A microscopic interaction model of maximum solubility of cholesterol in lipid bilayers

We recently reported the equilibrium maximum solubility of cholesterol in a lipid bilayer, chi*chol, to be 0.66 in four different phosphatidylcholines, and 0.51 in a phosphatidylethanolamine (Huang, J.,J.T. Buboltz, and G. W. Feigenson. 1999. Biochim. Biophys. Acta. in press). Here we present a model of cholesterol-phospholipid mixing that explains these observed values of chi*chol. Monte Carlo simulations show that pairwise-additivity of nearest-neighbor interactions is inadequate to describe all the chi*chol values. Instead, if cholesterol multibody interactions are assigned highly unfavorable energy, then jumps occur in cholesterol chemical potential that lead to its precipitation from the bilayer. Cholesterol precipitation is most likely to occur near three discrete values of cholesterol mole fraction, 0.50, 0.57, and 0.67, which correspond to cholesterol/phospholipid mole ratios of 1/1, 4/3, and 2/1, respectively. At these solubility limits, where cholesterol chemical potential jumps, the cholesterol-phospholipid bilayer mixture forms highly regular lipid distributions in order to minimize cholesterol-cholesterol contacts. This treatment shows that dramatic structural and thermodynamic changes can occur at particular cholesterol mole fractions without any stoichiometric complex formation. The physical origin of the unfavorable cholesterol multibody interaction is explained by an "umbrella model": in a bilayer, nonpolar cholesterol relies on polar phospholipid headgroup coverage to avoid the unfavorable free energy of cholesterol contact with water. Thus, at high cholesterol mole fraction, this unfavorable free energy, not any favorable cholesterol-phospholipid interaction, dominates the mixing behavior. This physical origin also explains the "cholesterol condensing effect" and the increase in acyl chain order parameter in cholesterol-phospholipid mixtures.     

Phosphatidylcholine and cholesterol interactions in model membranes

Various phosphatidylcholines differing either in the stereochemistry around their chiral center or in the position of a cis double bond along the acyl chains were synthesized in order to study critical contact regions in the phospholipid molecule with adjacent cholesterol in model membranes. Microviscosities calculated from fluorescence depolarization of diphenylhexatriene and chain order from spin label studies were measured to monitor physical membrane properties. The enhancing effect of cholesterol on the microviscosity of membranes containing phosphatidylcholines with comparable acyl chain length was largest when the two acyl chains were saturated and smallest when both were unsaturated. Membranes prepared from phosphatidylcholines having a single cis double bond at different positions along the sn-2 acyl chain showed roughly the same changes of microviscosity or chain order upon incorporation of cholesterol. No discrimination was evident in the interaction between cholesterol and enantiomeric phosphatidylcholines or between the enantiomeric phosphatidylcholine molecules themselves. We conclude that the rigidifying effect of cholesterol in membranes does not depend on specific sites of interaction and that with respect to physical membrane properties phosphatidylcholine behaves as an achiral molecule.     

Comparison of cholesterol-phospholipid interaction in bilayers of liposomes and the red cell membrane

The energetics of interactions of cholesterol with phospholipid in simple liposome bilayers were compared with those in the bilayer of the human erythrocyte membrane, by measuring cholesterol distribution between erythrocytes and liposomes prepared from their whole phospholipid extract. With liposomes of a range of initial cholesterol contents, the equilibrium value for $\text{r}$, the ratio of cholesterol/phospholipid in the liposomes to that in the cells, is in the range 1.1–1.2. The closeness of this value to 1.0 indicates that overall cholesterol-phospholipid interaction in the cell membrane is similar to that in liposomes. However, while the deviation from 1.0 is small, and could arise from average cholesterol-phospholipid interactions in the membrane being only 0.06 to 0.1 kcal · mol^?1 weaker than in liposomes, it could also result from 10 to 20% of the cell membrane phospholipid being unavailable to mix with cholesterol.     

Use of cytochrome P-450scc to measure cholesterol-lipid interactions

The interaction of cholesterol with phospholipids has been studied with a variety of techniques; however, the possible consequences of such interactions in vivo have not been demonstrated. In this study, the cholesterol-dependent absorbance spectrum of cytochrome P-450scc was used to monitor cholesterol availability in both micellar and vesicular environments. By use of this approach, in conjunction with titration of putative cholesterol binding species, a tight, approximately equimolar complex of cholesterol and digitonin was demonstrated. Sphingomyelin (SM) (both the synthetic N-palmitoyl and bovine brain forms) gave sigmoidal titration curves, suggesting a cooperative interaction between this lipid and cholesterol. The interaction of bovine brain glycerolipids and cholesterol was weaker than that of SM and showed no cooperativity. The importance of the phospholipid head group in these interactions was established by the differences in the ability of synthetic 1-palmitoyl-2-oleoylphosphatidylcholine, -phosphatidylethanolamine, and -phosphatidylserine to affect cholesterol availability. Comparison of these results with those of the bovine brain phospholipids indicates that the acyl chain composition of these molecules is also important to these interactions. Titrations of SM in phospholipid vesicles containing cytochrome P-450scc and different types of phosphatidylcholine established that the SM-cholesterol interactions also occur in a bilayer membrane. This study demonstrates that the association of cholesterol with cytochrome P-450scc is inhibited by concentrations of SM commonly found in biological membranes. Therefore, such cholesterol-lipid interactions can potentially affect the function of membrane enzymes.     

Side chain oxygenated cholesterol regulates cellular cholesterol homeostasis through direct sterol-membrane interactions

Side chain oxysterols exert cholesterol homeostatic effects by suppression of sterol regulatory element-binding protein maturation and promoting degradation of hydroxymethylglutaryl-CoA reductase. To examine whether oxysterol-membrane interactions contribute to the regulation of cellular cholesterol homeostasis, we synthesized the enantiomer of 25-hydroxycholesterol. Using this unique oxysterol probe, we provide evidence that oxysterol regulation of cholesterol homeostatic responses is not mediated by enantiospecific oxysterol-protein interactions. We show that side chain oxysterols, but not steroid ring-modified oxysterols, exhibit membrane expansion behavior in phospholipid monolayers and bilayers in vitro. This behavior is non-enantiospecific and is abrogated by increasing the saturation of phospholipid acyl chain constituents. Moreover, we extend these findings into cultured cells by showing that exposure to saturated fatty acids at concentrations that lead to endoplasmic reticulum membrane phospholipid remodeling inhibits oxysterol activity. These studies implicate oxysterol-membrane interactions in acute regulation of sterol homeostatic responses and provide new insights into the mechanism through which oxysterols regulate cellular cholesterol balance.     

Membrane properties of and cholesterol's interactions with a biologically relevant three-chain sphingomyelin: 3O-palmitoyl-N-palmitoyl-D-erythro-sphingomyelin

Sphingomyelins (SMs) are order-imposing phospholipids in cell membranes which interact favorably with cholesterol. The hydrophobic part of SM constitutes a long-chain base with an amide-linked acyl chain, whereas the polar head group is phosphocholine. The long-chain base has a free hydroxyl group in position 3, which is an important donor/acceptor in hydrogen bonding. In newborn mammals, a SM in which a palmitic acid is esterified to the 3-OH has been reported. We have synthesized this SM analog (3O-P-PSM) and studied its properties in bilayer membranes, and also determined its interactions with cholesterol. Fully hydrated 3O-P-PSM bilayers underwent a gel-to-liquid crystalline phase transition at 55.5 °C (ΔH 8 kcal/mol), which is about 15 °C higher than the phase transition temperature of PSM. The 3O-P-PSM displayed rather poor miscibility with PSM in mixed bilayers, suggesting that the third acyl chain interfered significantly with lateral interactions. Bilayers made from 3O-P-PSM were much more resistant to detergent-induced solubilization than bilayers made from PSM. In binary bilayers, cholesterol was able to destabilize the gel phase, and order the fluid phase of 3O-P-PSM, in a concentration-dependent manner. Cholesterol was also able to form sterol-enriched ordered domains with 3O-P-PSM in fluid POPC bilayers. The interaction between cholesterol and 3O-P-PSM was not, however, as favorable as the interaction between cholesterol and PSM. It is unclear what physiological role 3O-P-PSM could play in newborn mammalian membranes. However, it is clear that 3O-P-PSM will form more highly ordered domains than PSM while still having a limited ability to interact with cholesterol.     

Cholesterol interactions with tetracosenoic acid phospholipids in model cell membranes: role of the double-bond position

The synthesis and thermotropic properties of 1,2-di-(9Z)-9-tetracosenoylphosphatidylcholine [delta 9-PC(24:1,24:1), 1], 1,2-di-(5Z)-5-tetracosenoylphosphatidylcholine [delta 5-PC(24:1,24:1), 2], and 1,2-di-(15Z)-15- tetracosenoylphosphatidylcholine [delta 15-PC(24:1,24:1), 3] are reported. Liposomes prepared from these phospholipids differ from those of the natural sponge phospholipids, 1,2-di-(5Z,9Z)-5,9-hexacosadienoylphosphatidylcholine (4a) and the corresponding ethanolamine (4b), both of which virtually exclude cholesterol from their bilayers. The behavior of 1 and 2 is similar to that of 1,2-di-(6Z,9Z)-6,9-hexacosadienoylphosphatidylcholine (5), which exhibits a partial molecular interaction with cholesterol. In the case of 3, cholesterol appears to interact with the saturated acyl chain regions of this phospholipid in a manner similar to that of its interaction with DPPC acyl chains. This study delineates the effect of the double-bond location in long fatty acyl chains of phospholipids on their interactions with cholesterol.     

Trans fatty acid derived phospholipids show increased membrane cholesterol and reduced receptor activation as compared to their cis analogs

The consumption of trans fatty acid (TFA) is linked to the elevation of LDL cholesterol and is considered to be a major health risk factor for coronary heart disease. Despite several decades of extensive research on this subject, the underlying mechanism of how TFA modulates serum cholesterol levels remains elusive. In this study, we examined the molecular interaction of TFA-derived phospholipid with cholesterol and the membrane receptor rhodopsin in model membranes. Rhodopsin is a prototypical member of the G-protein coupled receptor family. It has a well-characterized structure and function and serves as a model membrane receptor in this study. Phospholipid-cholesterol affinity was quantified by measuring cholesterol partition coefficients. Phospholipid-receptor interactions were probed by measuring the level of rhodopsin activation. Our study shows that phospholipid derived from TFA had a higher membrane cholesterol affinity than their cis analogues. TFA phospholipid membranes also exhibited a higher acyl chain packing order, which was indicated by the lower acyl chain packing free volume as determined by DPH fluorescence and the higher transition temperature for rhodopsin thermal denaturation. The level of rhodopsin activation was diminished in TFA phospholipids. Since membrane cholesterol level and membrane receptors are involved in the regulation of cholesterol homeostasis, the combination of higher cholesterol content and reduced receptor activation associated with the presence of TFA-phospholipid could be factors contributing to the elevation of LDL cholesterol.     

Detection of liposomal cholesterol and monophosphoryl lipid A by QS-21 saponin and Limulus polyphemus amebocyte lysate

Liposomes containing cholesterol (Chol) have long been used as an important membrane system for modeling the complex interactions of Chol with adjacent phospholipids or other lipids in a membrane environment. In this study we utilize a probe composed of QS-21, a saponin molecule that recognizes liposomal Chol and causes hemolysis of erythrocytes. The interaction of QS-21 with liposomal Chol results in a stable formulation which, after injection into the tissues of an animal, lacks toxic effects of QS-21 on neighboring cells that contain Chol, such as erythrocytes. Here we have used liposomes containing different saturated phospholipid fatty acyl groups and Chol, with or without monophosphoryl lipid A (MPLA), as model membranes. QS-21 is then employed as a probe to study the interactions of liposomal lipids on the visibility of membrane Chol. We demonstrate that changes either in the mole fraction of Chol in liposomes, or with different chain lengths of phospholipid fatty acyl groups, can have a substantial impact on the detection of Chol by the QS-21. We further show that liposomal MPLA can partially inhibit detection of the liposomal Chol by QS-21. The Limulus amebocyte lysate assay is used for binding to and detection of MPLA. Previous work has demonstrated that sequestration of MPLA into the liposomal lipid bilayer can block detection by the Limulus assay, but the binding site on the MPLA to which the Limulus protein binds is unknown. Changes in liposomal Chol concentration and phospholipid fatty acyl chain length influenced the detection of the liposome-embedded MPLA.     

Effects of replacement of the hydroxyl group of cholesterol and tocopherol on the thermotropic behavior of phospholipid membranes

The role of the hydroxyl groups of cholesterol and tocopherol in mediating their interaction with phospholipid bilayers has been a subject of considerable interest. We have examined this question by using derivatives of cholesterol and tocopherol in which the hydroxyl group is esterified to succinate. The hemisuccinate esters of cholesterol and alpha-tocopherol can be readily incorporated into phospholipid membranes and in fact can by themselves form closed membrane vesicles as demonstrated by the encapsulation of [3H]sucrose. The thermotropic behavior of mixtures containing each succinate ester and phospholipid was studied by differential scanning calorimetry. The effect of cholesteryl hemisuccinate on the thermotropic properties of dipalmitoylphosphatidylcholine and dimyristoylphosphatidylethanolamine is very similar to that of cholesterol. This indicates that the 3 beta-OH is not required for the formation of a cholesterol-phospholipid complex. In mixtures of tocopherol acid succinate and phospholipids the peak transition temperature is progressively shifted to lower temperatures as the mole fraction of alpha-tocopherol succinate is increased, while the enthalpy of the transition is only slightly affected. At a tocopherol succinate/phospholipid molar ratio of 9/1 a phase transition is still detectable. A comparison between tocopherol succinate and tocopherol indicates that the substitution of the hydroxyl group reduces the interaction of tocopherol with phospholipids to a small but measurable extent. Thus, the hydroxyl group of tocopherol is more important than the hydroxyl group of cholesterol in influencing their interactions with phospholipids.     

Enzyme-catalyzed oxidation of cholesterol in mixed phospholipid monolayers reveals the stoichiometry at which free cholesterol clusters disappear.

In this study, we have used cholesterol oxidase as a probe to study cholesterol/phospholipid interactions in mixed monolayers at the air/water interface. Mixed monolayers, containing a single phospholipid class and cholesterol at differing cholesterol/phospholipid molar ratios, were exposed to cholesterol oxidase at a lateral surface pressure of 20 mN/m (at 22 degrees C). At equimolar ratios of cholesterol to phospholipid, the average rate of cholesterol oxidation was fastest in unsaturated phosphatidylcholine mixed monolayers (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine and egg yolk phosphatidylcholine), intermediate in 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, and slowest in sphingomyelin monolayers (egg yolk or bovine brain sphingomyelin). The average oxidation rate in mixed monolayers was not exclusively a function of monolayer packing density, since egg yolk and bovine brain sphingomyelin mixed monolayers occupied similar mean molecular areas even though the measured average oxidation rate was different with these two phospholipids. This suggests that the phospholipid acyl chain composition influenced the oxidation rate. The importance of the phospholipid acyl chain length on influencing the average oxidation rate was further examined in defined phosphatidylcholine mixed monolayers. The average oxidation rate decreased linearly with increasing acyl chain lengths (from di-8:0 to di-18:0). When the average oxidation rate was examined as a function of the cholesterol to phospholipid (C/PL) molar ratio in the monolayer, the otherwise linear function displayed a clear break at a 1:1 stoichiometry with phosphatidylcholine mixed monolayers, and at a 2:1 C/PL stoichiometry with sphingomyelin mixed monolayers.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lateral distribution of cholesterol in dioleoylphosphatidylcholine lipid bilayers: cholesterol-phospholipid interactions at high cholesterol limit

Lateral organization of cholesterol in dioleoyl-phosphatidylcholine (DOPC) lipid bilayers at high cholesterol concentration (>45 mol%) was investigated using steady-state fluorescence anisotropy and fluorescent resonance energy transfer techniques. The recently devised Low Temperature Trap method was used to prepare compositionally uniform cholesterol/DOPC liposomes to avoid the problem of lipid demixing. The fluorescence anisotropy of diphenylhexatrience chain-labeled phosphatidylcholine (DPH-PC) in these liposomes exhibited local maxima at cholesterol mol fractions of 0.50 and 0.57, and a sharp drop at 0.67. For the liposomes labeled with both dehydroergosterol and DPH-PC, the fluorescent resonance energy transfer efficiency from dehydroergosterol to DPH-PC displayed a steep jump at cholesterol mol fraction of 0.5, and dips at 0.57 and 0.68. These results indicate the presence of highly ordered cholesterol regular distribution domains at those observed critical compositions. The observed critical mol fraction at 0.67 agreed favorably with the solubility limit of cholesterol in DOPC bilayers as independently measured by light scattering and optical microscopy. The regular distribution at 0.57 was previously predicted from a Monte Carlo simulation based on the Umbrella model. The results strongly support the hypothesis that the primary requirement for cholesterol-phospholipid mixing is that the polar phospholipid headgroups need to cover the nonpolar body of cholesterol to avoid the exposure of cholesterol to water.     

Proton and carbon-13 nuclear magnetic resonance studies of rhodopsin-phospholipid interactions

Proton and carbon-13 nuclear magnetic resonance (1H and 13C NMR) spectra of rhodopsin-phospholipid membrane vesicles and sonicated disk membranes are presented and discussed. The presence of rhodopsin in egg phosphatidylcholine vesicles results in homogeneous broadening of the methylene and methyl resonances. This effect is enhanced with increasing rhodopsin content and decreased by increasing temperature. The proton NMR data indicate the phospholipid molecules exchange rapidly (less than 10(-3) s) between the bulk membrane lipid and the lipid in the immediate proximity of the rhodopsin. These interactions result in a reduction in either or both the frequency and amplitude of the tilting motion of the acyl chains. The 13C NMR spectra identify the acyl chains and the glycerol backbone as the major sites of protein lipid interaction. In the disk membranes the saturated sn-1 acyl chain is significantly more strongly immobilized than the polyunsaturated sn-2 acyl chain. This suggest a membrane model in which the lipid molecules preferentially solvate the protein with the sn-1 chain, which we term an edge-on orientation. The NMR data on rhodopsin-asolectin membrane vesicles demonstrate that the lipid composition is not altered during reconstitution of the membranes from purified rhodopsin and lipids in detergent.     

Hydrophobic membrane thickness and lipid-protein interactions of the leucine transport system of Lactococcus lactis

The effect of the phospholipid acyl chain carbon number on the activity of the branched-chain amino acid transport system of Lactococcus lactis has been investigated. Major fatty acids identified in a total lipid extract of L. lactis membranes are palmitic acid (16:0), oleic acid (18:1) and the cyclopropane-ring containing lactobacillic acid (19 delta). L. lactis membrane vesicles were fused with liposomes prepared from equimolar mixtures of synthetic phosphatidylethanolamine (PE) and phosphatidylcholine (PC) with cis mono-unsaturated acyl chains. The activity of the branched-chain amino acid carrier is determined by the bulk properties of the membrane (Driessen, A.J.M., Zheng, T., In 't Veld, G., Op den Kamp, J.A.F. and Konings, W.N. (1988) Biochemistry 27, 865-872). PE acts as an activator and PC is ineffective. Counterflow and protonmotive-force driven transport of leucine is sensitive to changes in the acyl chain carbon number of both phospholipids and maximal with dioleoyl-PE/dioleoyl-PC. Above the gel to liquid-crystalline phase transition temperature of the lipid species, membrane fluidity decreased with increasing acyl chain carbon number. Our data suggest that the carbon number of the acyl chains of PE and PC determine to a large extent the activity of the transport system. This might be relevant for the interaction of PE with the transport protein. Variations in the acyl chain composition of PC exert a more general effect on transport activity. The acyl chain composition of phospholipids determines the membrane thickness (Lewis, B.A. and Engelman, D.M. (1983) J. Mol. Biol. 166, 211-217). We therefore propose that the degree of matching between the lipid-bilayer and the hydrophobic thickness of the branched-chain amino acid carrier is an important parameter in lipid-protein interactions.     

The effect of variations in phospholipid and sterol structure on the nature of lipid–sterol interactions in lipid bilayer model membranes

This review deals with the effect of variations in phospholipid and sterol structure on the nature and magnitude of lipid-sterol interactions in lipid bilayer model membranes. The first portion of the review covers the effect of Chol itself on the thermotropic phase behavior and organization of a variety of different glycero- and sphingolipid membrane lipid classes, varying in the structure and charge of their polar headgroups and in the length and structure of their fatty acyl chains. The second part of this review deals with the effect of variations in sterol structure on the thermotropic phase behavior and organization primarily of the well studied DPPC model membrane system. In the third section, we focus on some of the contributions of sterol functional group chemistry, molecular conformation and dynamics, to sterol-lipid interactions. Using those studies, we re-examine the results of recently published experimental and computer-modeling studies to provide a new more dynamic molecular interpretation of sterol-lipid interactions. We suggest that the established view of the rigid sterol ring system and extended alkyl side-chain obtained from physical studies of cholesterol-phospholipid mixtures may not apply in lipid mixtures differing in their sterol chemical structure.     

Cholesterol affects spectrin-phospholipid interactions in a manner different from changes resulting from alterations in membrane fluidity due to fatty acyl chain composition

We previously showed that erythrocyte and brain spectrins bind phospholipid vesicles and monolayers prepared from phosphatidylethanolamine and phosphatidylserine and their mixtures with phosphatidylcholine (Review: A.F. Sikorski, B. Hanus-Lorenz, A. Jezierski, A. R. Dluzewski, Interaction of membrane skeletal proteins with membrane lipid domain, Acta Biochim. Polon. 47 (2000) 565). Here, we show how changes in the fluidity of the phospholipid monolayer affect spectrin-phospholipid interaction. The presence of up to 10%-20% cholesterol in the PE/PC monolayer facilitates the penetration of the monolayer by both types of spectrin. For monolayers constructed from mixtures of PI/PC and cholesterol, the effect of spectrins was characterised by the presence of two maxima (at 5 and 30% cholesterol) of surface pressure for erythroid spectrin, and a single maximum (at 20% cholesterol) for brain spectrin. The binding assay results indicated a small but easily detectable decrease in the affinity of erythrocyte spectrin for FAT-liposomes prepared from a PE/PC mixture containing cholesterol, and a 2- to 5-fold increase in maximal binding capacity (B(max)) depending on the cholesterol content. On the other hand, the results from experiments with a monolayer constructed from homogenous synthetic phospholipids indicated an increase in deltapi change with the increase in the fatty acyl chain length of the phospholipids used to prepare the monolayer. This was confirmed by the results of a pelleting experiment. Adding spectrins into the subphase of raft-like monolayers constructed from DOPC, SM and cholesterol (1/1/1) induced an increase in surface pressure. The deltapi change values were, however, much smaller than those observed in the case of a natural PE/PC (6/4) monolayer. An increased binding capacity for spectrins of liposomes prepared from a "raft-like" mixture of lipids could also be concluded from the pelleting assay. In conclusion, we suggest that the effect of membrane lipid fluidity on spectrin-phospholipid interactions is not simple but depends on how it is regulated, i.e., by cholesterol content or by the chemical structure of the membrane lipids.     

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.     

The lipopeptide antibiotic A21978C has a specific interaction with DMPC only in the presence of calcium ions

The A21978C group are lipopeptide antibiotics which kill Gram-positive bacteria only in the presence of calcium ions. The calcium requirement of the antibacterial activity of A21978C correlates well with an in vitro calcium-dependent insertion into phospholipid vesicles. In this paper the interaction of A21978C with phosphatidylcholine is investigated in mixed monomolecular films. The spontaneity of the antibiotic-lipid mixing was determined by calculating the free energy change. On a Ca2+ containing subphase there is a specific interaction between the components at all antibiotic-lipid ratios. This is not true on K+ subphases, where specific interactions never occur. On Mg2+ subphases specific interactions occur only in monolayers containing very little lipid. By analysing the fluorescence of the kynurenine residue we have followed the effects of two factors on the penetration of the antibiotic into lipid bilayer vesicles. Firstly, the phospholipid gel to liquid crystalline phase transition which in the absence of calcium leads to an exclusion of the antibiotic from the bilayer. This trend is completely reversed in the presence of Ca2+. Secondly, the role of this lipopeptide's lipid tail was clarified by use of a series of versions of increasing fatty acyl chain length. The results indicate that the interaction promoted by calcium is not simply a hydrophobic attraction between fatty acyl chains but is more likely to be a specific interaction between polar headgroups.     

Maximum solubility of cholesterol in phosphatidylcholine and phosphatidylethanolamine bilayers

In any lipid bilayer membrane, there is an upper limit on the cholesterol concentration that can be accommodated within the bilayer structure; excess cholesterol will precipitate as crystals of pure cholesterol monohydrate. This cholesterol solubility limit is a well-defined quantity. It is a first-order phase boundary in the phospholipid/cholesterol phase diagram. There are many different solubility limits in the literature, but no clear picture has emerged that can unify the disparate results. We have studied the effects that different sample preparation methods can have on the apparent experimental solubility limit. We find that artifactual demixing of cholesterol can occur during conventional sample preparation and that this demixed cholesterol may produce artifactual cholesterol crystals. Therefore, phospholipid/cholesterol suspensions which are prepared by conventional methods may manifest variable, falsely low cholesterol solubility limits. We have developed two novel preparative methods which are specifically designed to prevent demixing during sample preparation. For detection of the cholesterol crystals, X-ray diffraction has proven to be quantitative and highly sensitive. Experiments based on these methods yield reproducible and precise cholesterol solubility limits: 66 mol% for phosphatidylcholine (PC) bilayers and 51 mol% for phosphatidylethanolamine (PE) bilayers. We present evidence that these are true, equilibrium values. In contrast to the dramatic headgroup effect (PC vs. PE), acyl chain variations had no effect on the cholesterol solubility limit in four different PC/cholesterol mixtures.     

Sphingomyelin analogs with branched N-acyl chains: The position of branching dramatically affects acyl chain order and sterol interactions in bilayer membranes

Sphingolipids have been found to have single methyl branchings both in their long-chain base and in their N-linked acyl chains. In this study we determined how methyl-branching in the N-linked acyl chain of sphingomyelin (SM) affected their membrane properties. SM analogs with a single methyl-branching at carbon 15 (of a 17:0 acyl chain; anteiso) had a lower gel-liquid transition temperature as compared to an iso-branched SM analog. Phytanoyl SM (methyls at carbons 3, 7, 11 and 15) as well as a SM analog with a methyl on carbon 10 in a hexadecanoyl chain failed to show a gel-liquid transition above 10 °C. Only the two distally branched SM analogs (iso and anteiso) formed ordered domains with cholesterol in a 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) bilayer. However, domains formed by the branched SM analogs appeared to contain less sterol when compared to palmitoyl SM (PSM) as the saturated phospholipid. Sterol-enriched domains formed by the anteiso SM analog were also less stable against temperature than domains formed by PSM. Both the 10-methyl and phytanoyl SM analogs failed to form sterol-enriched domains in the POPC bilayer. Acyl chain branching weakened SM/sterol interactions markedly when compared to PSM, as also evidenced from the decreased affinity of cholestatrienol to bilayers containing branched SM analogs. Our results show that methyl-branching weakened intermolecular interactions in a position-dependent manner.