that of matched PC bilayers (17, 18). The presence of cholesterol at greater depths in SM bilayers compared to PC bilayers could partly explain why the desorption kinetics of cholesterol are much slower in SM bilayers than in matched PC bilayers (19). Recently, 13C and 15N NMR chemical shift anisotropy measurements revealed that cholesterol appeared to stabilize intermolecular hydrogen bonding involving the 2NH functional group of SM (20). The elimination of hydrogen bonding from SM by the methylation of 2NH or 3OH of the long-chain base was shown recently to markedly attenuate sterol/SM interactions in bilayer membranes (21), further supporting the view that hydrogen bonds involving SM have marked effects on their bilayer properties and colipid interactions.

Dihydro-sphingomyelin (dihydro-SM) lacks the trans Δ4 double bond in the long-chain base (22). Most cell types contain small amounts of dihydro-SM lipids (23, 24, 25). However, in lens cells, dihydro-SM lipids are the dominant SM species, with palmitic and stearic acid as predominant N-linked acyl chains (23, 24, 25). The lens membrane has a very high cholesterol content (∼60 mol % (25)). Dihydro-SM is also a major constituent of SM in bovine milk (26). Several studies have suggested that the hydrogen-bonding properties of SM and dihydro-SM differ (11, 27, 28). A recent study provided additional support for the notion that intermolecular hydrogen bonding appears to be much stronger in dihydro-SM bilayers than in comparable SM bilayers (29). Hydrogen bonding among dihydro-SM molecules contributed to the lateral segregation and stabilization of a macroscopic fluid dihydro-SM domain in disordered PC bilayers at high temperature (29). A similar segregation of a macroscopic fluid nondihydro-SM domain was not observed.

A detailed comparison of the bilayer properties of SM and dihydro-SM can reveal new aspects of the role of hydrogen bonding in SM/colipid interactions. Thus, in this study, we examined how differences in SM/dihydro-SM hydrogen bonding influenced interactions with cholesterol and ceramide. Our results point to significant differences in how cholesterol and ceramide interact with SM and dihydro-SM. Using quantum chemistry modeling, we propose how altered dynamic properties of the 3OH in dihydro-SM could explain the observed differences.

Materials and Methods

Materials

1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), egg SM, N-oleoyl SM, dipalmitoyl-glycerol (DPG), and dihydro lyso SM were obtained from Avanti Polar Lipids (Alabaster, AL). All the SMs in this study had a natural D-erythro configuration. Palmitoyl sphingomyelin (PSM) was isolated to 99% purity from egg SM by preparative reverse-phase high-performance liquid chromatography (HPLC) (Discovery C18 column, Supelco, Bellefonte, PA) using methanol as eluent (30). Dihydro-PSM was prepared from PSM by hydrogenation using 10% palladium on charcoal as a catalyst (Sigma-Aldrich, St. Louis, MO) and hydrogen gas (31). The product was purified by preparative reverse-phase HPLC with methanol as eluent.

Dihydro-oleoyl sphingomyelin (OSM) was prepared from dihydro-lyso SM and oleic anhydride (Sigma-Aldrich) using triethylamine (Sigma-Aldrich) as a catalyst, as described previously (32). Palmitoyl ceramide with a 20:1Δ4t long-chain base (PCer-20:1) was prepared by coupling palmitoyl anhydride (Sigma-Aldrich) to the 20:1Δ4t long-chain base (Larodan, Malmö, Sweden) using triethylamine as a catalyst, and it was purified by preparative HPLC (30). PCer with a 20:1 long-chain base has a similar chain length profile as DPG, and was therefore used for comparison (33). The identity of the synthesized lipids was ascertained by electrospray ionization mass spectrometry (Bruker Daltonics, Bremen, Germany). Cholestatrienol (cholesta-5,7,9(11)-trien-3β-ol; CTL) was synthesized from 7-dehydrocholesterol (Sigma-Aldrich) (34) and purified as described previously (35). The identity and purity of CTL were ascertained by atmospheric pressure chemical ionization mass spectrometry (Bruker Daltonics), analytical reverse-phase HPLC (coelution with authentic CTL), and absorbance and emission spectra, which were similar to published spectra.

Trans-parinaric acid (tPA) was synthesized from the methyl ester of alpha-linolenic acid (Sigma-Aldrich) according to the method described earlier (36). tPA was purified by crystallization from hexane (37) at −85°C to 99% purity. The identity and purity of tPA were ascertained by electrospray ionization mass spectrometry, analytical reverse-phase HPLC (coelution with authentic tPA), and absorbance and emission spectra. CTL and tPA, together with 0.5 mol % butylated hydroxytoluene, were stored dry under argon at –87°C until dissolved in argon-purged methanol.

Diphenylhexatriene and 1-palmitoyl-2-propionyl[DPH]-sn-glycero-3-phosphocholine (DPH-PC) were obtained from Molecular Probes (Eugene, OR). Working solutions of fluorophores were kept at −25°C and used within a week. All lipid stock solutions were prepared in methanol, stored at −25°C, and warmed to ambient temperature before use. The water used for sample preparation was purified by reverse osmosis, followed by passage through a Millipore UF Plus water-purification system (Millipore, Billerica, MA) to yield a product (MQ-water) with a final resistivity of 18.2 MΩcm. All organic and inorganic chemicals were of the highest purity available, and the solvents were of spectroscopic grade.

Steady-state fluorescence anisotropy: DPH and CTL

Multilamellar vesicles (MLVs) for fluorescence anisotropy measurements were prepared by mixing the lipids to the desired molar ratios (as indicated separately for each figure). The lipids, and the fluorescent probe (to give a final concentration of 1 mol %), were mixed, and then the solvent was evaporated under a stream of nitrogen. The dry lipid films were hydrated with argon-purged MQ-water in a water bath at 55°C for 30 min. Finally, the samples were vortex mixed and sonicated for 10 min in a water bath sonicator (FinnSonic M3 Bath Sonicator, FinnSonic Oy, Finland) at 55°C. The resulting MLVs (0.1 mM total lipid) ranged in size from 100 to 600 nm, and they were fairly polydispersed (data not shown). The samples were cooled to room temperature before the fluorescence measurements.

Steady-state fluorescence measurements were performed in quartz cuvettes on a PTI QuantaMaster-spectrofluorometer (Photon Technology International, Lawrenceville, NJ) operating in the T-format. The samples were constantly stirred, and the temperature in the samples was controlled by a Peltier element using a temperature probe immersed in the sample. The fluorescence emission was recorded continuously at 405 nm (tPA) or 430 nm (DPH) after excitation at 305 nm and 360 nm, respectively. The samples were heated at a rate of 2°C/min. The steady-state anisotropy, r, was determined as described previously (38), using the Felix32 software (Photon Technology International).

Fluorescence lifetime measurement of tPA

The fluorescence lifetimes of tPA in the MLVs (0.2 mM lipid final concentration) were measured at the temperature and lipid composition indicated in each relevant figure. Before fluorescence measurements, the samples were kept in the dark for at least 1 h at ambient temperature. The fluorescence decay of tPA was recorded with a FluoTime 200-spectrometer and a PicoHarp 300E time-correlated single photon counting module (PicoQuant GmbH, Berlin, Germany). tPA was excited with a 298 nm led laser source, and the emission was collected at 405 nm. During the measurements, the samples were kept at a constant temperature with constant stirring. The resulting data were analyzed with FluoFit Pro-software (PicoQuant). The decay was described by the sum of the exponentials, where αi was the normalized preexponential, and τi was the lifetime of the decay component i. The intensity-weighted lifetime was obtained by: 〈τ〉 = Σι αιτι2/Σι αιτι (38). The decay of tPA emissions in binary or more complex bilayers is often characterized by multiple (two or three) lifetime components (39). The longest lifetime component is associated with tPA localized in the most ordered environment, where non-radiative processes are lowest (40).

CTL partitioning between large unilamellar vesicles and cyclodextrin at low bilayer sterol content

The equilibrium distribution of CTL between methyl-β-cyclodextrin (CyD; Sigma-Aldrich) and large unilamellar phospholipid vesicles (LUVs) with a pore diameter of 200 nm was determined as described previously (41). Briefly, a constant amount of LUVs were incubated with increasing amounts of CyD in the reaction mixture, until equilibrium was reached. The concentration of CTL in the LUVs and CyD was determined from the CTL anisotropy signal, which was high in the LUVs but much lower in CyD. The assay yielded the molar fraction partition coefficient, Kx, of CTL. The CTL and thus the sterol concentration was 2 mol % in all the CTL partitioning experiments. A high Kx indicates that CTL has a higher affinity for the LUV bilayer than for CyD.

Cholesterol partitioning between the LUVs and CyD in bilayers with an intermediate cholesterol content

The equilibrium partitioning of cholesterol between the LUVs, containing the SM and cholesterol (an initial cholesterol concentration of 15 mol %), and CyD was determined. Briefly, constant amounts of the LUVs, made up of phospholipids in water (final concentration 0.05 mM), containing 1 mol % DPH-PC as reporter of the acyl chain order in the LUVs, were incubated until equilibrium with increasing amounts of CyD (0–2.5 mM) added to the reaction mixture. The concentration of the cholesterol in the LUVs was determined from the DPH-PC anisotropy signal in the LUVs by comparing it with the signal obtained from a standard curve with known variation in the cholesterol/phospholipid ratio. DPH-PC is nonexchangeable under the conditions used, and its anisotropy is sensitive to the amount of cholesterol in the LUVs. After first calculating the cholesterol concentration in the LUVs at each CyD concentration, the molar fraction partition coefficient, Kx, for cholesterol was next calculated as described previously (41, 42). A high Kx indicates a higher affinity of cholesterol for the bilayer as compared to CyD.

Computational optimization of SM and dihydro-SM fragment structures

Jaguar (43) in Maestro (Schrödinger, Portland, OR) was used in the optimization of fragments to understand the differences in the hydrogen bonding properties of 3OH in SM and dihydro-SM. Default settings of Jaguar with density function theory (B3LYP) with 6-31G∗∗ basis set was used.

Results

CTL partitioning between the LUVs and CyD

First, we determined the affinity of the fluorescent sterol, CTL, for bilayers prepared from unsaturated or saturated SM and dihydro-SM. CTL is slightly more polar than cholesterol, but otherwise its interactions with phospholipids are similar to cholesterol (44). The absolute bilayer affinity of CTL is ∼5–7 time less than that of cholesterol, although the relative difference in affinity for e.g., SM and PC bilayers is similar to that of cholesterol (42). As the acyl chain order of the bilayer has a marked effect on the affinity of cholesterol for the phospholipid bilayers (45), we carefully controlled the acyl chain order in our partition experiments, using CTL and DPH anisotropy measurements. In the binary bilayers with POPC/PSM or POPC/dihydro-PSM, we also measured the average tPA lifetimes. As shown in Fig. 1, the acyl chain order of the pure OSM and dihydro-OSM bilayers was different at any given temperature. This is also the case with PSM/dihydro-PSM bilayers (31), with the dihydro species being more ordered at a given temperature compared to the nondihydro SM. Based on the anisotropy data shown in Fig. 1, we measured CTL partitioning in the OSM and dihydro-OSM bilayers at 24°C and 28°C, respectively, to have equal acyl chain order. The POPC bilayers at 16°C also had equal acyl chain order as OSM and dihydro-OSM bilayers (see Fig. 2, B and C). Under these experimental conditions, both the CTL and DPH anisotropy values were similar in the three systems (Fig. 2 C).

Figure 1.

Figure 1

Open in a new tab

Bilayer acyl chain order measured from DPH anisotropy. Multilamellar vesicles prepared from pure OSM or pure dhOSM contained 1 mol % DPH. The steady-state anisotropy of DPH was measured as a temperature function (data shown for the 20–30°C temperature interval). Each value is the average ± SD from n = 3–5.

Figure 2.

Figure 2

Open in a new tab

CTL equilibrium partitioning between the LUVs and CyD. The LUVs (200 nm in diameter) were prepared from pure POPC, OSM, or dihydro-OSM (A–C); POPC/PSM or POPC/dihydro-PSM (4:1 molar ratio, D–F; or pure PSM or dihydro-PSM (G–I), and contained 2 mol % CTL at the start of the experiment. The Kx for CTL partitioning was determined at 16°C (POPC), 24°C (OSM), 28°C (dihydro-OSM), 37°C (POPC/PSM and POPC/dihydro-PSM), 50°C (PSM), or 53.5°C (dihydro-PSM) to achieve close to equal acyl chain order in the respective bilayers. (A, D, and G) Show the Kx values for each type of LUV, and (B, E, and H) show the measured CTL anisotropy values at the start of the experiment. Finally, (C and I) show the DPH anisotropy values at the start of the experiment, and (F) shows the average lifetime component of tPA. The asterisk indicates a statistically significant difference between the compared SM bilayers in each panel (p < 0.001 for A; p < 0.05 for D and G). The difference between CTL partitioning to POPC and the SM bilayers shown in (A) was also significant (p < 0.001). The value pairs in (B), (E), and (H) and (C), (F), and (I) were not significantly different (p > 0.05). Each value is the average ± SD from n = 3–5. Note that dihydro is indicated with dh within the figure.

The equilibrium partitioning of CTL between LUVs, prepared from POPC, OSM, or dihydro-OSM, and CyD showed that the measured Kx (partitioning coefficient, mM) was significantly higher (p < 0.001) in both the OSM and dihydro-OSM bilayers when compared to that of the POPC bilayers (see Fig. 2 A). However, the Kx of CTL was also significantly higher (p < 0.001) in the dihydro-OSM bilayers (Kx of ∼37) when compared to that of the OSM bilayer (Kx of ∼24; Fig. 2 A). Both the CTL and DPH steady-state anisotropies were the same under the conditions tested (Fig. 2, B and C, respectively), verifying that the acyl chain order was the same in both systems. However, the absolute anisotropy values of CTL and DPH were dissimilar, because CTL was more restricted at the interface, whereas DPH experiences more rotational freedom in the acyl chain region. The observed difference in the Kx of the POPC and OSM/dihydro-OSM bilayers can be explained by hydrogen bond stabilization in the OSM/dihydro-OSM bilayers that was not present in POPC bilayers.

To measure the affinity of CTL for binary mixed bilayers containing POPC (80 mol %) and either PSM or dihydro-PSM (at 20 mol %), equilibrium partitioning was performed at 37°C (Fig. 2 D). At this temperature, CTL anisotropy was not significantly different in the two types of bilayers (Fig. 2 E). As the lateral partitioning of DPH is almost the same in ordered and disordered phases (46), a DPH-based anisotropy measurement would not provide a reliable prediction of the acyl chain order in the SM-rich phase. Therefore, we used fluorescence lifetime analysis of tPA instead of DPH anisotropy analysis, to determine the acyl chain order of the more ordered SM-rich phase (39). The average fluorescence lifetime of tPA in POPC/PSM and POPC/dihydro-PSM at 37°C is given in Fig. 2 F. The values were not significantly different. With the experimental conditions used, we observed that the Kx for CTL was significantly higher (p < 0.05) in the POPC/dihydro-PSM bilayers (a Kx of ∼17.5) as compared to the POPC/PSM bilayers (a Kx of ∼13.5; Fig. 2 D).

To measure the partitioning coefficient of CTL in the pure PSM and dihydro-PSM bilayers at equal acyl chain order in both systems (based on both CTL and DPH anisotropy measurements, Fig. 2, H and I), the temperature was increased to 50°C for the PSM bilayers and to 53.5°C for the dihydro-PSM bilayers. The measured Kx of CTL was significantly higher (p < 0.05) in the dihydro-PSM bilayers (Kx ∼46.5) compared to that of the PSM bilayers (Kx ∼42; see Fig. 2 G).

Cholesterol partitioning between the LUVs and CyD

CTL partitioning has to be performed at a low bilayer sterol concentration because of technical limitations. At higher concentrations, CTL self-quenching can occur, and mixing of CTL with cholesterol can be nonideal, obscuring the partition results. However, it is possible that the bilayer sterol concentration influences the sterol affinity to bilayers because high cholesterol is known to induce the formation of a liquid-ordered phase (47, 48). To further determine the affinity of cholesterol for SM and dihydro-SM bilayers at a modest cholesterol concentration (15 mol %), we used a new, to our knowledge, partitioning approach in which the reported anisotropy of DPH-PC in donor LUVs was used to calculate the distribution of cholesterol between the LUVs and CyD. With this approach, we observed that cholesterol had a higher affinity for dihydro-PSM than PSM bilayers at an initial cholesterol concentration of 15 mol % (Fig. 3 A), whereas the initial acyl chain order was similar for both PSM and dihydro-PSM bilayers (Fig. 3, B and C). The difference was significant, with p < 0.05. The absolute values for the Kx of cholesterol and Kx of CTL were markedly different (Figs. 2 G and 3), reflecting the difference in the molecular polarity/hydrophobicity of the sterols. We showed previously that the relative differences in the affinities of sterol to phospholipids for different bilayers were similar (41, 42). We could not determine the affinity of cholesterol for OSM or dihydro-OSM bilayers at the initial 15 mol % cholesterol concentration, because the linear range of the cholesterol/LUV ratio was exceeded due to the higher CyD concentration needed in the partition assay (data not shown).

Figure 3.

Figure 3

Open in a new tab

Partitioning of cholesterol between CyD and the cholesterol-containing PSM and dihydro-PSM bilayers. LUVs (200 nm in diameter) were prepared by extrusion, and the equilibrium partitioning of cholesterol was determined using DPH-PC in the LUVs to register changes in the cholesterol concentration of the LUVs as the LUV/CyD ratio changed. The initial cholesterol content in the LUVs was 15 mol %. The experimental temperature was 50°C for the PSM and 53.5°C for the dihydro-PSM systems. The asterisk indicates a significant difference in Kx for PSM and dihydro-PSM (p < 0.05). Each value is the average ± SD from n = 3–5. (A) Shows the measured Kx, (B) gives the initial anisotropy determined reported by DPH-PC, and (C) shows the initial anisotropy reported by DPH. Note that dihydro is indicated with dh within the figure.

Cholesterol-induced ordering of the OSM and dihydro-OSM bilayers

To further examine the interaction of cholesterol with OSM and dihydro-OSM, we added cholesterol to the OSM/dihydro-OSM bilayers in small increments, and determined the formation of an increasingly ordered phase based on the fluorescence lifetime behavior of tPA. An increase in the fluorescence lifetime of tPA is indicative of tPA residing in an increasingly ordered environment. As shown in Fig. 4, the fluorescence lifetime of tPA increased almost linearly in response to the addition of cholesterol (between 3 and 21 mol %) to the bilayers. The longest fluorescence lifetime component was slightly longer in the dihydro-OSM bilayers as compared to that in the OSM bilayers at identical cholesterol concentrations (Fig. 4 A). For the average tPA fluorescence lifetime, there was no clear difference between dihydro-OSM and OSM bilayers (Fig. 4 B). Because the average fluorescence lifetime includes contributions from all phases present, the similarity in the average fluorescence lifetime of tPA for OSM and dihydro-OSM bilayers suggests that a fractional contribution from the more ordered cholesterol-rich phase was small to the average fluorescence lifetime of tPA. The lifetime of tPA in the OSM/dihydro-OSM bilayers was also not as long as it would be in a liquid-ordered phase composed of saturated SM and cholesterol (39). Unfortunately, we could not measure the effects of the addition of cholesterol on the tPA fluorescence lifetime in the saturated SM bilayers because the emission intensity of tPA is very low at the high temperatures needed to keep PSM and dihydro-PSM in a fluid state. The finding that the cholesterol/dihydro-OSM bilayers formed an ordered phase, with a longer tPA fluorescence lifetime as compared to the cholesterol/OSM bilayer, suggests that the cholesterol/dihydro-OSM ordered phase was more ordered than the cholesterol/OSM ordered phase. This finding may point to a more favorable interaction between cholesterol and dihydro-OSM than between cholesterol and OSM. As shown in the analysis of the OSM bilayers at 24°C and the dihydro-OSM bilayers at 28°C (Fig. 1), the initial acyl chain order in the bilayers was the same. However, as the cholesterol content of the bilayer increased, the acyl chain order changed.

Figure 4.

Figure 4

Open in a new tab

Cholesterol-induced ordering of the OSM and dihydro-OSM bilayers. MLVs containing OSM or dihydro-OSM and increasing amounts of cholesterol and 1 mol % tPA were prepared. A fluorescence lifetime analysis of tPA emission was performed at 24°C for the OSM bilayers (solid triangles) and 28°C for the dihydro-OSM bilayers (open circles). (A) Shows the longest lifetime of tPA, and (B) shows the average lifetime of tPA as a function of the cholesterol concentration. Each value is the average ± SD from n = 3–5. Note that dihydro is indicated with dh within the figure.

Ceramide and diacylglycerol segregation in OSM and dihydro-OSM bilayers

The presence of palmitoyl ceramide in a fluid POPC bilayer leads to efficient self-association into an increasingly ordered phase above 3–4 mol % ceramide (49). A chain-matched diacylglycerol will also segregate in a fluid POPC bilayer, but the concentration needed for the formation of the ordered phase is higher for the diacylglycerol than for ceramide (33). This observation was interpreted as showing that intermolecular hydrogen bonding among ceramides contributed to the stabilization of the ceramide ordered phase (33).

We determined the formation of a ceramide or DPG-induced ordered phase in the fluid OSM or dihydro-OSM bilayers using time-resolved analysis of tPA fluorescence emission. The initial conditions were selected so that the acyl chain order in the OSM and dihydro-OSM bilayers were equal. As shown in Fig. 5 A, where the longest lifetime component of the tPA emission is plotted against the ceramide or DPG concentration, PCer-20:1 formed an ordered phase in OSM bilayers at concentrations higher than 6 mol %. However, DPG failed to form an ordered phase in the same concentration interval (Fig. 5 A). In the dihydro-OSM bilayers, PCer-20:1 started to form an ordered phase in bilayers at an even lower concentration (>2 mol %) than was the case in the OSM bilayers (Fig. 5, A and B). DPG more readily formed an ordered phase in the dihydro-OSM bilayer than in the OSM bilayer. The difference in the ordered phase formation of PCer-20:1 and DPG supports the notion that hydrogen bonding stabilized the self-association of ceramide. Furthermore, the differences in the hydrogen bonding of the OSM and dihydro-OSM bilayers appeared to influence the phase formation of DPG (i.e., it more readily formed an ordered phase in the dihydro-OSM than in the OSM bilayers). A comparison of the concentration of PCer-20:1 required to produce a certain ordered phase packing, as deduced from the longest lifetime component (Fig. 5, A and B), showed that ∼4 mol % less PCer-20:1 was needed in dihydro-OSM bilayers than OSM bilayers. This suggests that the self-association of PCer-20:1 was better in the dihydro-OSM bilayers than in the OSM bilayers. Thus, it appears that the hydrogen bonding differences of the OSM and dihydro-OSM bilayers affected the self-association of PCer-20:1.

Figure 5.

Figure 5

Open in a new tab

Formation of a gel phase by PCer-20:1 and DPG in the OSM and dihydro-OSM bilayers. MLVs with the indicated lipid compositions were prepared, together with 1 mol % tPA. As a function of added PCer-20:1 (solid symbols) or DPG (open symbols), the longest tPA lifetime, indicative of the formation of a gel phase, of each sample was determined at 24°C (OSM, A) or 28°C (dihydro-OSM, B). At these temperatures, the acyl chain order of the pure OSM and pure dihydro-OSM bilayers were initially similar. Each value is the average ± SD from n = 3–5. Note that dihydro is indicated with dh within the figure.

Discussion

The bilayer affinity of cholesterol and sterols is influenced by the phospholipid acyl chain length (50), degree of unsaturation, and hence the acyl chain order (45). Effects of the phospholipid headgroup on cholesterol/phospholipid association are well documented (51, 52, 53). Hydrogen bonding has also been reported to affect cholesterol/colipid interactions (54). The relative contribution of all these factors to cholesterol/colipid interactions is difficult to approximate. In this study, we compared SM with its chain-matched dihydro-SM to elucidate the effect of the trans double bond of the sphingosine base in SM on SM/colipid interactions. We studied SM and dihydro-SM because several reports have suggested that their hydrogen bonding properties differed significantly (11, 27, 28, 29), with the trans double bond (or a lack thereof) having a strong influence on how SMs interact with each other and with other lipids in bilayer membranes.

To measure the sterol affinity of the unilamellar bilayers, we used two approaches. In the first, we determined the bilayer affinity of CTL at a low bilayer sterol concentration (2 mol %). In the second approach, we determined the bilayer affinity of cholesterol at an initial bilayer cholesterol concentration of 15 mol %. In both cases, we measured equilibrium partitioning under conditions where the bilayer acyl chain order of SM and dihydro-SM was the same (Figs. 2 and 3). This was accomplished by varying the experimental temperature. The distribution of CTL or cholesterol between the bilayers and CyD in the aqueous phase was determined from the CTL or DPH-PC anisotropy in the LUV fraction, respectively (41, 42). Both with CTL (at a low bilayer sterol concentration) and cholesterol (an initial concentration of 15 mol %), we observed that the bilayer affinity of CTL/cholesterol was higher with dihydro-SM than with SM bilayers having the same acyl chain order. Furthermore, we confirmed that the sterol affinity for SM bilayers was higher than for comparable PC bilayers (Fig. 2 A, this study, and Fig. 1 in (45)). As reported previously (11, 27, 28, 29), the most obvious explanation for the increased sterol affinity for the dihydro-SM bilayers compared to the matched SM bilayers is the difference in hydrogen bonding of the two types of SMs. The effect of increasing temperature on hydrogen bonding is likely to weaken the bond strength. Because dihydro-SM bilayers were measured at a slightly higher temperature than comparable SM bilayers, the difference in temperature-corrected Kx would probably be slightly larger than the one observed. Packing differences could also affect the measured partition of cholesterol. However, even though dihydro-ceramides have been reported to pack more densely than comparable ceramides in monolayer membranes (55), dihydro-SM and SM have been shown to pack similarly in monolayer membranes (31).

The systematic inclusion of increasing amounts of cholesterol in the OSM or dihydro-OSM bilayers led to the formation of an ordered cholesterol-enriched phase. The presence of the cholesterol-rich phase was determined from the time-resolved emission of tPA, a fluorescent fatty acid that favors ordered phases to disordered ones. The longest tPA fluorescence lifetime component measured in the dihydro-OSM bilayers suggested that the cholesterol-enriched phase was slightly more ordered than the corresponding phase that formed in the OSM bilayers (Fig. 4). The longest tPA fluorescence lifetime represents the most ordered phase present in a bilayer. Therefore, it is more sensitive than the average fluorescence lifetime value, which also contains information about shorter fluorescence lifetime components resulting from tPA in more disordered phases. The finding that the dihydro-OSM bilayers formed a more ordered cholesterol-rich phase than the OSM bilayers does suggest a more favorable and closer association between cholesterol and dihydro-OSM. This association is most likely in part a result of the hydrogen-bonding properties of dihydro-OSM, which were different from those in the OSM bilayers.

Although both cholesterol and ceramide are amphiphilic molecules, with small polar functions at their water interface, their hydrogen-bonding properties are different. Cholesterol has a hydrogen bond-donating hydroxyl in the C3-position, whereas ceramide has two hydroxyls (on C1 and C3). In addition, the acyl chain of ceramide is amide-linked, and this functional group plays a major role in hydrogen bonding. In addition, whereas the cholesterol has a rough beta surface and a smoother alpha surface, saturated ceramides do not have these properties, and thus close van der Waals interactions are likely to differ for cholesterol and saturated ceramides. Based on these structural considerations, it is perhaps not surprising that the sensitivity of cholesterol and ceramide to the headgroup properties of their interacting partner molecules differs (51, 56). In addition to more favorable van der Waals interactions, it is likely that also hydrogen bonding appears to contribute to the stabilization of ceramide/SM interaction in a way that is not possible with cholesterol. In light of the aforementioned, the difference in the lateral segregation of PCer-20:1 into an ordered phase in the dihydro-OSM and OSM bilayers is perhaps not surprising (Fig. 5). The observation that DPG required a much higher bilayer concentration to form an ordered phase is consistent with our recent report on the lateral segregation of PCer-20:1 and DPG in POPC bilayers (33). It suggests that hydrogen-bonding differences between ceramides and matched diglycerides have marked effects on their molecular interactions and lateral segregation. The difference in DPG segregation in OSM and dihydro-OSM bilayers can be understood if hydrogen bonding among dihydro-OSM molecules is stronger than between OSM-molecules—in that situation DPG would more efficiently be excluded from dihydro-SM interactions, and would consequently segregate to their own phase more readily.

Given the clear differences between SM and dihydro-SM, with increased gel phase stabilization in dihydro-PSM (31), more favorable sterol (Figs. 2 and 3) and ceramide (Fig. 5) interactions in the dihydro-SM bilayers than in the SM bilayers, and the reported lower Prodan partitioning into dihydro-PSM bilayers compared to PSM bilayers (57), one can start to consider how the trans double bond (or its absence) might affect the properties of the adjacent 3OH. The trans double bond has been reported to allow much better hydrogen bonding among SMs than a cis double bond in the same position (58). However, the double bond itself is not a partner in hydrogen bonding, so the effect must be indirect, via changed packing properties or by altered hydrogen bonding. Based on quantum chemical calculations, the trans double bond of the sphingoid base on SM restricts the rotational freedom of the neighboring C-C bond, where the 3OH is attached, into two likely conformations (Fig. 6, A and B), which are nearly equally possible. The conformation shown in Fig. 6 A is favored over that shown in Fig. 6 B only by 0.21 kcal/mol. In case of dihydro-SM, similar bond rotation restriction does not apply due to the absence of the trans double bond, leading to an increased rotational freedom of 3OH (Fig. 6, C and D). As hydrogen bonds are directional, the more flexible 3OH in dihydro-SM has more possibilities to form a hydrogen bond than a sterically restricted 3OH in SM. The intramolecular hydrogen bonds between the 3OH and phosphate oxygens (8, 11, 59, 60), are also likely to be affected by the different rotational freedom of the 3OH in SM and dihydro-SM bilayers. It is also possible that a restricted flexibility of this interfacial region in dihydro-SM influences order in the direction of the hydrocarbon tail.

Figure 6.

Figure 6

Open in a new tab

Energy-minimized molecular structures of a partial SM and dihydro-SM fragment. Structures (A) and (B) represent SM, and fragments (C) and (D) depict dihydro-SM. The blue dotted arrow (A) indicates the restricted rotation of the C-C bond. The blue complete arrow (C and D) indicates less hindered rotation of the C-C bond. The orange dotted arrow (B) indicates the different orientation of the bond in (B) compared to (A), but its rotation is still hindered. The complete orange arrow in (D) indicates the changed orientation compared to (C), with the rotation of the C-C bond less hindered. The energy difference between configuration (A and B) is only ∼0.21 kcal/mol, which means that both configurations are likely. To see this figure in color, go online.

In conclusion, we have used the different hydrogen bonding propensity of SM and dihydro-SM to experimentally demonstrate consequences of such differences for SM/colipid interactions. We have previously demonstrated how differing hydrogen bonding of glycerophospholipids and SM affect phospholipid/cholesterol interactions in bilayer membranes (54). Now we show that hydrogen bonding differences between SM molecular species also give rise to measurable differences in bilayer interactions involving these lipids. All these results together demonstrate that hydrogen bonding affect lipid interactions to a marked extent, although it is clear that other types of interactions (hydrophobic or van der Waals interactions, steric effects, headgroup effects, etc.) are at least equally important for lipid interactions in hydrated bilayers.

Author Contributions

All authors contributed to the execution of the study and the writing of the article. All authors have approved the final version of the article.

Acknowledgments

We thank Dr. Thomas Nyholm for valuable suggestions regarding this project.

Financial support was provided by the Academy of Finland, the Sigrid Juselius Foundation, and the Åbo Akademi Foundation. CSC, The Finnish IT Center for Science is acknowledged for computational resources (project No. jyy2516). T.Y. was supported by the International Collaboration Promotion Program from Osaka University.

Editor: Tobias Baumgart.

References

  • 1.Barenholz Y. Sphingomyelin-lecithin balance in membranes: composition, structure, and function relationships. In: Shinitzky M., editor. Physiology of Membrane Fluidity. CRC Press; Boca Raton, FL: 1984. pp. 131–174. [Google Scholar]
  • 2.Pick L., Bielschowsky M. Verhandlungen ärztlicher gesellschaften. Klin. Wschr. 1927;6:1631–1637. [Google Scholar]
  • 3.Shapiro D., Flowers H.M. Studies on sphingolipids. VII. Synthesis and configuration of natural sphingomyelins. J. Am. Chem. Soc. 1962;84:1047–1050. [Google Scholar]
  • 4.Boggs J.M. Lipid intermolecular hydrogen bonding: influence on structural organization and membrane function. Biochim. Biophys. Acta. 1987;906:353–404. doi: 10.1016/0304-4157(87)90017-7. [DOI] [PubMed] [Google Scholar]
  • 5.Fong J.W., Tirri L.J., Brockerhoff H. Studies on the hydrogen belts of membranes: I. Diester, diether, and dialkyl phosphatidycholines and polyoxyethylene glycerides in monolayers with cholesterol. Lipids. 1977;12:857–862. doi: 10.1007/BF02533276. [DOI] [PubMed] [Google Scholar]
  • 6.Tirri L.J., Schmidt P.C., Brockerhoff H. Studies on the hydrogen belts of membranes: II. Non-electrolyte permeability of liposomes of diester, diether, and dialkyl phosphatidylcholine and cholesterol. Lipids. 1977;12:863–868. doi: 10.1007/BF02533277. [DOI] [PubMed] [Google Scholar]
  • 7.Tirri L.J., Ayengar N.K., Brockerhoff H. Studies on the hydrogen belts of membranes: III. Glycerol permeability of dihydrosphingomyelin-cholesterol membranes. Lipids. 1978;13:267–269. doi: 10.1007/BF02533668. [DOI] [PubMed] [Google Scholar]
  • 8.Schmidt C.F., Barenholz Y., Thompson T.E. A nuclear magnetic resonance study of sphingomyelin in bilayer systems. Biochemistry. 1977;16:2649–2656. doi: 10.1021/bi00631a011. [DOI] [PubMed] [Google Scholar]
  • 9.Villalaín J., Ortiz A., Gómez-Fernández J.C. Molecular interactions between sphingomyelin and phosphatidylcholine in phospholipid vesicles. Biochim. Biophys. Acta. 1988;941:55–62. doi: 10.1016/0005-2736(88)90213-1. [DOI] [PubMed] [Google Scholar]
  • 10.Lamba O.P., Borchman D., Lou M.F. Structure and molecular conformation of anhydrous and of aqueous sphingomyelin bilayers determined by infrared and Raman spectroscopy. J. Mol. Struct. 1991;248:1–24. [Google Scholar]
  • 11.Talbott C.M., Vorobyov I., Yappert M.C. Conformational studies of sphingolipids by NMR spectroscopy. II. Sphingomyelin. Biochim. Biophys. Acta. 2000;1467:326–337. doi: 10.1016/s0005-2736(00)00229-7. [DOI] [PubMed] [Google Scholar]
  • 12.Róg T., Pasenkiewicz-Gierula M. Cholesterol-sphingomyelin interactions: a molecular dynamics simulation study. Biophys. J. 2006;91:3756–3767. doi: 10.1529/biophysj.106.080887. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Niemelä P., Hyvönen M.T., Vattulainen I. Structure and dynamics of sphingomyelin bilayer: insight gained through systematic comparison to phosphatidylcholine. Biophys. J. 2004;87:2976–2989. doi: 10.1529/biophysj.104.048702. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Sodt A.J., Pastor R.W., Lyman E. Hexagonal substructure and hydrogen bonding in liquid-ordered phases containing palmitoyl sphingomyelin. Biophys. J. 2015;109:948–955. doi: 10.1016/j.bpj.2015.07.036. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Kan C.-C., Ruan Z.S., Bittman R. Interaction of cholesterol with sphingomyelin in bilayer membranes: evidence that the hydroxy group of sphingomyelin does not modulate the rate of cholesterol exchange between vesicles. Biochemistry. 1991;30:7759–7766. doi: 10.1021/bi00245a013. [DOI] [PubMed] [Google Scholar]
  • 16.Guo W., Kurze V., Hamilton J.A. A solid-state NMR study of phospholipid-cholesterol interactions: sphingomyelin-cholesterol binary systems. Biophys. J. 2002;83:1465–1478. doi: 10.1016/S0006-3495(02)73917-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Matsumori N., Yasuda T., Murata M. Comprehensive molecular motion capture for sphingomyelin by site-specific deuterium labeling. Biochemistry. 2012;51:8363–8370. doi: 10.1021/bi3009399. [DOI] [PubMed] [Google Scholar]
  • 18.Yasuda T., Kinoshita M., Matsumori N. Detailed comparison of deuterium quadrupole profiles between sphingomyelin and phosphatidylcholine bilayers. Biophys. J. 2014;106:631–638. doi: 10.1016/j.bpj.2013.12.034. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Clejan S., Bittman R. Decreases in rates of lipid exchange between Mycoplasma gallisepticum cells and unilamellar vesicles by incorporation of sphingomyelin. J. Biol. Chem. 1984;259:10823–10826. [PubMed] [Google Scholar]
  • 20.Matsumori N., Yamaguchi T., Murata M. Orientation and order of the amide group of sphingomyelin in bilayers determined by solid-state NMR. Biophys. J. 2015;108:2816–2824. doi: 10.1016/j.bpj.2015.05.011. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Björkbom A., Róg T., Slotte J.P. N- and O-methylation of sphingomyelin markedly affects its membrane properties and interactions with cholesterol. Biochim. Biophys. Acta. 2011;1808:1179–1186. doi: 10.1016/j.bbamem.2011.01.009. [DOI] [PubMed] [Google Scholar]
  • 22.Schneider P.B., Kennedy E.P. Metabolism of labeled dihydrosphingomyelin in vivo. J. Lipid Res. 1968;9:58–64. [PubMed] [Google Scholar]
  • 23.Borchman D., Yappert M.C. Lipids and the ocular lens. J. Lipid Res. 2010;51:2473–2488. doi: 10.1194/jlr.R004119. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Ferguson S.R., Borchman D., Yappert M.C. Confirmation of the identity of the major phospholipid in human lens membranes. Invest. Ophthalmol. Vis. Sci. 1996;37:1703–1706. [PubMed] [Google Scholar]
  • 25.Yappert M.C., Borchman D. Sphingolipids in human lens membranes: an update on their composition and possible biological implications. Chem. Phys. Lipids. 2004;129:1–20. doi: 10.1016/j.chemphyslip.2003.12.003. [DOI] [PubMed] [Google Scholar]
  • 26.Byrdwell W.C., Borchman D. Liquid chromatography/mass-spectrometric characterization of sphingomyelin and dihydrosphingomyelin of human lens membranes. Ophthalmic Res. 1997;29:191–206. doi: 10.1159/000268014. [DOI] [PubMed] [Google Scholar]
  • 27.Ferguson-Yankey S.R., Borchman D., Yappert M.C. Conformational studies of sphingolipids by NMR spectroscopy. I. Dihydrosphingomyelin. Biochim. Biophys. Acta. 2000;1467:307–325. doi: 10.1016/s0005-2736(00)00228-5. [DOI] [PubMed] [Google Scholar]
  • 28.Epand R.M. Cholesterol in bilayers of sphingomyelin or dihydrosphingomyelin at concentrations found in ocular lens membranes. Biophys. J. 2003;84:3102–3110. doi: 10.1016/S0006-3495(03)70035-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Kinoshita M., Matsumori N., Murata M. Coexistence of two liquid crystalline phases in dihydrosphingomyelin and dioleoylphosphatidylcholine binary mixtures. Biochim. Biophys. Acta. 2014;1838:1372–1381. doi: 10.1016/j.bbamem.2014.01.017. [DOI] [PubMed] [Google Scholar]
  • 30.Térová B., Slotte J.P., Nyholm T.K. Miscibility of acyl-chain defined phosphatidylcholines with N-palmitoyl sphingomyelin in bilayer membranes. Biochim. Biophys. Acta. 2004;1667:182–189. doi: 10.1016/j.bbamem.2004.10.003. [DOI] [PubMed] [Google Scholar]
  • 31.Kuikka M., Ramstedt B., Slotte J.P. Membrane properties of D-erythro-N-acyl sphingomyelins and their corresponding dihydro species. Biophys. J. 2001;80:2327–2337. doi: 10.1016/S0006-3495(01)76203-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Cohen R., Barenholz Y., Dagan A. Preparation and characterization of well defined D-erythro sphingomyelins. Chem. Phys. Lipids. 1984;35:371–384. doi: 10.1016/0009-3084(84)90079-3. [DOI] [PubMed] [Google Scholar]
  • 33.Ekman P., Maula T., Slotte J.P. Formation of an ordered phase by ceramides and diacylglycerols in a fluid phosphatidylcholine bilayer--correlation with structure and hydrogen bonding capacity. Biochim. Biophys. Acta. 2015;1848(10 Pt A):2111–2117. doi: 10.1016/j.bbamem.2015.06.019. [DOI] [PubMed] [Google Scholar]
  • 34.Fischer R.T., Stephenson F.A., Schroeder F. delta 5,7,9(11)-Cholestatrien-3 beta-ol: a fluorescent cholesterol analogue. Chem. Phys. Lipids. 1984;36:1–14. doi: 10.1016/0009-3084(84)90086-0. [DOI] [PubMed] [Google Scholar]
  • 35.Björkqvist Y.J., Nyholm T.K., Ramstedt B. Domain formation and stability in complex lipid bilayers as reported by cholestatrienol. Biophys. J. 2005;88:4054–4063. doi: 10.1529/biophysj.104.054718. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Kuklev D.V., Smith W.L. Synthesis of four isomers of parinaric acid. Chem. Phys. Lipids. 2004;131:215–222. doi: 10.1016/j.chemphyslip.2004.06.001. [DOI] [PubMed] [Google Scholar]
  • 37.Sklar L.A., Hudson B.S., Diamond J. Conjugated polyene fatty acids on fluorescent probes: spectroscopic characterization. Biochemistry. 1977;16:813–819. doi: 10.1021/bi00624a001. [DOI] [PubMed] [Google Scholar]
  • 38.Lakowicz J.R. Kluwer Academic/Plenum Publishers; New York: 1999. Principles of Fluorescence Spectroscopy. [Google Scholar]
  • 39.Nyholm T.K., Lindroos D., Slotte J.P. Construction of a DOPC/PSM/cholesterol phase diagram based on the fluorescence properties of trans-parinaric acid. Langmuir. 2011;27:8339–8350. doi: 10.1021/la201427w. [DOI] [PubMed] [Google Scholar]
  • 40.Sklar L.A., Hudson B.S., Simoni R.D. Conjugated polyene fatty acids as fluorescent probes: synthetic phospholipid membrane studies. Biochemistry. 1977;16:819–828. doi: 10.1021/bi00624a002. [DOI] [PubMed] [Google Scholar]
  • 41.Nyholm T.K., Grandell P.M., Slotte J.P. Sterol affinity for bilayer membranes is affected by their ceramide content and the ceramide chain length. Biochim. Biophys. Acta. 2010;1798:1008–1013. doi: 10.1016/j.bbamem.2009.12.025. [DOI] [PubMed] [Google Scholar]
  • 42.Nyström J.H., Lönnfors M., Nyholm T.K. Transmembrane peptides influence the affinity of sterols for phospholipid bilayers. Biophys. J. 2010;99:526–533. doi: 10.1016/j.bpj.2010.04.052. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 43.Bochevarov A.D., Harder E., Friesner R.A. Jaguar: a high-performance quantum chemistry software program with strengths in life and material sciences. Int. J. Quantum Chem. 2013;113:2110–2142. [Google Scholar]
  • 44.Scheidt H.A., Muller P., Huster D. The potential of fluorescent and spin-labeled steroid analogs to mimic natural cholesterol. J. Biol. Chem. 2003;278:45563–45569. doi: 10.1074/jbc.M303567200. [DOI] [PubMed] [Google Scholar]
  • 45.Halling K.K., Ramstedt B., Nyholm T.K. Cholesterol interactions with fluid-phase phospholipids: effect on the lateral organization of the bilayer. Biophys. J. 2008;95:3861–3871. doi: 10.1529/biophysj.108.133744. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 46.Kaiser R.D., London E. Location of diphenylhexatriene (DPH) and its derivatives within membranes: comparison of different fluorescence quenching analyses of membrane depth. Biochemistry. 1998;37:8180–8190. doi: 10.1021/bi980064a. [DOI] [PubMed] [Google Scholar]
  • 47.Ipsen J.H., Karlström G., Zuckermann M.J. Phase equilibria in the phosphatidylcholine-cholesterol system. Biochim. Biophys. Acta. 1987;905:162–172. doi: 10.1016/0005-2736(87)90020-4. [DOI] [PubMed] [Google Scholar]
  • 48.Sankaram M.B., Thompson T.E. Modulation of phospholipid acyl chain order by cholesterol. A solid-state 2H nuclear magnetic resonance study. Biochemistry. 1990;29:10676–10684. doi: 10.1021/bi00499a015. [DOI] [PubMed] [Google Scholar]
  • 49.Silva L., de Almeida R.F., Prieto M. Ceramide-platform formation and -induced biophysical changes in a fluid phospholipid membrane. Mol. Membr. Biol. 2006;23:137–148. doi: 10.1080/09687860500439474. [DOI] [PubMed] [Google Scholar]
  • 50.Jaikishan S., Slotte J.P. Effect of hydrophobic mismatch and interdigitation on sterol/sphingomyelin interaction in ternary bilayer membranes. Biochim. Biophys. Acta. 2011;1808:1940–1945. doi: 10.1016/j.bbamem.2011.04.004. [DOI] [PubMed] [Google Scholar]
  • 51.Björkbom A., Róg T., Slotte J.P. Effect of sphingomyelin headgroup size on molecular properties and interactions with cholesterol. Biophys. J. 2010;99:3300–3308. doi: 10.1016/j.bpj.2010.09.049. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 52.Huang J., Buboltz J.T., Feigenson G.W. Maximum solubility of cholesterol in phosphatidylcholine and phosphatidylethanolamine bilayers. Biochim. Biophys. Acta. 1999;1417:89–100. doi: 10.1016/s0005-2736(98)00260-0. [DOI] [PubMed] [Google Scholar]
  • 53.Huang J., Feigenson G.W. A microscopic interaction model of maximum solubility of cholesterol in lipid bilayers. Biophys. J. 1999;76:2142–2157. doi: 10.1016/S0006-3495(99)77369-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 54.Lönnfors M., Doux J.P., Slotte J.P. Sterols have higher affinity for sphingomyelin than for phosphatidylcholine bilayers even at equal acyl-chain order. Biophys. J. 2011;100:2633–2641. doi: 10.1016/j.bpj.2011.03.066. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 55.Pascher I. Molecular arrangements in sphingolipids. Conformation and hydrogen bonding of ceramide and their implication on membrane stability and permeability. Biochim. Biophys. Acta. 1976;455:433–451. doi: 10.1016/0005-2736(76)90316-3. [DOI] [PubMed] [Google Scholar]
  • 56.Artetxe I., Sergelius C., Maula T. Effects of sphingomyelin headgroup size on interactions with ceramide. Biophys. J. 2013;104:604–612. doi: 10.1016/j.bpj.2012.12.026. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 57.Nyholm T., Nylund M., Slotte J.P. Properties of palmitoyl phosphatidylcholine, sphingomyelin, and dihydrosphingomyelin bilayer membranes as reported by different fluorescent reporter molecules. Biophys. J. 2003;84:987–997. doi: 10.1016/S0006-3495(03)74915-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 58.Janosi L., Gorfe A. Importance of the sphingosine base double-bond geometry for the structural and thermodynamic properties of sphingomyelin bilayers. Biophys. J. 2010;99:2957–2966. doi: 10.1016/j.bpj.2010.09.020. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 59.Bruzik K.S. Conformation of the polar headgroup of sphingomyelin and its analogues. Biochim. Biophys. Acta. 1988;939:315–326. doi: 10.1016/0005-2736(88)90076-4. [DOI] [PubMed] [Google Scholar]
  • 60.Venable R.M., Sodt A.J., Klauda J.B. CHARMM all-atom additive force field for sphingomyelin: elucidation of hydrogen bonding and of positive curvature. Biophys. J. 2014;107:134–145. doi: 10.1016/j.bpj.2014.05.034. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Biophysical Journal are provided here courtesy of The Biophysical Society

RESOURCES