Formation of ceramide/sphingomyelin gel domains in the presence of an unsaturated phospholipid: a quantitative multiprobe approach

To better understand how ceramide modulates the biophysical properties of the membrane, the interactions between palmitoyl-ceramide (PCer) and palmitoyl-sphingomyelin (PSM) were studied in the presence of the fluid phospholipid palmitoyl-oleoyl-phosphatidylcholine (POPC) in membrane model systems. The use of two fluorescent membrane probes distinctly sensitive to lipid phases allowed a thorough biophysical characterization of the ternary system. In these mixtures, PCer recruits POPC and PSM in the fluid phase to form extremely ordered and compact gel domains. Gel domain formation by low PCer mol fraction (up to 12 mol %) is enhanced by physiological PSM levels (approximately 20-30 mol % total lipid). For higher PSM content, a three-phase situation, consisting of fluid (POPC-rich)/gel (PSM-rich)/gel (PCer-rich) coexistence, is clearly shown. To determine the fraction of each phase a quantitative method was developed. This allowed establishing the complete ternary phase diagram, which helps to predict PCer-rich gel domain formation and explains its enhancement through PSM/PCer interactions.     

Ceramide-domain formation and collapse in lipid rafts: membrane reorganization by an apoptotic lipid

The effect of physiologically relevant ceramide concentrations (< or = 4 mol %) in raft model membranes with a lipid composition resembling that of cell membranes, i.e., composed of different molar ratios of an unsaturated glycerophospholipid, sphingomyelin, and cholesterol (Chol) along a liquid-disordered-liquid-ordered tie line was explored. The application of a fluorescence multiprobe and multiparameter approach, together with multiple fluorescence resonance energy transfer (FRET) pairs, in the well-characterized palmitoyl-oleoyl-phosphocholine (POPC)/palmitoyl-sphingomyelin (PSM)/Chol ternary mixture, revealed that low palmitoyl-ceramide (PCer) concentrations strongly changed both the biophysical properties and lipid lateral organization of the ternary mixtures in the low-to-intermediate Chol/PSM-, small raft size range (<25 mol % Chol). For these mixtures, PCer recruited up to three PSM molecules for the formation of very small ( approximately 4 nm) and highly ordered gel domains, which became surrounded by rafts (liquid-ordered phase) when Chol/PSM content increased. However, the size of these rafts did not change, showing that PCer did not induce the formation of large platforms or the coalescence of small rafts. In the high Chol/PSM-, large raft domains range (>33 mol % Chol), Chol completely abolished the effect of PCer by competing for PSM association. Lipid rafts govern the biophysical properties and lateral organization in these last mixtures.     

Effects of sphingosine 2N- and 3O-methylation on palmitoyl ceramide properties in bilayer membranes

To study the role of the interfacial properties of ceramides in their interlipid interactions, we synthesized palmitoylceramide (PCer) analogs in which a methyl group was introduced to the amide-nitrogen or the C3-oxygen of the sphingosine backbone. A differential scanning calorimetry analysis of equimolar mixtures of palmitoylsphingomyelin (PSM) and PCer showed that these sphingolipids formed a complex gel phase that melted between 67°C and 74°C. The PCer analogs also formed gel phases with PSM, but they melted at lower temperatures compared with the system with PCer. In complex bilayers composed of an unsaturated glycerophospholipid, PSM, and cholesterol, the 3O-methylated ceramide formed a cholesterol-poor ordered phase with PSM. However, the 2N-methylated and doubly methylated (2N and 3O) PCer analogs failed to displace sterol from interactions with PSM. Like PCer, the analogs reduced sterol affinity for the complex bilayers, but this effect was most pronounced for the 3O-methylated ceramide. Taken together, our results show that 2N-methylation weakened the ceramide-PSM interactions, whereas the 3O-methylated ceramide behaved more like PCer in interactions with PSM. Our findings are compatible with the view that interlipid interactions between the amide-nitrogen and neighboring lipids are important for the cohesive properties of sphingolipids in membranes, and this also appears to be a valid model for ceramide.     

Miscibility of Sphingomyelins and Phosphatidylcholines in Unsaturated Phosphatidylcholine Bilayers

Polyunsaturated phospholipids are common in biological membranes and affect the lateral structure of bilayers. We have examined how saturated sphingomyelin (SM; palmitoyl and stearoyl SM (PSM and SSM, respectively)) and phosphatidylcholine (PC; dipalmitoyl PC and 1-palmitoyl-2-stearoyl PC (DPPC and PSPC, respectively)) segregate laterally to form ordered gel phases in increasingly unsaturated PC bilayers (sn-1: 16:0 and sn-2: 18:1...22:6; or sn-1 and sn-2: 18:1…22:6). The formation of gel phases was determined from the lifetime analysis of trans-parinaric acid. Using calorimetry, we also determined gel phase formation by PSM and DPPC in unsaturated PC mixed bilayers. Comparing PSM with DPPC, we observed that PSM formed a gel phase with less order than DPPC at comparable bilayer concentrations. The same was true when SSM was compared with PSPC. Furthermore, we observed that at equal saturated phospholipid concentration, the gel phases formed were less ordered in unsaturated PCs having 16:0 in sn-1, as compared to PCs having unsaturated acyl chains in both sn-1 and sn-2. The gel phases formed by the saturated phospholipids in unsaturated PC bilayers did not appear to achieve properties similar to pure saturated phospholipid bilayers, suggesting that complete lateral phase separation did not occur. Based on scanning calorimetry analysis, the melting of the gel phases formed by PSM and DPPC in unsaturated PC mixed bilayers (at 45 mol % saturated phospholipid) had low cooperativity and hence most likely were of mixed composition, in good agreement with trans-parinaric acid lifetime data. We conclude that both interfacial properties of the saturated phospholipids and their chain length, as well as the presence of 16:0 in sn-1 of the unsaturated PCs and the total number of cis unsaturations and acyl chain length (18 to 22) of the unsaturated PCs, all affected the formation of gel phases enriched in saturated phospholipids, under the conditions used.     

Nonlamellar-Phase-Promoting Colipids Enhance Segregation of Palmitoyl Ceramide in Fluid Bilayers

Ceramide is an important intermediate in sphingolipid homeostasis. We examined how colipids, with negative intrinsic curvature and which may induce curvature stress in the bilayers, affected the segregation of palmitoyl ceramide (PCer). Such colipids include 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), and tetra-linoleoyl cardiolipin (CL). In 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) bilayers, PCer formed ordered, gel-like domains at concentrations above 10 mol% at 23°C, as evidenced by the change in the average lifetime of the trans-parinaric acid emission. When POPE or DOPE were included in the DOPC bilayer (at 20:80 or 40:60 POPE or DOPE to DOPC, by mol), the lateral segregation of PCer was facilitated in a concentration-dependent manner, and less PCer was required for the formation of the ordered ceramide-rich domains. Inclusion of CL in the DOPE bilayer (at 10:90 or 20:80 CL to PC, by mol) also caused a similar facilitation of the lateral segregation of PCer. The PCer-rich domains formed in the presence of POPE, DOPE, or CL in DOPC bilayers were slightly more thermostable (by 2–10°C) when compared to PCer-rich domains in DOPC-only bilayers. Nonlamellar phases were not present in bilayers in which the effects of POPE or DOPE on PCer segregation were the largest, as verified by ³¹P NMR. When palmitoyl sphingomyelin was added to the different bilayer compositions at 5 mol%, relative to the phospholipids, PCer segregated into gel domains at lower concentrations (2–3 mol% PCer), and the effect of POPE on PCer segregation was eliminated. We suggest that the effects of POPE, DOPE, and CL on PCer segregation was in part influenced by their effects on membrane curvature stress and in part because of unfavorable interactions with PCer due to their unsaturated acyl chains. These lipids are abundant in mitochondrial membranes and are likely to affect functional properties of saturated ceramides in them.     

On the importance of the phosphocholine methyl groups for sphingomyelin/cholesterol interactions in membranes: a study with ceramide phosphoethanolamine

In this study, we have examined how the headgroup size and properties affect the membrane properties of sphingomyelin and interactions with cholesterol. We prepared N-palmitoyl ceramide phosphoethanolamine (PCPE) and compared its membrane behavior with D-erythro-N-palmitoyl-sphingomyelin (PSM), both in monolayers and bilayers. The pure PCPE monolayer did not show a phase transition at 22 degrees C (in contrast to PSM), but displayed a much higher inverse isothermal compressibility as compared to the PSM monolayer, indicating stronger intermolecular interactions between PCPEs than between PSMs. At 37 degrees C the PCPE monolayer was more expanded (than at 22 degrees C) and displayed a rather poorly defined phase transition. When cholesterol was comixed into the monolayer, a condensing effect of cholesterol on the lateral packing of the lipids in the monolayer could be observed. The phase transition from an ordered to a disordered state in bilayer membranes was determined by diphenylhexatriene steady-state anisotropy. Whereas the PSM bilayer became disordered at 41 degrees C, the PCPE bilayer main transition occurred around 64 degrees C. The diphenylhexatriene steady-state anisotropy values were similar in both PCPE and PSM bilayers before and after the phase transition, suggesting that the order in the hydrophobic core in both bilayer types was rather similar. The emission from Laurdan was blue shifted in PCPE bilayers in the gel phase when compared to the emission spectra from PSM bilayers, and the blue-shifted component in PCPE bilayers was retained also after the phase transition, suggesting that Laurdan molecules sensed a more hydrophobic environment at the PCPE interface compared to the PSM interface both below and above the bilayer melting temperature. Whereas PSM was able to form sterol-enriched domains in dominantly fluid bilayers (as determined from cholestatrienol dequenching experiments), PCPE failed to form such domains, suggesting that the size and/or properties of the headgroup was important for stabilizing sphingolipid/sterol interaction. In conclusion, our study has highlighted how the headgroup in sphingomyelin affect its membrane properties and interactions with cholesterol.     

Cholesterol stimulates and ceramide inhibits Sticholysin II-induced pore formation in complex bilayer membranes

The pore forming capacity of Sticholysin II (StnII; isolated from Stichodactyla helianthus) in bilayer membranes containing 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), palmitoylsphingomyelin (PSM) and either cholesterol or palmitoyl ceramide (PCer) has been examined. The aim of the study was to elucidate how the presence of differently ordered PSM domains affected StnII oligomerization and pore formation. Cholesterol is known to enhance pore formation by StnII, and our results confirmed this and provide kinetic information for the process. The effect of cholesterol on bilayer permeabilization kinetics was concentration-dependent. In the concentration regime used (2.5–10 nmol cholesterol in POPC:PSM 80:20 by nmol), cholesterol also increased the acyl chain order in the fluid PSM domain and thus decreased bilayer fluidity, suggesting that fluidity per se was not responsible for cholesterol's effect. Addition of PCer (2.5–10 nmol) to the POPC:PSM (80:20 by nmol) bilayers attenuated StnII-induced pore formation, again in a concentration-dependent fashion. This addition also led to the formation of a PCer-rich gel phase. Addition of cholesterol to PCer-containing membranes could partially reduce the inhibitory effect of PCer on StnII pore formation. We conclude that the physical state of PSM (as influenced by either cholesterol or PCer) affected StnII binding and pore formation under the conditions examined.     

Characterization of the ternary mixture of sphingomyelin, POPC, and cholesterol: support for an inhomogeneous lipid distribution at high temperatures

A ternary lipid mixture of palmitoyl-oleoyl-phosphatidylcholine (POPC), palmitoyl-erythro-sphingosylphosphorylcholine (PSM), and cholesterol at a mixing ratio of 37.5:37.5:25 mol/mol/mol was characterized using fluorescence microscopy, ²H NMR, and electron paramagnetic resonance spectroscopy. The synthetic PSM provides an excellent molecule for studying the molecular properties of raft phases. It shows a narrow phase transition at a temperature of 311 K and is commercially available with a perdeuterated sn-2 chain. Fluorescence microscopy shows that large inhomogeneities in the mixed membranes are observed in the coexistence region of liquid-ordered and liquid-disordered lipid phases. Above 310 K, no optically detectable phase separation was shown. Upon decrease in temperature, a redistribution of the cholesterol into large liquid-ordered PSM/cholesterol domains and depletion of cholesterol from liquid-disordered POPC domains was observed by ²H NMR and electron paramagnetic resonance experiments. However, there is no complete segregation of the cholesterol into the liquid-ordered phase and also POPC-rich domains contain the sterol in the phase coexistence region. We further compared order parameters and packing properties of deuterated PSM or POPC in the raft mixture at 313 K, i.e., in the liquid crystalline phase state. PSM shows significantly larger ²H NMR order parameters in the raft phase than POPC. This can be explained by an inhomogeneous interaction of cholesterol between the lipid species and the mutual influence of the phospholipids on each other. These observations point toward an inhomogeneous distribution of the lipids also in the liquid crystalline phase at 313 K. From the prerequisite that order parameters are identical in a completely homogeneously mixed membrane, we can determine a minimal microdomain size of 45-70 nm in PSM/POPC/cholesterol mixtures above the main phase transition of all lipids.     

Melting studies of dangling-ended DNA hairpins: effects of end length,loop sequence and biotinylation of loop bases

The effects of 3′ single-strand dangling-ends of different lengths, sequence identity of hairpin loop, and hairpin loop biotinylation at different loop residues on DNA hairpin thermodynamic stability were investigated. Hairpins contained 16 bp stem regions and five base loops formed from the sequence, 5′-TAGTCGACGTGGTCC-N₅-GGACCACGTCGACTAG-E_n-3′. The length of the 3′ dangling-ends (E_n) was n = 13 or 22 bases. The identities of loop bases at positions 2 and 4 were varied. Biotinylation was varied at loop base positions 2, 3 or 4. Melting buffers contained 25 or 115 mM Na⁺. Average t_m values for all molecules were 73.5 and 84.0°C in 25 and 115 mM Na⁺, respectively. Average two-state parameters evaluated from van’t Hoff analysis of the melting curve shapes in 25 mM Na⁺ were ΔH_vH = 84.8 ± 15.5 kcal/mol, ΔS_vH = 244.8 ± 45.0 cal/K·mol and ΔG_vH = 11.9 ± 2.1 kcal/mol. In 115 mM Na⁺, two-state parameters were not very different at ΔH_vH = 80.42 ± 12.74 kcal/mol, ΔS_vH = 225.24 ± 35.88 cal/K·mol and ΔG_vH = 13.3 ± 2.0 kcal/mol. Differential scanning calorimetry (DSC) was performed to test the validity of the two-state assumption and evaluated van’t Hoff parameters. Thermodynamic parameters from DSC measurements (within experimental error) agreed with van’t Hoff parameters, consistent with a two-state process. Overall, dangling-end DNA hairpin stabilities are not affected by dangling-end length, loop biotinylation or sequence and vary uniformly with [Na⁺]. Consider able freedom is afforded when designing DNA hairpins as probes in nucleic acid based detection assays, such as microarrays.     

Cholesterol favors phase separation of sphingomyelin

The phase behavior of mixed lipid dispersions representing the inner leaflet of the cell membrane has been characterized by X-ray diffraction. Aqueous dispersions of phosphatidylethanolamine:phosphatidylserine (4:1 mole/mole) have a heterogeneous structure comprising an inverted hexagonal phase H(II) and a lamellar phase. Both phases coexist in the temperature range 20-45 degrees C. The fluid-to-gel mid-transition temperature of the lamellar phase assigned to phosphatidylserine is decreased from 27 to 24 degrees C in the presence of calcium. Addition of sphingomyelin to phosphatidylethanolamine/phosphatidylserine prevents phase separation of the hexagonal H(II) phase of phosphatidylethanolamine but the ternary mixture phase separates into two lamellar phases of periodcity 6.2 and 5.6 nm, respectively. The 6.2-nm periodicity is assigned to the gel phase enriched in sphingomyelin of molecular species comprising predominantly long saturated hydrocarbon chains because it undergoes a gel-to-fluid phase transition above 40 degrees C. The coexisting fluid phase we assign to phosphatidylethanolamine and phosphatidylserine and low melting point molecular species of sphingomyelin which suppresses the tendency of phosphatidylethanolamine to phase-separate into hexagonal H(II) structure. There is evidence for considerable hysteresis in the separation of lamellar fluid and gel phases during cooling. The addition of cholesterol prevents phase separation of the gel phase of high melting point sphingomyelin in mixtures with phosphatidylserine and phosphatidylethanolamine. In the quaternary mixture the lamellar fluid phase, however, is phase separated into two lamellar phases of periodicities of 6.3 and 5.6 nm (20 degrees C), respectively. The lamellar phase of periodicity 5.6 nm is assigned to a phase enriched in aminoglycerophospholipids and the periodicity 6.3 nm to a liquid-ordered phase formed from cholesterol and high melting point molecular species of sphingomyelin characterized previously by ESR. Substituting 7-dehydrocholesterol for cholesterol did not result in evidence for lamellar phase separation in the mixture within the temperature range 20-40 degrees C. The specificity of cholesterol in creation of liquid-ordered lamellar phase is inferred.     

Effects of Sphingomyelin Headgroup Size on Interactions with Ceramide

Sphingomyelins (SMs) and ceramides are known to interact favorably in bilayer membranes. Because ceramide lacks a headgroup that could shield its hydrophobic body from unfavorable interactions with water, accommodation of ceramide under the larger phosphocholine headgroup of SM could contribute to their favorable interactions. To elucidate the role of SM headgroup for SM/ceramide interactions, we explored the effects of reducing the size of the phosphocholine headgroup (removing one, two, or three methyls on the choline moiety, or the choline moiety itself). Using differential scanning calorimetry and fluorescence spectroscopy, we found that the size of the SM headgroup had no marked effect on the thermal stability of ordered domains formed by SM analog/palmitoyl ceramide (PCer) interactions. In more complex bilayers composed of a fluid glycerophospholipid, SM analog, and PCer, the thermal stability and molecular order of the laterally segregated gel domains were roughly identical despite variation in SM headgroup size. We suggest that that the association between PCer and SM analogs was stabilized by ceramide’s aversion for disordered phospholipids, by interfacial hydrogen bonding between PCer and the SM analogs, and by attractive van der Waals’ forces between saturated chains of PCer and SM analogs.     

Palmitoyl ceramide promotes milk sphingomyelin gel phase domains formation and affects the mechanical properties of the fluid phase in milk-SM/DOPC supported membranes

Ceramides are minor structural components of membranes involved in biological functions. In the milk fat globule membrane (MFGM), ceramides are susceptible to affect the lateral packing of polar lipids, especially the milk sphingomyelin (MSM). To investigate this, palmitoylceramide (PCer) was added to MSM/DOPC (dioleoylphosphatidylcholine) in order to form hydrated lipid bilayers. Differential scanning calorimetry evidenced interactions of PCer with the MSM in the solid-ordered phase to form MSM/PCer structures with a higher thermostability than MSM. Atomic force microscopy revealed that PCer modified lipid packing in both the liquid-disordered DOPC phase where it increased thickness and mechanical stability, and the solid-ordered MSM phase where it recruited MSM molecules yet initially in the liquid phase at 26 °C and then increased the area of the MSM/PCer domains. The effect of PCer on the mechanical properties of the MSM-rich domains remains to be elucidated. These results bring new insights on the role of ceramides in the control of biophysical and biological properties of the MFGM. They also open perspectives for the design of emulsions and liposomes, using milk polar lipids as food-grade ingredients.     

Ergosterol in POPC membranes: physical properties and comparison with structurally similar sterols

The physical properties of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)/ergosterol bilayers in the liquid-crystalline phase were determined using deuterium nuclear magnetic resonance (²H NMR) and vesicle extrusion. For the ²H NMR experiments, the sn-1 chain of POPC was perdeuterated, and spectra were taken as a function of ergosterol concentration and temperature. Analysis of the liquid-crystalline spectra provides clear evidence that two types of liquid-crystalline domains, neither of which is a liquid-ordered phase, having distinct average chain conformations coexist in 80:20 and 75:25 POPC/ergosterol membranes over a wide temperature range (from −2 to at least 31°C). Adding ergosterol to a concentration of 25 mol % increases POPC-d₃₁ chain ordering as measured by the NMR spectral first moment M₁ and also increases the membrane lysis tension, obtained from vesicle extrusion. Further addition of ergosterol had no effect on either chain order or lysis tension. This behavior is in marked contrast to the effect of cholesterol on POPC membranes: POPC/cholesterol membranes have a linear dependence of chain order on sterol concentration to at least 40 mol %. To investigate further we compared the dependence on sterol structure and concentration of the NMR spectra and lysis tension for several POPC/sterol membranes at 25°C. For all POPC/sterol membranes investigated in this study, we observed a universal linear relation between lysis tension and M₁. This suggests that changes in acyl chain ordering directly affect the tensile properties of the membrane.     

Mobility of Core Water in Bacillus subtilis Spores by 2H NMR

Bacterial spores in a metabolically dormant state can survive long periods without nutrients under extreme environmental conditions. The molecular basis of spore dormancy is not well understood, but the distribution and physical state of water within the spore is thought to play an important role. Two scenarios have been proposed for the spore’s core region, containing the DNA and most enzymes. In the gel scenario, the core is a structured macromolecular framework permeated by mobile water. In the glass scenario, the entire core, including the water, is an amorphous solid and the quenched molecular diffusion accounts for the spore’s dormancy and thermal stability. Here, we use ²H magnetic relaxation dispersion to selectively monitor water mobility in the core of Bacillus subtilis spores in the presence and absence of core Mn²⁺ ions. We also report and analyze the solid-state ²H NMR spectrum from these spores. Our NMR data clearly support the gel scenario with highly mobile core water (∼25 ps average rotational correlation time). Furthermore, we find that the large depot of manganese in the core is nearly anhydrous, with merely 1.7% on average of the maximum sixfold water coordination.     

Thermodynamic characterization of the multivalent binding of chartreusin to DNA

Characterization of the thermodynamics of DNA– drug interactions is a very useful part in rational drug design. Isothermal titration calorimetry (ITC), differential scanning calorimetry (DSC) and UV melting experiments have been used to analyze the multivalent (intercalation plus minor groove) binding of the antitumor antibiotic chartreusin to DNA. Using DNA UV melting studies in the presence of the ligand and the binding enthalpy determined by ITC, we determined that the binding constant for the interaction was 3.6 × 10⁵ M^–1 at 20°C, in a solution containing 18 mM Na⁺. The DNA–drug interaction was enthalpy driven, with a ΔH_b of –7.07 kcal/mol at 20°C. Binding enthalpies were determined by ITC in the 20–35°C range and used to calculate a binding-induced change in heat capacity (ΔCp) of –391 cal/mol K. We have obtained a detailed thermodynamic profile for the interaction of this multivalent drug, which makes possible a dissection of ΔG_obs into the component free energy terms. The hydrophobic transfer of the chartreusin chromophore from the solution to the DNA intercalating site is the main contributor to the free energy of binding.     

Partial purification and characterization of platelet factors stimulating the multiplication of normal human glial cells

Multiplication-stimulating activity for human glial cells was purified from human outdated platelets. By ion exchange chromatography anionic activity was separated from cationic activity. The former could be further separated by Sephadex G-200 gel chromatography into two peaks, whose molecular weights were 40 000 and < 10 000. The cationic activity was partially purified by concanavalin A (ConA) Sepharose chromatography, hydroxylapatite chromatography and SDS-polyacrylamide gel electrophoresis. The cationic activity was heterogeneous as demonstrated by isoelectric focusing (Ip 9.5–10.4), gel filtration on Bio-Gel P-150 and SDS-polyacrylamide gel electrophoresis (mol. wt 26 000–33 000). Less than 50 ng/ml was required of the factor to give a glial cell stimulation corresponding to that afforded by 1 % of human serum. A thymidine-degrading enzyme, present in human platelets and to a low degree also in human serum, was found to interfere with the assay for multiplication-stimulating activity. The enzyme (probably a thymidine phosphorylase) converted [³H]thymidine to [³H]thymine, causing a reduced incorporation of ³H into cellular DNA. This difficulty was circumvented by use of an autoradiographic estimation (per cent labelled nuclei) of the multiplication-stimulating activity.     

Monounsaturated PE does not phase-separate from the lipid raft molecules sphingomyelin and cholesterol: role for polyunsaturation?

We investigated interactions of the lipid raft molecules sphingomyelin (SM) and cholesterol (CHOL) in monolayers and bilayers composed of 1-palmitoyl-2-oleoyl-sn-glycerophosphatidylethanolamine (POPE) or 1-palmitoyl-2-docosahexaenoyl-sn-glycerophosphatidylethanolamine (PDPE) at 35 degrees C. Techniques employed were pressure-area (pi-A) isotherms generated from Langmuir-Blodgett films, solid-state (2)H and (31)P NMR spectroscopies, and differential scanning calorimetry (DSC). Condensation calculated from pi-A isotherms and reduction in the enthalpy of the gel-liquid-crystalline transition in DSC scans showed CHOL has a strong affinity for POPE, comparable to that observed between SM-CHOL. Order parameters derived from (2)H NMR spectra of the perdeuterated sn-1 chain of POPE-d(31) increased by >50% upon addition of equimolar CHOL to POPE-d(31)/SM (1:1 mol) bilayers. Close proximity of CHOL to POPE even in the presence of SM is indicated. Chemical shift anisotropy (Deltasigma(csa)) measured from (1)H-decoupled (31)P NMR spectra also implied intimate lipid mixing in POPE/SM/CHOL (1:1:1 mol). In contrast, pi-A isotherms and corroborating DSC studies of PDPE/SM (1:1 mol) indicate phase separation between SM and PDPE, which was maintained in the presence of CHOL. The cholesterol-associated increase in order of the perdeuterated sn-1 chain of PDPE determined by (2)H NMR was 2-fold less for PDPE-d(31)/SM/CHOL (1:1:1 mol) than POPE-d(31)/SM/CHOL (1:1:1 mol). Our findings support the notion that acyl chain dependent lateral phase separation occurs in the presence of a docosahexaenoic acid (DHA)-containing phospholipid (PDPE), but not an oleic acid-containing phospholipid (POPE). We propose that monounsaturated lipids do not promote formation of stable lipid rafts and that polyunsaturation may be important for raft stability.     

Using P MAS NMR to monitor a gel phase thermal disorder transition in sphingomyelin/cholesterol bilayers

The impact of low cholesterol concentrations on an egg sphingomyelin bilayer is investigated using ³¹P magic angle spinning (MAS) NMR spectroscopy. The magnitude of the isotropic ³¹P MAS NMR line width is used to monitor the main gel to liquid crystalline phase transition, along with a unique gel phase pretransition. In addition, the ³¹P chemical shift anisotropy (CSA) and spin-spin relaxation times (T₂), along with the effects of spinning speed, proton decoupling and magnetic field strength, are reported. The variation of this unique gel phase thermal pretransition with the inclusion of 5 through 21 mol% cholesterol is presented and discussed.     

Protein composition and structure of human placental microvillous membrane. External surface, sialic acid containing, extrinsic and intrinsic components.

The microvillous membrane of human placenta is in direct contact with maternal blood and thus plays a vital role in many essential functions of the placenta. As an initial step in understanding the membrane proteins, and their relationship to these functions and to the structure of the membrane, we have investigated an isolated membrane preparation. Ten major peptide bands and an approximately equal number of minor bands were seen with sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Sialoglycoproteins were labeled with periodate (PA-³H) borohydride and external surface components with lactoperoxidase-[¹²⁵I] (LP-¹²⁵I). One principal (69 000 mol. wt) and several minor (100 000, 45 000, and 38–40 000 mol. wt) bands were labeled as Sialoglycoproteins and found to be exposed on the surface of the membrane. Approx. 50% of the membrane protein and all of the sialic acid was tightly bound to membrane lipid and resistant to extraction with dimethyl maleic anhydride (DMMA). Electron microscopy demonstrated extraction by DMMA of microfilaments presumptively identified as actin and other electron dense components from the villous core. The extracted supernate and the residual pellet differed markedly in protein composition. The supernatant contained bands of 180 000, 115 000, 85 000, 70–72 000, 45 000, and 38–40 000 mol. wt whereas the lipid pellet contained components of 200 000, 150 000, 100 000, 69 000, and 64 000 mol. wt. The lipid matrix with which these proteins were associated contained phosphatidyl choline and sphingomyelin and was similar in composition to other plasma membranes. Thus by using a variety of experimental approaches the proteins of the human placental microvillous membrane can be divided into groups based on their sialic acid content, exposure on the external surface, tightness of binding to the membrane lipid, and relation to membrane structure.     

Characterization of the Lipid-Binding Site of Equinatoxin II by NMR and Molecular Dynamics Simulation

Equinatoxin II (EqtII) is a soluble, 20 kDa pore-forming protein toxin isolated from the sea anemone Actinia equina. Although pore formation has long been known to occur in distinct stages, including monomeric attachment to phospholipid membranes followed by detachment of the N-terminal helical domain and oligomerization into the final pore assembly, atomistic-level detail of the protein-lipid interactions underlying these events remains elusive. Using high-resolution solution state NMR of uniformly-¹⁵N-labeled EqtII at the critical micelle concentration of dodecylphosphocholine, we have mapped the lipid-binding site through chemical shift perturbations. Subsequent docking of an EqtII monomer onto a dodecylphosphocholine micelle, followed by 400 ns of all-atom molecular dynamics simulation, saw several high-occupancy lipid-binding pockets stabilized by cation-π, hydrogen bonding, and hydrophobic interactions; and stabilization of the loop housing the conserved arginine-glycine-aspartate motif. Additional simulation of EqtII with an N-acetyl sphingomyelin micelle, for which high-resolution NMR data cannot be obtained due to aggregate formation, revealed that sphingomyelin specificity might occur via hydrogen bonding to the 3-OH and 2-NH groups unique to the ceramide backbone by side chains of D109 and Y113; and main chains of P81 and W112. Furthermore, a binding pocket formed by K30, K77, and P81, proximate to the hinge region of the N-terminal helix, was identified and may be implicated in triggering pore formation.