Synthesis and fluorescence properties of new fluorescent, polymerizable, metal-chelating lipids

Liposomes incorporating fluorescent, metal-chelating lipids find applications in molecular recognition of peptides, 2D protein recrystallization, protein targeting, and biological sensing. It would be advantageous to combine the usefulness of polymerizable, metal-chelating lipids and fluorescent lipids. Herein, we report the synthesis and fluorescence properties of several fluorescent, polymerizable, metal-chelating lipids. They have been successfully incorporated into liposomes and then polymerized. These lipids can be used as membrane probes to study the polymerizable liposomes in the unpolymerized state and to investigate lipid redistribution during polymerization. In addition, if a luminescent metal ion (e.g., Eu(3+), Tb(3+), etc.) is used to complex the headgroup, the lipids can probe the membrane interior and exterior simultaneously.     

Patterned monomolecular films from polymerizable and fluorinated lipids for the presentation of glycosylated lipids

This paper deals with pattern formation in Langmuir monolayers of two sets of lipid mixtures that include (1) a fluorinated lipid for phase separation, (2) a polymerizable lipid for stabilization of the patterned structure, and (3) a unit for the presentation of biological recognition units. Differences in the distribution of these functionalities allow a polymerization of dispersed or continuous phase and a placement of the recognition units in crystalline or solid analogue phase. Also, a ternary mixture including a lipid modified with the tandem repeat domain of MUC1 plus a T_N-antigen was studied. Based on the biphasic pattern obtained (starlike crystals of up to 50 μm with a fine structure of some micrometers), we also verified the potential of the laterally patterned monolayer to stimulate the immune system (quartz crystal microbalance). The second set of lipids combines a highly fluorinated itaconic ester (polymerizable unit) with the natural phospholipid 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine.     

Photopolymerization of mixed monolayers and black lipid membranes containing gramicidin A and diacetylenic phospholipids

We formed monolayers and black lipid membranes (BLMs) of photopolymerizable lipids mixed with the channel-forming protein gramicidin A to evaluate their miscibility and the potential for improved stability of the BLM scaffold through polymerization. Analyses of surface pressure vs area isotherms indicated that gramicidin A dispersed with three different synthetic, polymerizable, diacetylene-containing phospholipids, 1,2-di-10,12-tricosadiynoyl-sn-glycero-3-phosphocholine (DTPC), 1,2-di-10,12-tricosadiynoyl-sn-glycero-3-phosphoethanolamine (DTPE), and 1-palmitoyl-2,10,12-tricosadiynoyl-sn-glycero-3-phosphoethanolamine (PTPE) to form mixed monolayers at the air-water interface on a Langmuir-Blodgett (LB) trough. Conductance measurements across a diacetylenic lipid-containing BLM confirmed dispersion of the gramicidin channel with the lipid layer and demonstrated gramicidin ion-channel activity before and after UV exposure. Polymerization kinetics of the diacetylenic films were monitored by film pressure changes at constant LB trough area and by UV-vis absorption spectroscopy of polymerized monolayers deposited onto quartz. An initial increase in film pressure of both the pure diacetylene lipid monolayers and mixed films upon exposure to UV light indicated a change in the film structure. Over the time scale of the pressure increase, an absorbance peak indicative of polymerization evolved, suggesting that the structural change in the lipid monolayer was due to polymerization. Film pressure and absorbance kinetics also revealed degradation of the polymerized chains at long exposure times, indicating an optimum time of UV irradiation for maximized polymerization in the lipid layer. Accordingly, exposure of polymerizable lipid-containing black lipid membranes to short increments of UV light led to an increase in the bilayer lifetime.     

Polymer nanostructure development of fluorinated and aliphatic monoacrylates in smectic liquid crystals via photopolymerization

To develop viable polymer stabilized liquid crystal systems, it is crucial to understand the factors that affect polymer nanostructure evolution. This work examines the influence of the photopolymerization of aliphatic and fluorinated monoacrylate monomer within a room temperature smectic liquid crystal (LC). Additionally, the effect of LC order on polymerization kinetics, monomer and polymer organization, and the effect of the polymer on LC properties have been examined. Through this work, insight has been gained regarding the impact that the introduction of a fluorinated monoacrylate monomer has on polymerization kinetics, LC organization, and monomer/polymer segregation and organization within a polymer/LC system. Fluorinated moieties lower the surface energy of the monomer to enhance segregation between the smectic layers of the LC as compared with an analogous aliphatic monomer. Additionally, the enhanced segregation significantly increases the polymerization rate in the smectic phase and drives the continued segregation of the fluorinated polymer during and after polymerization. Fluorination also leads to the formation of an ordered polymer nanostructure if polymerized in ordered LC phases. This ordering is particularly evident when the fluorinated monomer is polymerized in the smectic phase in which the monomer is organized between the smectic layers of the LC. In addition, the ordered polymer structure found with the fluorinated monomer in the smectic phase leads to continued birefringence above the clearing point of the LC due to surface interactions between the LC and the ordered fluorinated polymer. The continued birefringence offers an exceptional opportunity to examine how factors such as polymer molecular mass and UV light intensity affect the overall polymer morphology of these polymer/LC systems. As the initiator concentration and UV light intensity are decreased, longer polymer chains form lattice-type morphologies; whereas, shorter polymer chains form smoother morphologies that more closely mirror the texture of the LC smectic phase.     

Polymer nanostructure development of fluorinated and aliphatic monoacrylates in smectic liquid crystals via photopolymerization

To develop viable polymer stabilized liquid crystal systems, it is crucial to understand the factors that affect polymer nanostructure evolution. This work examines the influence of the photopolymerization of aliphatic and fluorinated monoacrylate monomer within a room temperature smectic liquid crystal (LC). Additionally, the effect of LC order on polymerization kinetics, monomer and polymer organization, and the effect of the polymer on LC properties have been examined. Through this work, insight has been gained regarding the impact that the introduction of a fluorinated monoacrylate monomer has on polymerization kinetics, LC organization, and monomer/polymer segregation and organization within a polymer/LC system. Fluorinated moieties lower the surface energy of the monomer to enhance segregation between the smectic layers of the LC as compared with an analogous aliphatic monomer. Additionally, the enhanced segregation significantly increases the polymerization rate in the smectic phase and drives the continued segregation of the fluorinated polymer during and after polymerization. Fluorination also leads to the formation of an ordered polymer nanostructure if polymerized in ordered LC phases. This ordering is particularly evident when the fluorinated monomer is polymerized in the smectic phase in which the monomer is organized between the smectic layers of the LC. In addition, the ordered polymer structure found with the fluorinated monomer in the smectic phase leads to continued birefringence above the clearing point of the LC due to surface interactions between the LC and the ordered fluorinated polymer. The continued birefringence offers an exceptional opportunity to examine how factors such as polymer molecular mass and UV light intensity affect the overall polymer morphology of these polymer/LC systems. As the initiator concentration and UV light intensity are decreased, longer polymer chains form lattice-type morphologies; whereas, shorter polymer chains form smoother morphologies that more closely mirror the texture of the LC smectic phase.     

Gamma-ray polymerization of phospholipids having diene or triene groups as liposomes

Small unilamellar liposomes were prepared in an aqueous medium by the sonication of phospholipids containing diene or triene groups in their hydrocarbon acyl chains. These liposomes were polymerized by gamma-ray irradiation. Conversion of polymerization was successively followed by UV spectrometry. Diene-type lipid liposomes were revealed for which a gamma-ray dose of 0.8 Mrad was required for complete polymerization and which were polymerized more easily than triene-type lipid liposomes. Triene-type lipids required 2.3 Mrad gamma ray to polymerize them completely. Contrary to UV-irradiation polymerization, there was no concentration dependence on the polymerization. Structure of the polymerized liposomes were confirmed by electron microscopy as small unilamellar liposomes. Study on the leakage of fluorescein from inner aqueous phase of the polymerized liposomes revealed that polymerized triene-type liposomes were relatively more stable than the polymerized diene-type liposomes.     

Synthesis of a polymerizable metal-ion-chelating lipid for fluid bilayers.

Hydrated lipid structures, such as liposomes, that display tethered metal-ion-chelating groups have proven useful in peptide and protein binding, as well as 2D protein crystallization through molecular recognition of accessible histidine sites in proteins and peptides. Polymerizable metal-ion-chelating lipids bearing a reactive diacetylene group have been described. These interesting compounds can be polymerized in the solid-analogous phase. Here we describe the design of the first polymerizable metal-ion-chelating lipid that can be used in the fluid, i.e., liquid analogous, phase of lipid bilayers. The synthesis of 1-palmitoyl-2-[8-[(E,E)-2',4'-hexadienoyloxy]octanoyl]-sn-glycero-3-N-[11-[N',N'-bis[carboxymethyl]imino]-3,6,9-trioxaundecanoyl] phosphatidylethanolamine (1) is described. The chelator moiety, iminodiacetate (IDA), was linked to the polymerizable phosphatidylethanolamine (PE) with a terminal 2,4-hexadienoyl (sorbyl) group through an oligo(ethylene glycol)-based spacer. Lipid 1-Cu complex is designed to be combined with the corresponding polymerizable matrix lipids (bis-SorbPC) to form functionalized liposomes that can be stabilized by various polymerization methods.     

Reconstitution of rhodopsin into polymerizable planar supported lipid bilayers: influence of dienoyl monomer structure on photoactivation

G-protein-coupled receptors (GPCRs) play key roles in cellular signal transduction and many are pharmacologically important targets for drug discovery. GPCRs can be reconstituted in planar supported lipid bilayers (PSLBs) with retention of activity, which has led to development of GPCR-based biosensors and biochips. However, PSLBs composed of natural lipids lack the high stability desired for many technological applications. One strategy is to use synthetic lipid monomers that can be polymerized to form robust bilayers. A key question is how lipid polymerization affects GPCR structure and activity. Here we have investigated the photochemical activity of bovine rhodopsin (Rho), a model GPCR, reconstituted into PSLBs composed of lipids having one or two polymerizable dienoyl moieties located in different regions of the acyl chains. Plasmon waveguide resonance spectroscopy was used to compare the degree of Rho photoactivation in fluid and poly(lipid) PSLBs. The position of the dienoyl moiety was found to have a significant effect: polymerization near the glycerol backbone significantly attenuates Rho activity whereas polymerization near the acyl chain termini does not. Differences in cross-link density near the acyl chain termini also do not affect Rho activity. In unpolymerized PSLBs, an equimolar mixture of phosphatidylethanolamine and phosphatidylcholine (PC) lipids enhances activity relative to pure PC; however after polymerization, the enhancement is eliminated which is attributed to stabilization of the membrane lamellar phase. These results should provide guidance for the design of robust lipid bilayers functionalized with transmembrane proteins for use in membrane-based biochips and biosensors.     

Molecular packing of functionalized fluorinated lipids in Langmuir monolayers

Fluorinated amphipaths are a fascinating class of compounds, which, despite significant challenges associated with their syntheses, have found use across a number of areas of biotechnology. Applications range from the in vitro stabilization of membrane proteins to the development of enhanced stability intravenous drug and gene delivery systems. More recently, monolayer-forming fluorinated lipids have found use in the 2D crystallization of detergent-solubilized hydrophobic or partially hydrophobic proteins at the air-water interface. In this study, we investigate the surface properties of a novel suite of monolayer forming, partially fluorinated lipids. These modular lipid structures contain a densely fluorinated insertion in the hydrocarbon tail and a synthetically modifiable headgroup. Analyses of surface-pressure area isotherms and X-ray reflectometry profiles reveal that the lipids spread into fluid monolayers and are more compressible than their non-fluorinated counterparts. Furthermore, the data support a model whereby the partially fluorinated chains of the lipid tails form a film which is fundamentally incompatible with detergents and other destabilizing amphipaths.     

Spectroscopic studies of polymerized surfactants: 1,2-bis(10,12-tricosadiynoyl)-sn-glycero-3-phosphocholine

Spectroscopic studies have been performed on aqueous dispersions of the surfactant 1,2-bis(10,12-tricosadiynoyl)-sn-glycero-3-phosphocholine before and after polymerization with ul-traviolet light. Monomers of this lipid can, under certain conditions, convert from the expected spherical liposomal form to a unique phase consisting of hollow tubules. To determine the molecular conformation of these structures we have used Raman and infrared spectroscopies to probe the structure of the hydrocarbon chains and head groups of the lipids, and used absorption spectroscopy and resonance enhanced Raman scattering of the colored polymer to monitor the length and structure of the diacetylenic polymer backbone. Unusual C? H stretch-ing Raman bands imply that the hydrocarbon chain packing in the monomeric bilayers is different from that observed in other phosphatidylcholines, and that a distrubance in alkyl chain packing occurs on polymerization. Depending on irradiation conditions and the dispersal state of the lipid the polymer chains may be of at least three different colors, from which distinct resonance Raman spectra are obtained. The effective bond conjugation lengths range from quite short in the yellow polymer produced in sonicated vesicles to extremely long in a blue component seen in polymerized tubules.     

Liposome formation of selectively-polymerized diene-containing phospholipids and their postpolymerization

Small and large unilamellar liposomes composed of 1,2-bis(2,4-octadecadienoyl)-sn-glycero-3-phosphorylcholine (DODPC) are prepared by sonication and extrusion, respectively. They are polymerized with water-insoluble radical initiator, azobis(isobutyronitrile) (AIBN) which can selectively polymerize diene groups in 1-acyl chains of the lipids. Polymerized liposomes are freeze-dried to obtain the polymerized liposome powder. There are two methods to redisperse lyophilized liposomes into water. The extrusion is an effective method to disperse them because the energy at extrusion is necessary only for redispersion, whereas the excess energy at sonication gives damage on liposome structure. There is no difference in stability between polymerized liposomes before and after redispersion with extrusion. DODPC polymers, obtained from free radical-initiated polymerization with AIBN, are linear and have polymerizable diene groups in 2-acyl chains. The liposome powder is therefore soluble in organic solvents. Reconstruction of polymerized liposomes is performed with lipid polymers having low or high molecular weight. The lipid polymers having high molecular weight provide stable large unilamellar liposomes by ethanol injection, but unstable small unilamellar liposomes are formed by sonication. The liposomes reconstructed from lipid polymers having low molecular weight are unstable regardless of their size. After reconstruction of liposomes selectively polymerized by AIBN, diene groups in 2-acyl chains are polymerized by water-soluble radical initiator or UV-irradiation to yield highly crosslinked structure. Their stability is improved remarkably by this postpolymerization.     

Microstructure and lateral diffusion in monolayers of polymerizable amphiphiles

Lipid analogue amphiphilic molecules containing polymerizable units were investigated in monolayers at the air/water interface by using film balance measurements, fluorescence microscopy, and photobleaching techniques. The polymerizable groups (diene-, diyne-, and methacrylate units) were introduced into the hydrophobic alkyl chains or into the polar head of the amphiphilic molecules.In the case of the diene- and diyne-containing compounds the polymerizable units are incorporated into the hydrophobic alkyl chains, enabling them to form a two-dimensional network. Due to the free chain flexibility of the monomers the lateral mobility was comparable to that of saturated lipid analogues and decreases upon polymerization proportionally to the dose of UV irradiation. In addition, fluid/solid phase transitions of compounds with polymerizable groups in the hydrophobic part tend to vanish during the formation of the polymers. However, the direct observation of the growth of polymeric crystalline domains can be followed by using diacetylene lipid analogues.In the case of the methacrylate derivatives the polymerizable unit was coupled to the polar part via a flexible spacer. For these systems the characteristics of the monomeric phase transition are retained after polymerization. However, it shows a significant, strong decrease of the in-plane mobility already in the fluid-expanded phase of the polymer. The quantitative measurements of the lateral diffusion in the monolayers can be correlated with fluorescence microscopic images of their structure.     

Polymerized fluorescent liposomes incorporating lanthanide ions

This paper describes the design and synthesis of a polymerizable lipid capable of complexing lanthanide ions. The lipid has been successfully incorporated into liposomes and then polymerized. Fluorescence studies indicate that the diacetylene (unpolymerized lipid) and the conjugated alkenes (after polymerization) can be used as sensitizers for the lanthanide ion.     

FTIR spectroscopic analyses of the temperature and pH influences on stratum corneum lipid phase behaviors and interactions

A thermotropic investigation of different lipid dispersions containing ceramide 3 (CER3) or sphingomyelin (SPM), perdeuterated palmitic acid (PA-d(31)) and cholesterol (CH) or cholesterol sulfate (CS) at pH 5.2 and 7.4 used as model membranes, has been carried out by Fourier transform infrared (FTIR) spectroscopy in order to estimate the importance of these lipids and of the temperature and pH for the maintenance of the structural organization of the stratum corneum (SC) lipid lamellae. The results obtained for the CER3 and SPM mixtures at pH 5.2 and 7.4 indicated that the little size of the polar headgroup of CER3 compared with that of SPM could permit a more closely packing in the CER3 acyl chains. Moreover, the CH and CS induced an increase of the order in the CER3 acyl chains over the physiological temperatures while a disordering was seen above 60 degrees C. In addition, the thermal phase behaviors observed for the CER3/PA-d(31) dispersion at pH 5.2 and 7.4, suggested a phase separation between the CER3 and PA-d(31) molecules in this mixture. Nevertheless, the miscibility between the CER3 and PA-d(31) was raised in the presence of CH or CS at pH 5.2. In particular, the incorporation of these sterols into the CER3/PA-d(31) dispersion at pH 5.2 appeared to result in an increase of the order in the acyl chains of CER3 and PA-d(31) at about 37 degrees C. In contrast, a phase separation was observed between the CER3 and PA-d(31) in the CER3/PAd(31)/CH and CER3/PA-d(31)/CS dispersions at pH 7.4. Interestingly, the pH change from 5.2 to 7.4 in these tertiary dispersions was also accompanied by a substantial deprotonation of the PA-d(31) molecules which seemed more pronounced in the presence of CH as compared with CS. Altogether, the results suggested that the ceramides, fatty acids and sterols could play an important structural role in the SC cohesion.     

Lipid fluorination enables phase separation from fluid phospholipid bilayers

To probe the effect of lipid fluorination on the formation of lipid domains in phospholipid bilayers, several new fluorinated and non-fluorinated synthetic lipids were synthesised, and the extent of phase separation of these lipids from phospholipid bilayers of different compositions was determined. At membrane concentrations as low as 1% mol/mol, both fluorinated and non-fluorinated lipids were observed to phase separate from a gel-phase (solid ordered) phospholipid matrix, but bilayers in a liquid disordered state caused no phase separation; if the gel-phase samples were heated above the transition temperature, then phase separation was lost. We found incorporation of perfluoroalkyl groups into the lipid enhanced phase separation, to such an extent that phase separation was observed from cholesterol containing bilayers in the liquid ordered phase.     

Investigation in monolayers of double-chain fluorocarbon amphiphiles Effect of headgroups on molecular packing and interaction between hydrocarbon and fluorocarbon monolayers

The surface pressure-area diagrams of double-chain fluorocarbon amphiphiles with different headgroup compositions show that the amphiphiles arrange almost perpendicularly to the water subphase and the structure of headgroups exerts significant influence on the amphiphile packing. Strong hydrogen bonding and weak electrostatic interaction favor the formation of stable monolayers. Perfluorooctanoic acid (FOA) cannot form monolayer at water/air interface and can only form liquid monolayer in subphase of calcium nitrate solution. Complete phase separation of palmitic acid and a fluorocarbon amphiphile with shorter hydrocarbon spacer group, 1, could be demonstrated in monolayers by using the phase rule of Crisp. The creation of phase-separated monolayers is possible when the monolayer is composed of a mixture of palmitic acid and a fluorocarbon amphiphile with longer hydrocarbon spacer group, 2. It can be suggested that the miscibility of hydrocarbon amphiphiles with fluorocarbon amphiphiles is determined by the hydrocarbon fraction of fluorocarbon amphiphiles.     

Patterned biomimetic membranes: effect of concentration and pH

Planar-supported lipid bilayers have attracted enormous attention because of their properties as model cell membranes, which can be employed in a variety of fundamental biological studies and medical devices. Furthermore, the development of patterned biological interfaces is of great practical and scientific interest because of their potential applications in the field of biosensors, drug screening, tissue engineering, and medical implants. In this study, mica-supported membranes were constructed from biomimetic peptide-amphiphiles and their mixtures with lipidated poly(ethylene glycol) (PEG120) molecules or 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) phospholipids using the Langmuir-Blodgett technique. The two peptide-amphiphiles used in this study were a fibronectin-mimetic with the PHSRN(SG)(3)SGRGDSP headgroup (referred to as PHSRN-GRGDSP) that contains both the primary (GRGDSP) and the synergy (PHSRN) recognition sites for alpha(5)beta(1) integrins and a peptide-amphiphile that mimics a fragment of the N-terminus of the fractalkine receptor (referred to as NTFR). Compression isotherms of the peptide-amphiphiles and their mixtures with PEG120 at the air/water interface were recorded and analyzed to evaluate the extent of miscibility in the two-component LB films. Domain formation in mica-supported bilayers constructed from mixtures of peptide-amphiphiles and lipidated PEG120 or DPPC was observed using atomic force microscopy. In PHSRN-GRGDSP/PEG120 mixtures deposited from an aqueous subphase at pH 7, concentration-dependent phase separation was observed on the AFM images. The NTFR/PEG120 and NTFR/DPPC mixtures deposited at pH 10 exhibited extensive lateral phase separation at all mixture compositions, whereas at deposition pH 7 the concentrations of NTFR/DPPC examined here were well mixed.     

Dye induced great enhancement of broadband reflection from polymer stabilized cholesteric liquid crystals

A series of polymer stabilized cholesteric liquid crystals (PSCLCs) films were prepared from cholesteric liquid crystal (Ch‐LC) mixtures containing different components such as non‐reactive LC monomer, polymerizable monomer, chiral dopant, dye, and photoinitiator upon polymerization. The influence of the polymerizable monomer and dye of Ch‐LC mixtures on the reflection properties was investigated. The reflection bandwidth for all the samples can be increased by photo‐polymerization, and the network upon polymerization derived from two different polymerizable monomers with both one and two functional groups is more effective than that from one polymerizable monomer for broadening the reflection band. Especially, a dye‐doped Ch‐LC film can reflect incident light with the bandwidth over the wavelength range of 550–2350 nm, which is due to a greater pitch gradient formed inside of Ch‐LC film. The gradient pitch network structure was firstly demonstrated by scanning electron microscopy (SEM) with the film prepared from high diacrylate monomer concentration and subsequently proved by using a wash‐out/refill method. The nematic liquid crystals monomers was infiltrated into the polymer network that was prefabricated by removing the low molar weight LCs from the original PSCLCs film, and SEM exhibited the existence of a pitch gradient across the film thickness. The refilled nematic liquid crystals film showed broadband reflection after polymerzition, too. Copyright © 2011 John Wiley & Sons, Ltd.     

Phase separation in polystyrene latex interpenetrating polymer networks

Polystyrene/polystyrene latex interpenetrating polymer networks (IPNs) were prepared by seeded emulsion polymerzation of styrene–divinylbenzene mixtures in crosslinked monodisperse polystyrene seed latexes. The resulting latexes comprised uniform nonspherical particles, which were formed by separation of the second-stage monomer from the crosslinked seed network during swelling and polymerization. The kinetics of phase separation were investigated by examining the changes in particle morphology using optical microscopy, which revealed that the phase separation was induced by the relaxation of the polymer chains before polymerization began and was enhanced by increased conversion. The thermodynamics of phase separation were investigated by analysis of the free-energy changes during swelling and polymerization, and the phase separation was described by a nucleation-and-growth mechanism. The results of this study have been applied to the design and synthesis of a series of uniform nonspherical particles of different morphology.     

Raftlike mixtures of sphingomyelin and cholesterol investigated by solid-state 2H NMR spectroscopy

Sphingomyelin is a lipid that is abundant in the nervous systems of mammals, where it is associated with putative microdomains in cellular membranes and undergoes alterations due to aging or neurodegeneration. We investigated the effect of varying the concentration of cholesterol in binary and ternary mixtures with N-palmitoylsphingomyelin (PSM) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) using deuterium nuclear magnetic resonance ((2)H NMR) spectroscopy in both macroscopically aligned and unoriented multilamellar dispersions. In our experiments, we used PSM and POPC perdeuterated on the N-acyl and sn-1 acyl chains, respectively. By measuring solid-state (2)H NMR spectra of the two lipids separately in mixtures with the same compositions as a function of cholesterol mole fraction and temperature, we obtained clear evidence for the coexistence of two liquid-crystalline domains in distinct regions of the phase diagram. According to our analysis of the first moments M1 and the observed (2)H NMR spectra, one of the domains appears to be a liquid-ordered phase. We applied a mean-torque potential model as an additional tool to calculate the average hydrocarbon thickness, the area per lipid, and structural parameters such as chain extension and thermal expansion coefficient in order to further define the two coexisting phases. Our data imply that phase separation takes place in raftlike ternary PSM/POPC/cholesterol mixtures over a broad temperature range but vanishes at cholesterol concentrations equal to or greater than a mole fraction of 0.33. Cholesterol interacts preferentially with sphingomyelin only at smaller mole fractions, above which a homogeneous liquid-ordered phase is present. The reasons for these phase separation phenomena seem to be differences in the effects of cholesterol on the configurational order of the palmitoyl chains in PSM-d31 and POPC-d31 and a difference in the affinity of cholesterol for sphingomyelin observed at low temperatures. Hydrophobic matching explains the occurrence of raftlike domains in cellular membranes at intermediate cholesterol concentrations but not saturating amounts of cholesterol.