Hexagonal Substructure and Hydrogen Bonding in Liquid-Ordered Phases Containing Palmitoyl Sphingomyelin

All-atom simulation data are presented for ternary mixtures of palmitoyl sphingomyelin (PSM), cholesterol, and either palmitoyl oleoyl phosphatidyl choline or dioleoyl phosphatidyl choline (DOPC). For comparison, data for a mixture of dipalmitoyl phosphatidyl choline (DPPC), cholesterol, and DOPC are also presented. Compositions corresponding to the liquid-ordered phase, the liquid-disordered phase, and coexistence of the two phases are simulated for each mixture. Within the liquid-ordered phase, cholesterol is preferentially solvated by DOPC if it is available, but if DOPC is replaced by POPC, cholesterol is preferentially solvated by PSM. In the DPPC mixtures, cholesterol interacts preferentially with the saturated chains via its smooth face, whereas in the PSM mixtures, cholesterol interacts preferentially with PSM via its rough face. Interactions between cholesterol and PSM have a very particular character: hydrogen bonding between cholesterol and the amide of PSM rotates the tilt of the amide plane, which primes it for more robust hydrogen bonding with other PSM. Cholesterol-PSM hydrogen bonding also locally modifies the hexagonal packing of hydrocarbon chains in the liquid-ordered phase of PSM mixtures.     

Phase diagrams and lipid domains in multicomponent lipid bilayer mixtures

Understanding the phase behavior of biological membranes is helped by the study of more simple systems. Model membranes that have as few as 3 components exhibit complex phase behavior that can be well described, providing insight for biological membranes. A number of different studies are in agreement on general findings for some compositional phase diagrams, in particular, those that model the outer leaflet of animal cell plasma membranes. These model mixtures include cholesterol, together with one high-melting lipid and one low-melting lipid. An interesting finding is of two categories of such 3-component mixtures, leading to what we term Type I and Type II compositional phase diagrams. The latter have phase regions of macroscopic coexisting domains of {Lα + Lβ + Lo} and of {Lα + Lo}, with domains resolved under the light microscope. Type I mixtures have the same phase coexistence regions, but the domains seem to be nanoscopic. Type I mixtures are likely to be better models for biological membranes.     

Interaction of cholesterol with sphingomyelin in mixed membranes containing phosphatidylcholine, studied by spin-label ESR and IR spectroscopies. A possible stabilization of gel-phase sphingolipid domains by cholesterol

The ESR spectra from different positional isomers of sphingomyelin and phosphatidylcholine spin-labeled in their acyl chain have been studied in sphingomyelin(cerebroside)-phosphatidylcholine mixed membranes that contain cholesterol. The aim was to investigate mechanisms by which cholesterol could stabilize possible domain formation in sphingolipid-glycerolipid membranes. The outer hyperfine splittings in the ESR spectra of sphingomyelin and phosphatidylcholine spin-labeled on the 5 C atom of the acyl chain were consistent with mixing of the components, but the perturbations on adding cholesterol were greater in the membranes containing sphingomyelin than in those containing phosphatidylcholine. Infrared spectra of the amide I band of egg sphingomyelin were shifted and broadened in the presence of cholesterol to a greater extent than the carbonyl band of phosphatidylcholine, which was affected very little by cholesterol. Two-component ESR spectra were observed from lipids spin-labeled on the 14 C atom of the acyl chain in cholesterol-containing membranes composed of sphingolipids, with or without glycerolipids (sphingomyelin/cerebroside and sphingomyelin/cerebroside/phosphatidylcholine mixtures). These results indicate the existence of gel-phase domains in otherwise liquid-ordered membranes that contain cholesterol. In the gel phase of egg sphingomyelin, the outer hyperfine splittings of sphingomyelin spin-labeled on the 14-C atom of the acyl chain are smaller than those for the corresponding spin-labeled phosphatidylcholine. In the presence of cholesterol, this situation is reversed; the outer splitting of 14-C spin-labeled sphingomyelin is then greater than that of 14-C spin-labeled phosphatidylcholine. This result provides some support for the suggestion that transbilayer interdigitation induced by cholesterol stabilizes the coexistence of gel-phase and "liquid-ordered" domains in membranes containing sphingolipids.     

Sphingolipid symmetry governs membrane lipid raft structure

Lipid domain formation in membranes underlies the concept of rafts but their structure is controversial because the key role of cholesterol has been challenged. The configuration of glycosphingolipid receptors for agonists, bacterial toxins and enveloped viruses in plasma membrane rafts appears to be an important factor governing ligand binding and infectivity but the details are as yet unresolved. I have used X-ray diffraction methods to examine how cholesterol affects the distribution of glycosphingolipid in aqueous dispersions of an equimolar mixture of cholesterol and egg-sphingomyelin containing different proportions of glucosylceramide from human extracts. Three coexisting liquid-ordered bilayer structures are observed at 37 °C in mixtures containing up to 20 mol% glycosphingolipid. All the cholesterol was sequestered in one bilayer with the minimum amount of sphingomyelin (33 mol%) to prevent formation of cholesterol crystals. The other two bilayers consisted of sphingomyelin and glucosylceramide. Asymmetric molecular species of glucosylceramide with N-acyl chains longer than 20 carbons form an equimolar complex with sphingomyelin in which the glycosidic residues are arranged in hexagonal array. Symmetric molecular species mix with sphingomyelin in proportions less than equimolar to form quasicrystalline bilayers. When the glycosphingolipid exceeds equimolar proportions with sphingomyelin cholesterol is incorporated into the structure and formation of a gel phase of glucosylceramide is prevented. The demonstration of particular structural features of ceramide molecular species combined with the diversity of sugar residues of glycosphingolipid classes paves the way for a rational approach to understanding the functional specificity of lipid rafts and how they are coupled across cell membranes.     

Phase studies of model biomembranes: Macroscopic coexistence of Lα + Lβ, with light-induced coexistence of Lα + Lo Phases

Phase diagrams of 3-component lipid bilayer mixtures containing cholesterol reveal major differences among the different types of lipids. Here we report that mixtures of cholesterol together with POPC and a high-melting temperature PC or sphingomyelin show different phase behavior from similar mixtures that contain DOPC or di-phytanoyl-PC instead of POPC. In particular, only one region of macroscopic phase coexistence occurs with POPC, a region of coexisting liquid disordered and solid phases, {Lα + Lβ}. Fluorescence microscopy imaging is useful for these studies, but is subject to artifactual light-induced domain formation, as reported by Ayuyan and Cohen [A.G. Ayuyan, F.S. Cohen, Lipid peroxides promote large rafts: Effects of excitation of probes in fluorescence microscopy and electrochemical reactions during vesicle formation, Biophys. J. 91 (2006) 2172-2183.]. This artifact can be attenuated by decreased illumination and low dye concentration. The use of the free radical scavenger n-propyl gallate can reduce the artifact, but this molecule enters the bilayer and itself perturbs the phase behavior. We suggest that the light-induced domain separation artifact might actually arise from pre-existing lipid clusters that are induced to coalesce, and therefore indicates highly nonrandom mixing of the lipid components.     

Asymmetric structural features in single supported lipid bilayers containing cholesterol and GM1 resolved with synchrotron X-Ray reflectivity

The cell membrane comprises numerous protein and lipid molecules capable of asymmetric organization between leaflets and liquid-liquid phase separation. We use single supported lipid bilayers (SLBs) to model cell membranes, and study how cholesterol and asymmetrically oriented ganglioside receptor G_M1 affect membrane structure using synchrotron x-ray reflectivity. Using mixtures of cholesterol, sphingomyelin, and 1,2-dioleoyl-sn-glycero-3-phosphocholine, we characterize the structure of liquid-ordered and liquid-disordered SLBs in terms of acyl-chain density, headgroup size, and leaflet thickness. SLBs modeling the liquid-ordered phase are 10 Å thicker and have a higher acyl-chain electron density (〈ρ_chain〉 = 0.33 e⁻/ų) compared to SLBs modeling the liquid-disordered phase, or pure phosphatidylcholine SLBs (〈ρ_chain〉 = 0.28 e⁻/ų). Incorporating G_M1 into the distal bilayer leaflet results in membrane asymmetry and thickening of the leaflet of 4-9 Å. The structural effect of G_M1 is more complex in SLBs of cholesterol/sphingomyelin/1,2-dioleoyl-sn-glycero-3-phosphocholine, where the distal chains show a high electron density (〈ρ_chain〉 = 0.33 e⁻/ų) and the lipid diffusion constant is reduced by ∼50%, as measured by fluorescence microscopy. These results give quantitative information about the leaflet asymmetry and electron density changes induced by receptor molecules that penetrate a single lipid bilayer.     

Hydration and lateral organization in phospholipid bilayers containing sphingomyelin: a 2H-NMR study

Interfacial properties of lipid bilayers were studied by (2)H nuclear magnetic resonance spectroscopy, with emphasis on a comparison between phosphatidylcholine and sphingomyelin. Spectral resolution and sensitivity was improved by macroscopic membrane alignment. The motionally averaged quadrupolar interaction of interlamellar deuterium oxide was employed to probe the interfacial polarity of the membranes. The D(2)O quadrupolar splittings indicated that the sphingomyelin lipid-water interface is less polar above the phase transition temperature T(m) than below T(m). The opposite behavior was found in phosphatidylcholine bilayers. Macroscopically aligned sphingomyelin bilayers also furnished (2)H-signals from the amide residue and from the hydroxyl group of the sphingosine moiety. The rate of water-hydroxyl deuteron exchange could be measured, whereas the exchange of the amide deuteron was too slow for the inversion-transfer technique employed, suggesting that the amide residue is involved in intermolecular hydrogen bonding. Order parameter profiles in mixtures of sphingomyelin and chain-perdeuterated phosphatidylcholine revealed an ordering effect as a result of the highly saturated chains of the sphingolipids. The temperature dependence of the (2)H quadrupolar splittings was indicative of lateral phase separation in the mixed systems. The results are discussed with regard to interfacial structure and lateral organization in sphingomyelin-containing biomembranes.     

Self-assembly of two fluorescent supramolecular frameworks constructed from unsymmetrical benzene tricarboxylate and bipyridine

Two new supramolecular compounds, [Ag(4,4′-bipy)]_n [Ag(HBTC)]_n (1) and [Cu(H₂BTC)(2,2′-bipy)] (2) (HBTC/H₂BTC = 1,2,4-benzenetricarboxylate, 4,4′-bipy/2,2′-bipy = 4,4′/2,2′-bipyridine), have been synthesized and characterized by elemental analyses, IR spectra, ultraviolet-visible diffuse reflection integral spectra (UV-Vis DRIS), fluorescent spectra, thermogravimetric analysis and single crystal X-ray diffraction analysis. It is noteworthy that there were two kinds of one-dimensional stairs-chain including cationic [Ag(4,4′-bipy)]_n chain and anionic [Ag(HBTC)]_n chain in 1. Furthermore, a two-dimensional double layer supramolecular framework was constructed through coordination bonds, hydrogen bonds, π-π stacking interactions and Ag?O weak coordinative interactions. The one-dimensional supramolecular chain of 2 was built from combining mononuclear [Cu(H₂BTC)(2,2′-bipy)] by inter- and intra-molecular hydrogen bonding interactions. Additionally, the two complexes exhibit intense blue or olivine luminescence at room temperature.     

Conformational changes of chicken liver bile acid-binding protein bound to anionic lipid membrane are coupled to the lipid phase transitions

Chicken liver bile acid-binding protein (L-BABP) binds to anionic lipid membranes by electrostatic interactions and acquires a partly folded state [Nolan, V., Perduca, M., Monaco, H., Maggio, B. and Montich, G. G. (2003) Biochim. Biophys. Acta 1611, 98-106]. We studied the infrared amide I′ band of L-BABP bound to dipalmitoylphosphatidylglycerol (DPPG), dimyristoylphosphatidylglycerol (DMPG) and palmitoyloleoylphosphatidylglycerol (POPG) in the range of 7 to 60 °C. Besides, the thermotrophic behaviour of DPPG and DMPG was studied in the absence and in the presence of bound-protein by differential scanning calorimetry (DSC) and infrared spectra of the stretching vibration of methylene and carbonyl groups. When L-BABP was bound to lipid membranes in the liquid-crystalline state (POPG between 7 and 30 °C) acquired a more unfolded conformation that in membranes in the gel state (DPPG between 7 and 30 °C). Nevertheless, this conformational change of the protein in DMPG did not occur at the temperature of the lipid gel to liquid-crystalline phase transition detected by infrared spectroscopy. Instead, the degree of unfolding in the protein was coincident with a phase transition in DMPG that occurs with heat absorption and without change in the lipid order.     

Self-assembly of three novel mixed-ligand coordination polymers: Formation of 2-D grids, 1-D chains and ladders and crystal band structure

Hydrothermal reaction of the carboxylate-based ligands with metal salts (or oxide) and 4,4′-bipyridine as a second linker, afforded three new coordination polymers, namely, [Co(PCPA)₂(4,4′-bpy)]_n (1) with 2-D rectangle grids, Cu(PCPA)₂(4,4′-bpy)]_n (2) with a linear chain, [Ag(PCPA)(PCPAH)(4,4′-bpy) · H₂O]_n (3) with 1-D molecular ladder (4,4′-bpy = 4,4′-bipyridine; PCPA = p-chlorophenoxyacetate; PCPAH = p-chlorophenoxyacetic acid). It is noticeable that compound 3 is also a supramolecular framework built by coordination bonds, weak interactions between Ag ions, π-π stacking interactions and hydrogen-bonded interactions. The three compounds with different structure motifs have been characterized by elemental analyses, IR spectra, ultraviolet-visible diffuse reflection integral spectra, fluorescent spectra and single crystal X-ray diffraction analysis. Furthermore, the bonding properties of compound 3 were investigated in terms of the absorption spectrum, as well as the calculated band structures and density of states.     

Differential lipid packing abilities and dynamics in giant unilamellar vesicles composed of short-chain saturated glycerol-phospholipids, sphingomyelin and cholesterol

The ability of membrane components to arrange themselves heterogeneously within the bilayer induces the formation of microdomains. Much work has been devoted to mimicking domain-assembly in artificial bilayers and characterizing their physico-chemical properties. Ternary lipid mixtures composed of unsaturated phospholipids, sphingomyelin and cholesterol give rise to large, round domains. Here, we replaced the unsaturated phospholipid in the ternary mixture with sphingomyelin and cholesterol by saturated glycero-phospholipids of different chain length and characterized the critical role of cholesterol in sorting these lipids by confocal imaging and fluorescence correlation spectroscopy (FCS). More cholesterol is needed to obtain phase segregation in ternary mixtures, in which the unsaturated phospholipid is replaced by a saturated one. Finally, lipid dynamics in distinct phases is very low and astonishingly similar, thereby suggesting the poor ability of cholesterol in sorting short-chain saturated glycero-phospholipids and sphingomyelin.     

Comparison of the liquid-ordered bilayer phases containing cholesterol or 7-dehydrocholesterol in modeling Smith-Lemli-Opitz syndrome

The phase behavior of egg sphingomyelin (ESM) mixtures with cholesterol or 7-dehydrocholesterol (7-DHC) has been investigated by independent methods: fluorescence microscopy, X-ray diffraction, and electron spin resonance spectroscopy. In giant vesicles, cholesterol-enriched domains appeared as large and clearly delineated domains assigned to a liquid-ordered (L_o) phase. The domains containing 7-DHC were smaller and had more diffuse boundaries. Separation of a gel phase assigned by X-ray examination to pure sphingomyelin domains coexisting with sterol-enriched domains was observed at temperatures less than 38°C in binary mixtures containing 10-mol% sterol. At higher sterol concentrations, the coexistence of liquid-ordered and liquid-disordered phases was evidenced in the temperature range 20°–50°C. Calculated electron density profiles indicated the location of 7-DHC was more loosely defined than cholesterol, which is localized precisely at a particular depth along the bilayer normal. ESR spectra of spin-labeled fatty acid partitioned in the liquid-ordered component showed a similar, high degree of order for both sterols in the center of the bilayer, but it was higher in the coexisting disordered phase for 7-DHC. The differences detected in the models of the lipid membrane matrix are said to initiate the deleterious consequences of the Smith-Lemli-Opitz syndrome.     

Probing lipid mobility of raft-exhibiting model membranes by fluorescence correlation spectroscopy

Confocal fluorescence microscopy and fluorescence correlation spectroscopy (FCS) have been employed to investigate the lipid spatial and dynamic organization in giant unilamellar vesicles (GUVs) prepared from ternary mixtures of dioleoyl-phosphatidylcholine/sphingomyelin/cholesterol. For a certain range of cholesterol concentration, formation of domains with raft-like properties was observed. Strikingly, the lipophilic probe 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI-C18) was excluded from sphingomyelin-enriched regions, where the raft marker ganglioside GM1 was localized. Cholesterol was shown to promote lipid segregation in dioleoyl-phosphatidylcholine-enriched, liquid-disordered, and sphingomyelin-enriched, liquid-ordered phases. Most importantly, the lipid mobility in sphingomyelin-enriched regions significantly increased by increasing the cholesterol concentration. These results pinpoint the key role, played by cholesterol in tuning lipid dynamics in membranes. At cholesterol concentrations >50 mol%, domains vanished and the lipid diffusion slowed down upon further addition of cholesterol. By taking the molecular diffusion coefficients as a fingerprint of membrane phase compositions, FCS is proven to evaluate domain lipid compositions. Moreover, FCS data from ternary and binary mixtures have been used to build a ternary phase diagram, which shows areas of phase coexistence, transition points, and, importantly, how lipid dynamics varies between and within phase regions.     

Distinguishing individual lipid headgroup mobility and phase transitions in raft-forming lipid mixtures with 31P MAS NMR

A model membrane system composed of egg sphingomyelin (SM), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and cholesterol was studied with static and magic angle spinning (31)P NMR spectroscopy. This model membrane system is of significant biological relevance since it is known to form lipid rafts. (31)P NMR under magic angle spinning conditions resolves the SM and DOPC headgroup resonances allowing for extraction of the (31)P NMR parameters for the individual lipid components. The isotropic chemical shift, chemical shift anisotropy, and asymmetry parameter can be extracted from the spinning side band manifold of the individual components that form liquid-ordered and liquid-disordered domains. The magnitude of the (31)P chemical shift anisotropy and the line width is used to determine headgroup mobility and monitor the gel-to-gel and gel-to-liquid crystalline phase transitions of SM as a function of temperature in these mixtures. Spin-spin relaxation measurements are in agreement with the line width results, reflecting mobility differences and some heterogeneities. It will be shown that the presence of DOPC and/or cholesterol greatly impacts the headgroup mobility of SM both above and below the liquid crystalline phase transition temperature, whereas DOPC displays only minor variations in these lipid mixtures.     

Raw and boiled garlic enhances plasma antioxidant activity and improves plasma lipid metabolism in cholesterol-fed rats

In the present study the effect of garlic, in a form more similar to how most people eat garlic, on lipid and antioxidant metabolism in rats was investigated. The antioxidant activity was determined by the efficacy to scavenge 2, 2′-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) derived radicals in garlic samples. The highest results were estimated in aqueous fraction in comparison with other extracts divided on the basis of polarity. Wistar male rats were randomly divided into 10 diet groups, each with seven animals. The groups were named: Control, RG (raw garlic), BG (boiled garlic for 20 min), AERG (aqueous extract of raw garlic), AEBG (aqueous extract of boiled garlic), Ch (Cholesterol), Ch/RG, Ch/BG, Ch/AERG and Ch/AEBG. All experimental diets were supplemented with 25 mg of lyophilized garlic/kg body weight obtained from raw, boiled and their aqueous extracts over a period of 30 days.Serum lipid (total cholesterol, LDL-cholesterol and triglycerides) concentrations were higher in all groups fed cholesterol (Ch); however, the increase was significant only in Ch group, without garlic supplementation. In groups of rats fed diets with cholesterol, garlic samples significantly hindered the rise of TC and LDL-C (P < 0.05). A significant increase (P < 0.05) in the plasma antioxidant activity was registered in experimental groups of rats fed cholesterol-free diets supplemented with garlic; oppositely, a significant decrease was only in group of rats given food containing cholesterol without garlic.The protein spectra has shown that during short boiling some proteins change their functional properties such as solubility and mobility, resulting in a number of protein bands in SDS-electrophoresis.

Conclusions

Raw and boiled garlic improved plasma lipid metabolism and plasma antioxidant activity in an experiment on rats. Thus, dietary hypolipidemic garlic was effective in reducing the oxidant stress, which was indicated by an increase of antioxidant activity and a decrease of lipids in the rats' blood. It was found that garlic boiled for 20 min has the same bioactivity as raw garlic in its antioxidant and protein spectra. Therefore it should be added at this time to foods. The selenium and copper content of raw garlic is not altered by boiling. The protein electrophoretic pattern of raw garlic is altered by boiling.     

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.     

Molecular double chains and 3-D supramolecular frameworks with open channels assembled from 1D copper(II) maleate coordination polymers with chelating aromatic amine ligands by noncovalent interactions

Two 1D coordination polymers, [Cu(mal)(Hdpa)]_n (1) and {[Cu(mal)(tpy)] · 4H₂O}_n (2) (mal = maleate, Hdpa = 2, 2′-dipyridylamine and tpy = 2,2′:6′,2″-terpyridine), have been synthesized and their crystal structures, IR spectra, thermal analyses, magnetic properties were determined. In 1 and 2, the maleate ligands link adjacent metal centers to form 1D coordination polymeric chains. Furthermore, the carboxylate oxygen atoms and the aromatic chelate ligands provide potential supramolecular recognition sites for noncovalent interactions to form higher dimensionality. In 1, each pair of 1D chains recognize each other through strong hydrogen bond formed by active amine (-NH-) hydrogen and aromatic π-π stacking interactions to generate a zipperlike double-stranded chain. In contrast, when the substitutions of tpy for dpa further enhance the π-π aromatic stacking interactions, complex 2 is directly constructed into 2D zipperlike layers by the unprecedented aromatic π-π stacking interactions that same orientated aromatic chelate ligands are simultaneously attached to both sides of the chain and the significant C-H?O hydrogen bonds. These layers are constructed through hydrogen bonds and result in a 3D supramolecular network with 1D open channels, in which exists an unprecedented one-dimensional water chain. Both were magnetically characterized, showing weak ferromagnetic behavior.     

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.