Formation of Raft-Like Assemblies within Clusters of Influenza Hemagglutinin Observed by MD Simulations

The association of hemagglutinin (HA) with lipid rafts in the plasma membrane is an important feature of the assembly process of influenza virus A. Lipid rafts are thought to be small, fluctuating patches of membrane enriched in saturated phospholipids, sphingolipids, cholesterol and certain types of protein. However, raft-associating transmembrane (TM) proteins generally partition into Ld domains in model membranes, which are enriched in unsaturated lipids and depleted in saturated lipids and cholesterol. The reason for this apparent disparity in behavior is unclear, but model membranes differ from the plasma membrane in a number of ways. In particular, the higher protein concentration in the plasma membrane may influence the partitioning of membrane proteins for rafts. To investigate the effect of high local protein concentration, we have conducted coarse-grained molecular dynamics (CG MD) simulations of HA clusters in domain-forming bilayers. During the simulations, we observed a continuous increase in the proportion of raft-type lipids (saturated phospholipids and cholesterol) within the area of membrane spanned by the protein cluster. Lateral diffusion of unsaturated lipids was significantly attenuated within the cluster, while saturated lipids were relatively unaffected. On this basis, we suggest a possible explanation for the change in lipid distribution, namely that steric crowding by the slow-diffusing proteins increases the chemical potential for unsaturated lipids within the cluster region. We therefore suggest that a local aggregation of HA can be sufficient to drive association of the protein with raft-type lipids. This may also represent a general mechanism for the targeting of TM proteins to rafts in the plasma membrane, which is of functional importance in a wide range of cellular processes.     

Phospholipid Chain Interactions with Cholesterol Drive Domain Formation in Lipid Membranes

Cholesterol is a key component of eukaryotic membranes, but its role in cellular biology in general and in lipid rafts in particular remains controversial. Model membranes are used extensively to determine the phase behavior of ternary mixtures of cholesterol, a saturated lipid, and an unsaturated lipid with liquid-ordered and liquid-disordered phase coexistence. Despite many different experiments that determine lipid-phase diagrams, we lack an understanding of the molecular-level driving forces for liquid phase coexistence in bilayers with cholesterol. Here, we use atomistic molecular dynamics computer simulations to address the driving forces for phase coexistence in ternary lipid mixtures. Domain formation is directly observed in a long-timescale simulation of a mixture of 1,2-distearoyl-sn-glycero-3-phosphocholine, unsaturated 1,2-dilinoleoyl-sn-glycero-3-phosphocholine, and cholesterol. Free-energy calculations for the exchange of the saturated and unsaturated lipids between the ordered and disordered phases give insight into the mixing behavior. We show that a large energetic contribution to domain formation is favorable enthalpic interactions of the saturated lipid in the ordered phase. This favorable energy for forming an ordered, cholesterol-rich phase is opposed by a large unfavorable entropy. Martini coarse-grained simulations capture the unfavorable free energy of mixing but do not reproduce the entropic contribution because of the reduced representation of the phospholipid tails. Phospholipid tails and their degree of unsaturation are key energetic contributors to lipid phase separation.     

How cholesterol is distributed between monolayers in asymmetric lipid membranes

The distribution of cholesterol in asymmetric lipid bilayers was studied by extensive coarse-grained molecular dynamics simulations. The effects of the lipid head group charge, acyl chain saturation, spontaneous membrane curvature and surface tension of the membrane were investigated. Four asymmetric bilayers containing DOPC, DOPS, DSPC or DSPS lipids were simulated on a time scale extended to tens of microseconds. We show that cholesterol strongly prefers anionic lipids to neutral and saturated lipid tails to unsaturated with a distribution ratio of ~0.7 in neutral/anionic bilayers and of ~0.4 in unsaturated/saturated bilayers. Multiple flip-flop transitions of cholesterol were observed directly, and their mean times ranged from 80 to 250?ns. It was shown that the distribution of cholesterol in the asymmetric membrane depends not only on the type of lipid, but also on the local membrane curvature and the surface tension. The membrane curvature enhances the influence of the lipid head groups on cholesterol distribution, while non-optimal surface tension caused by different areas per lipid in different monolayers increases the effect of the lipid tail saturation. It was clearly seen that the monolayers of asymmetric bilayers are interdependent. Mean distances from the bilayer center to cholesterol molecules depend not only on the type of the lipid in the considered monolayer but also on the composition of the opposite monolayer.     

Partitioning of lipidated peptide sequences into liquid-ordered lipid domains in model and biological membranes.

We have used a fluorescence assay and detergent fractionation to examine the partitioning of different fluorescent lipidated peptides, with sequences and lipid substituents matching those found in various classes of lipidated cellular proteins, into liquid-ordered (raft-like) domains in lipid bilayers. Peptides incorporating isoprenyl groups, or multiple unsaturated acyl chains, show negligible affinity for liquid-ordered domains in mixed-phase liquid-ordered/liquid-disordered (l(o)/l(d)) bilayers composed of dipalmitoylphosphatidylcholine, a spin-labeled unsaturated phosphatidylcholine, and cholesterol. By contrast, peptides incorporating multiple S- and/or N-acyl chains, or a cholesterol residue plus an N-terminal palmitoyl chain, show significant partitioning into liquid-ordered domains under the same conditions. Interestingly, the affinity of a lipidated peptide for l(o) domains can be strongly influenced, not only by the structures of the lipid substituents but also by the nature and the positions of their attachment to the peptide chain. These results are well correlated with those obtained from parallel assays based on low-temperature detergent fractionation. Using the latter approach, we further demonstrate that a truly minimal l(o) domain partitioning motif [myristoylGlyCys(palmitoyl)-] can mediate efficient incorporation into the "raft" fraction of COS-7 cell membranes.     

Separation of liquid phases in giant vesicles of ternary mixtures of phospholipids and cholesterol

We use fluorescence microscopy to directly observe liquid phases in giant unilamellar vesicles. We find that a long list of ternary mixtures of high melting temperature (saturated) lipids, low melting temperature (usually unsaturated) lipids, and cholesterol produce liquid domains. For one model mixture in particular, DPPC/DOPC/Chol, we have mapped phase boundaries for the full ternary system. For this mixture we observe two coexisting liquid phases over a wide range of lipid composition and temperature, with one phase rich in the unsaturated lipid and the other rich in the saturated lipid and cholesterol. We find a simple relationship between chain melting temperature and miscibility transition temperature that holds for both phosphatidylcholine and sphingomyelin lipids. We experimentally cross miscibility boundaries both by changing temperature and by the depletion of cholesterol with beta-cyclodextrin. Liquid domains in vesicles exhibit interesting behavior: they collide and coalesce, can finger into stripes, and can bulge out of the vesicle. To date, we have not observed macroscopic separation of liquid phases in only binary lipid mixtures.     

Permeabilization of raft-containing lipid vesicles by delta-lysin: a mechanism for cell sensitivity to cytotoxic peptides

Delta-lysin is a linear, 26-residue peptide that adopts an alpha-helical, amphipathic structure upon binding to membranes. Delta-lysin preferentially binds to mammalian cell membranes, the outer leaflets of which are enriched in sphingomyelin, cholesterol, and unsaturated phosphatidylcholine. Mixtures including these lipids have been shown to exhibit separation between liquid-disordered (l(d)) and liquid-ordered (l(o)) domains. When rich in sphingomyelin and cholesterol, these ordered domains have been called lipid "rafts". We found that delta-lysin binds poorly to the l(o) (raft) domains; therefore, in mixed-phase lipid vesicles, delta-lysin preferentially binds to the l(d) domains. This leads to the concentration of delta-lysin in l(d) domains, enhancing peptide aggregation and, consequently, the rate of peptide-induced dye efflux from lipid vesicles. The efficient lysis of eukaryotic cells by delta-lysin can thus be attributed not to specific delta-lysin-cholesterol or delta-lysin-sphingomyelin interactions but, rather, to the exclusion of delta-lysin from ordered rafts. The degree to which the kinetics of dye efflux are enhanced in mixed-phase vesicles over those observed in pure, unsaturated phosphatidylcholine vesicles directly reflects the amount of l(d) phase present in mixed-phase systems. This effect of lipid domains has broader consequences, beyond the hemolytic efficiency of delta-lysin. We discuss the hypothesis that bacterial sensitivity to antimicrobial peptides may be determined by a similar mechanism.     

Structural dynamics of Smoothened (SMO) in the ciliary membrane and its interaction with membrane lipids

The Smoothened receptor (SMO, a 7 pass transmembrane domain, Class F GPCR family protein) plays a crucial role in the Hedgehog (HH) signaling pathway, which is involved in embryonic development and is implicated in various types of cancer throughout the animal kingdom. In the absence of HH signaling, SMO is inhibited by Patched 1 (PTC1; a 12 pass transmembrane domain protein), which is localized in the primary cilia. HH binding leads to the dislocation of PTC1 from the cilia, thus making way for SMO to localize in the primary cilia, as an essential prerequisite for its activation. We have carried out MARTINI coarse-grained molecular dynamics simulations of SMO in POPC and in ciliary membrane models, respectively, to study the interactions of SMO with cholesterol and other lipid molecules in the ciliary membrane, and to gain molecular-level insights into the role of the primary cilia in shaping the functional dynamics of SMO. We are able to identify the interaction of membrane cholesterols with definite sites and domains within SMO and relate them with known cholesterol-binding sequence and structure motifs. We show that cholesterol interactions with the transmembrane domain TMD, unlike those with the cysteine-rich domain (CRD) and the intracellular domain (ICD), are through residues belonging to known cholesterol-binding motifs. Notably, a few persistent interactions of cholesterol with lower TM cholesterol-binding domains are governed by the presence of multiple cholesterol-binding motifs. These analyses have also helped to identify and define a strict cholesterol consensus motif (CCM), which may well steer cholesterol into the hitherto identified binding sites within the TMD of SMO. We have also reported the interaction of phosphatidylinositol 4-phosphate with the intracellular region of transmembrane (TM) helices (TM1, TM3, TM4, and TM5), intracellular loop1, helix8, and Arg/Lys clusters of the ICD. Structural analysis of SMO domains shows significant changes in the CRD and ICD, during the course of the simulation. Further detailed analysis of the dynamics of the TMD reveals the movements of TM5, TM6, and TM7, linked with the helix8, which are possibly involved in shaping the conformational disposition of the ICD. The movement of these TM helices could possibly be a consequence of interactions involving the extracellular domain and extracellular loops. In addition, our analysis also shows that phosphatidylinositol-4-phosphate (PI4P), along with some ICD cholesterols, are implicated in anchoring SMO in the membrane.     

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.     

The Mechanism of Collapse of Heterogeneous Lipid Monolayers

Collapse of homogeneous lipid monolayers is known to proceed via wrinkling/buckling, followed by folding into bilayers in water. For heterogeneous monolayers with phase coexistence, the mechanism of collapse remains unclear. Here, we investigated collapse of lipid monolayers with coexisting liquid-liquid and liquid-solid domains using molecular dynamics simulations. The MARTINI coarse-grained model was employed to simulate monolayers of ∼80 nm in lateral dimension for 10–25 μs. The monolayer minimum surface tension decreased in the presence of solid domains, especially if they percolated. Liquid-ordered domains facilitated monolayer collapse due to the spontaneous curvature induced at a high cholesterol concentration. Upon collapse, bilayer folds formed in the liquid (disordered) phase; curved domains shifted the nucleation sites toward the phase boundary. The liquid (disordered) phase was preferentially transferred into bilayers, in agreement with the squeeze-out hypothesis. As a result, the composition and phase distribution were altered in the monolayer in equilibrium with bilayers compared to a flat monolayer at the same surface tension. The composition and phase behavior of the bilayers depended on the degree of monolayer compression. The monolayer-bilayer connection region was enriched in unsaturated lipids. Percolation of solid domains slowed down monolayer collapse by several orders of magnitude. These results are important for understanding the mechanism of two-to-three-dimensional transformations in heterogeneous thin films and the role of lateral organization in biological membranes. The study is directly relevant for the function of lung surfactant, and can explain the role of nanodomains in its surface activity and inhibition by an increased cholesterol concentration.     

Interplay of unsaturated phospholipids and cholesterol in membranes: effect of the double-bond position

The structural and dynamical properties of lipid membranes rich in phospholipids and cholesterol are known to be strongly affected by the unsaturation of lipid acyl chains. We show that not only unsaturation but also the position of a double bond has a pronounced effect on membrane properties. We consider how cholesterol interacts with phosphatidylcholines comprising two 18-carbon long monounsaturated acyl chains, where the position of the double bond is varied systematically along the acyl chains. Atomistic molecular dynamics simulations indicate that when the double bond is not in contact with the cholesterol ring, and especially with the C18 group on its rough β-side, the membrane properties are closest to those of the saturated bilayer. However, any interaction between the double bond and the ring promotes membrane disorder and fluidity. Maximal disorder is found when the double bond is located in the middle of a lipid acyl chain, the case most commonly found in monounsaturated acyl chains of phospholipids. The results suggest a cholesterol-mediated lipid selection mechanism in eukaryotic cell membranes. With saturated lipids, cholesterol promotes the formation of highly ordered raft-like membrane domains, whereas domains rich in unsaturated lipids with a double bond in the middle remain highly fluid despite the presence of cholesterol.     

Fluorescence-based evaluation of the partitioning of lipids and lipidated peptides into liquid-ordered lipid microdomains: a model for molecular partitioning into "lipid rafts"

A fluorescence-quenching assay is described that can directly monitor the relative extents of partitioning of different but structurally homologous fluorescent molecules into liquid-ordered (l(o)) domains in lipid vesicles exhibiting liquid-ordered/liquid-disordered (l(o)/l(d)) phase coexistence. Applying this assay to a series of bimane-labeled diacyl phospholipid probes in cholesterol-containing ternary lipid mixtures exhibiting l(o)/l(d) phase separation, we demonstrate that partitioning into l(o)-phase domains is negligible for diunsaturated species and greatest for long-chain disaturated species. These conclusions agree well with those derived from previous studies of the association of lipids and lipid-anchored molecules with l(o)-phase domains, using methods based on the isolation of a detergent-insoluble fraction from model or biological membranes at low temperatures. However, we also find that monounsaturated and shorter-chain saturated species partition into l(o) phases with significant, albeit modest affinities, and that the level of partitioning of these latter species into l(o)-phase domains is significantly underestimated (relative to that of their long-chain saturated counterparts) by the criterion of low-temperature detergent insolubility. Finally, applying the fluorescence-quenching method to a family of lipid-modified peptides, we demonstrate that the S-palmitoyl/S-isoprenyl dual-lipidation motif found in proteins such as H- and N-ras and yeast Ste18p does not promote significant association with l(o) domains in l(o)/l(d)-phase-separated bilayers.     

A helix heterodimer in a lipid bilayer: prediction of the structure of an integrin transmembrane domain via multiscale simulations

Dimerization of transmembrane (TM) α helices of membrane receptors plays a key role in signaling. We show that molecular dynamics simulations yield models of integrin TM helix heterodimers, which agree well with available NMR structures. We use?a multiscale simulation approach, combining coarse-grained and subsequent atomistic simulation, to model the dimerization of wild-type (WT) and mutated sequences of the αIIb and β3 integrin TM helices. The WT helices formed a stable, right-handed dimer with the same helix-helix interface as in the published NMR structure (PDB: 2K9J). In contrast, the presence of disruptive mutations perturbed the interface between the helices, altering the conformational stability of the dimer. The αIIb/β3 interface was more flexible than that of, e.g., glycophorin A. This is suggestive of a role for alternative packing modes of the TM helices in transbilayer signaling.     

Fusion activity of HIV gp41 fusion domain is related to its secondary structure and depth of membrane insertion in a cholesterol-dependent fashion

The human immunodeficiency virus (HIV) gp41 fusion domain plays a critical role in membrane fusion during viral entry. A thorough understanding of the relationship between the structure and the activity of the fusion domain in different lipid environments helps to formulate mechanistic models on how it might function in mediating membrane fusion. The secondary structure of the fusion domain in small liposomes composed of different lipid mixtures was investigated by circular dichroism spectroscopy. The fusion domain formed an α-helix in membranes containing less than 30?mol% cholesterol and formed β-sheet secondary structure in membranes containing ≥30?mol% cholesterol. EPR spectra of spin-labeled fusion domains also indicated different conformations in membranes with and without cholesterol. Power saturation EPR data were further used to determine the orientation and depth of α-helical fusion domains in lipid bilayers. Fusion and membrane perturbation activities of the gp41 fusion domain were measured by lipid mixing and contents leakage. The fusion domain fused membranes in both its helical form and its β-sheet form. High cholesterol, which induced β-sheets, promoted fusion; however, acidic lipids, which promoted relatively deep membrane insertion as an α-helix, also induced fusion. The results indicate that the structure of the HIV gp41 fusion domain is plastic and depends critically on the lipid environment. Provided that their membrane insertion is deep, α-helical and β-sheet conformations contribute to membrane fusion.     

Defining raft domains in the plasma membrane

Many plasma membrane (PM) functions depend on the cholesterol concentration in the PM in strikingly nonlinear, cooperative ways: fully functional in the presence of physiological cholesterol levels (35~45 mol%), and nonfunctional below 25 mol% cholesterol; namely, still in the presence of high concentrations of cholesterol. This suggests the involvement of cholesterol‐based complexes/domains formed cooperatively. In this review, by examining the results obtained by using fluorescent lipid analogs and avoiding the trap of circular logic, often found in the raft literature, we point out the fundamental similarities of liquid‐ordered (Lo)‐phase domains in giant unilamellar vesicles, Lo‐phase‐like domains formed at lower temperatures in giant PM vesicles, and detergent‐resistant membranes: these domains are formed by cooperative interactions of cholesterol, saturated acyl chains, and unsaturated acyl chains, in the presence of >25 mol% cholesterol. The literature contains evidence, indicating that the domains formed by the same basic cooperative molecular interactions exist and play essential roles in signal transduction in the PM. Therefore, as a working definition, we propose that raft domains in the PM are liquid‐like molecular complexes/domains formed by cooperative interactions of cholesterol with saturated acyl chains as well as unsaturated acyl chains, due to saturated acyl chains' weak multiple accommodating interactions with cholesterol and cholesterol's low miscibility with unsaturated acyl chains and TM proteins. Molecules move within raft domains and exchange with those in the bulk PM. We provide a logically established collection of fluorescent lipid probes that preferentially partition into raft and non‐raft domains, as defined here, in the PM.     

Line Active Hybrid Lipids Determine Domain Size in Phase Separation of Saturated and Unsaturated Lipids

A simple model of the line activity of a hybrid lipid (e.g., POPC) with one fully saturated chain and one partially unsaturated chain demonstrates that these lipids preferentially pack at curved interfaces between phase-separated saturated and unsaturated domains. We predict that the domain sizes typically range from tens to hundreds of nm, depending on molecular interactions and parameters such as molecular volume and area per headgroup in the bulk fluid phase. The role of cholesterol is taken into account by an effective change in the headgroup areas and the domain sizes are predicted to increase with cholesterol concentration.     

Role of cholesterol in the formation and nature of lipid rafts in planar and spherical model membranes

Sterols play a crucial regulatory and structural role in the lateral organization of eukaryotic cell membranes. Cholesterol has been connected to the possible formation of ordered lipid domains (rafts) in mammalian cell membranes. Lipid rafts are composed of lipids in the liquid-ordered (l(o)) phase and are surrounded with lipids in the liquid-disordered (l(d)) phase. Cholesterol and sphingomyelin are thought to be the principal components of lipid rafts in cell and model membranes. We have used fluorescence microscopy and fluorescence recovery after photobleaching in planar supported lipid bilayers composed of porcine brain phosphatidylcholine (bPC), porcine brain sphingomyelin (bSM), and cholesterol to map the composition-dependence of l(d)/l(o) phase coexistence. Cholesterol decreases the fluidity of bPC bilayers, but disrupts the highly ordered gel phase of bSM, leading to a more fluid membrane. When mixed with bPC/bSM (1:1) or bPC/bSM (2:1), cholesterol induces the formation of l(o) phase domains. The fraction of the membrane in the l(o) phase was found to be directly proportional to the cholesterol concentration in both phospholipid mixtures, which implies that a significant fraction of bPC cosegregates into l(o) phase domains. Images reveal a percolation threshold, i.e., the point where rafts become connected and fluid domains disconnected, when 45-50% of the total membrane is converted to the l(o) phase. This happens between 20 and 25 mol % cholesterol in 1:1 bPC/bSM bilayers and between 25 and 30 mol % cholesterol in 2:1 bPC/bSM bilayers at room temperature, and at approximately 35 mol % cholesterol in 1:1 bPC/bSM bilayers at 37 degrees C. Area fractions of l(o) phase lipids obtained in multilamellar liposomes by a fluorescence resonance energy transfer method confirm and support the results obtained in planar lipid bilayers.     

The Mechanism of Collapse of Heterogeneous Lipid Monolayers

Collapse of homogeneous lipid monolayers is known to proceed via wrinkling/buckling, followed by folding into bilayers in water. For heterogeneous monolayers with phase coexistence, the mechanism of collapse remains unclear. Here, we investigated collapse of lipid monolayers with coexisting liquid-liquid and liquid-solid domains using molecular dynamics simulations. The MARTINI coarse-grained model was employed to simulate monolayers of ∼80 nm in lateral dimension for 10–25 μs. The monolayer minimum surface tension decreased in the presence of solid domains, especially if they percolated. Liquid-ordered domains facilitated monolayer collapse due to the spontaneous curvature induced at a high cholesterol concentration. Upon collapse, bilayer folds formed in the liquid (disordered) phase; curved domains shifted the nucleation sites toward the phase boundary. The liquid (disordered) phase was preferentially transferred into bilayers, in agreement with the squeeze-out hypothesis. As a result, the composition and phase distribution were altered in the monolayer in equilibrium with bilayers compared to a flat monolayer at the same surface tension. The composition and phase behavior of the bilayers depended on the degree of monolayer compression. The monolayer-bilayer connection region was enriched in unsaturated lipids. Percolation of solid domains slowed down monolayer collapse by several orders of magnitude. These results are important for understanding the mechanism of two-to-three-dimensional transformations in heterogeneous thin films and the role of lateral organization in biological membranes. The study is directly relevant for the function of lung surfactant, and can explain the role of nanodomains in its surface activity and inhibition by an increased cholesterol concentration.     

Leukocyte integrin αLβ2 transmembrane association dynamics revealed by coarse-grained molecular dynamics simulations

Integrins are transmembrane (TM) proteins that mediate bidirectional mechanical signaling between the extracellular matrix and the cellular cytoskeletal network. Each integrin molecule consists of non-covalently associated α- and β-subunits, with each subunit consisting of a large ectodomain, a single-pass TM helix, and a short cytoplasmic tail. Previously we found evidence for a polar interaction (hydrogen bond) in the outer membrane clasp (OMC) of the leukocyte integrin αLβ2 TMs that is absent in the platelet integrin αIIβ3 OMC. Here, we compare the self-assembly dynamics of αLβ2 and αIIβ3 TM helices in a model membrane using coarse-grained molecular dynamics simulations. We found that although αIIβ3 TM helices associate more easily, packing is suboptimal. In contrast, αLβ2 TM helices achieve close-to-optimal packing. This suggests that αLβ2 TM packing is more specific, possibly due to the interhelix hydrogen bond. Theoretical association free energy profiles show a deeper minimum at a smaller helix-helix separation for αLβ2 compared with αIIβ3. The αIIβ3 profile is also more rugged with energetic barriers whereas that of αLβ2 is almost without barriers. Disruption of the interhelix hydrogen bond in αLβ2 via the β2T686G mutation results in poorer association and a similar profile as αIIβ3. The OMC polar interaction in αLβ2 thus plays a significant role in the packing of the TM helices.     

Differential ability of cholesterol-enriched and gel phase domains to resist benzyl alcohol-induced fluidization in multilamellar lipid vesicles

Benzyl alcohol (BA) has a well-known fluidizing effect on both artificial and cellular membranes. BA is also likely to modulate the activities of certain membrane proteins by decreasing the membrane order. This phenomenon is presumably related to the ability of BA to interrupt interactions between membrane proteins and the surrounding lipids by fluidizing the lipid bilayer. The components of biological membranes are laterally diversified into transient assemblies of varying content and order, and many proteins are suggested to be activated or inactivated by their localization in or out of membrane domains displaying different physical phases. We studied the ability of BA to fluidize artificial bilayer membranes representing liquid-disordered, cholesterol-enriched and gel phases. Multilamellar vesicles were studied by steady-state fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene and trans-parinaric acid, which display different phase partitioning. Domains of different degree of order and thermal stability showed varying abilities to resist fluidization by BA. In bilayers composed of mixtures of an unsaturated phosphatidylcholine, a saturated high melting temperature lipid (sphingomyelin or phosphatidylcholine) and cholesterol, BA fluidized and lowered the melting temperature of the ordered and gel phase domains. In general, cholesterol-enriched domains were more resistant to BA than pure gel phase domains. In contrast, bilayers containing high melting temperature gel phase domains containing a ceramide or a galactosylceramide proved to be the most effective in resisting fluidization. The results of our study suggest that the ability of BA to affect the fluidity and lateral organization of the membranes was dependent on the characteristic features of the membrane compositions studied and related to the intermolecular cohesion in the domains.