Cholesterol supports headgroup superlattice domain formation in fluid phospholipid/cholesterol bilayers

Fluorescence and Fourier transform infrared (FTIR) spectroscopic techniques were used to explore the effect of added cholesterol on the composition-dependent formation of putative phospholipid headgroup superlattices in fluid 1-palmitoyl-2-oleoyl-phosphatidylethanolamine/1-palmitoyl-2-oleoyl-phosphatidylcholine/cholesterol (POPE/POPC/CHOL) bilayers. Steady-state fluorescence anisotropy measurements of diphenylhexatriene (DPH) chain-labeled phosphatidylcholine (DPH-PC) revealed significant dips at several POPE-to-phospholipid mole fractions (X(PE)'s) when the cholesterol-to-lipid mole fraction (X(CHOL)) was fixed at 0.00, 0.35, 0.40, and 0.50. Most of the observed dips occur at or close to critical X(PE)'s predicted by the Headgroup Superlattice (SL) model, suggesting that phospholipid headgroups of different structures tend to adopt regular distributions even in the presence of cholesterol. Time-resolved fluorescence anisotropy measurements revealed that DPH-PC senses a disordered and highly mobile microenvironment in the POPE/POPC/CHOL bilayers at those critical X(PE)'s, indicating that this probe may partition to defect regions in the bilayers. The presence of coexisting packing defect regions and regularly distributed SL domains is a key feature predicted by the Headgroup SL model. Importantly, probe-free FTIR measurements of acyl chain C-H, interfacial carbonyl, and headgroup phosphate stretching peak frequencies revealed the presence of abrupt changes at X(PE)'s close to those observed in the fluorescence data. When X(PE) was varied from 0.60 to 0.72 and X(CHOL) from 0.34 to 0.46, a clear dip at the lipid composition coordinates (X(PE), X(CHOL)) approximately (0.68, 0.40) was observed in the three-dimensional surface plots of DPH-PC anisotropy as well as the carbonyl and phosphate stretching frequencies. The critical X(CHOL) at 0.40 agrees with the Cholesterol SL model, which assumes that cholesterol and phospholipid form SL domains at the lipid acyl chain level. In conclusion, this study provides evidence that cholesterol supports formation of phospholipid headgroup SLs in fluid state ternary lipid bilayers. The feasibility of the parallel existence of SLs at the lipid headgroup and acyl chain levels supports the relevance of the lipid SL model for the membranes of eukaryotic cells that typically contain significant amounts of cholesterol. We speculate that lipid SL formation may play a central role in the regulation of membrane lipid compositions, maintenance of organelle boundaries, and other crucial phenomena in those cells.     

Complementary matching in domain formation within lipid bilayers

Domain structure and formation in lipid bilayers are investigated by molecular dynamics simulations using a coarse-grained lipid model. The lipid bilayers consist of two lipid types that are identical except for tail length. At a temperature intermediate to the two melting temperatures of the constituent lipid types, gel domains spontaneously form from an initial random structure. The simulations reveal that the gel domains consist of both lipid types in a complementary match. If a long lipid is in the top monolayer, then a short lipid is underneath and vice versa. The gel domains have a larger thickness than the surrounding liquid phase. The thickness of the gel domains is close to that of the pure long lipid gel phase bilayers. However, since in the mixed gel domains the lipids are not tilted and in the pure gel phase the lipids are tilted, the two thicknesses are similar, and the underlying structure is therefore not distinguishable solely by thickness measurements.     

Phase behavior and the partitioning of caveolin-1 scaffolding domain peptides in model lipid bilayers

The membrane binding and model lipid raft interaction of synthetic peptides derived from the caveolin scaffolding domain (CSD) of the protein caveolin-1 have been investigated. CSD peptides bind preferentially to liquid-disordered domains in model lipid bilayers composed of cholesterol and an equimolar ratio of dioleoylphosphatidylcholine (DOPC) and brain sphingomyelin. Three caveolin-1 peptides were studied: the scaffolding domain (residues 83-101), a water-insoluble construct containing residues 89-101, and a water-soluble construct containing residues 89-101. Confocal and fluorescence microscopy investigation shows that the caveolin-1 peptides bind to the more fluid cholesterol-poor phase. The binding of the water-soluble peptide to lipid bilayers was measured using fluorescence correlation spectroscopy (FCS). We measured molar partition coefficients of 10(4) M(-1) between the soluble peptide and phase-separated lipid bilayers and 10(3) M(-1) between the soluble peptide and bilayers with a single liquid phase. Partial phase diagrams for our phase-separating lipid mixture with added caveolin-1 peptides were measured using fluorescence microscopy. The water-soluble peptide did not change the phase morphology or the miscibility transition in giant unilamellar vesicles (GUVs); however, the water-insoluble and full-length CSD peptides lowered the liquid-liquid melting temperature.     

Pore formation in phospholipid bilayers by amphiphilic cavitands

Five new cavitands were prepared that have four pendant n-undecyl chains and "headgroups" connected by 2-carbon spacers. The headgroups were ~OCH(2)CONH-Ala-OCH(3), 1; ~OCH(2)CONH-Phe-OCH(3), 2; ~OCH(2)CONH-Ala-OH, 3; ~OCH(2)CONH-Phe-OH, 4; and ~OCH(2)CONHCH(2)CH(2)-thyminyl, 5. Pore formation by each cavitand was studied by use of the planar bilayer conductance experiment. All five compounds were found to form pores in asolectin bialyer membranes. Compounds 1-3 behaved in a generally similar fashion and exhibited open-close dynamics. Compounds 4 and 5 formed pores more rapidly, were more dynamic, and led more quickly to membrane rupture. Differences in the ion transport activity are rationalized in terms of structure and aggregate cavitand assemblies.     

Phase behavior of model lipid bilayers

We investigated the phase behavior of double-tail lipids, as a function of temperature, headgroup interaction and tail length. At low values of the head-head repulsion parameter a(hh), the bilayer undergoes with increasing temperature the transitions from the subgel phase L(c) via the flat gel phase L(beta) to the fluid phase L(alpha). For higher values of a(hh), the transition from the L(c) to the L(alpha) phase occurs via the tilted gel phase L(beta)(') and the rippled phase P(beta)('). The occurrence of the L(beta)(') phase depends on tail length. We find that the rippled structure (P(beta)(')) occurs if the headgroups are sufficiently surrounded by water and that the ripple is a coexistence between the L(c) or L(beta)(') phase and the L(alpha) phase. The anomalous swelling, observed at the P(beta)(') --> L(alpha) transition, is not directly related to the rippled phase, but a consequence of conformational changes of the tails.     

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.     

Simulation of domain formation in DLPC-DSPC mixed bilayers

Binary mixtures of two phosphatidylcholines of different chain lengths are simulated in the bilayer state. We find a phase transition between a liquid state and a gel state at all concentrations. This phase transition is characterized by the area per lipid headgroup, the order parameter, and a change in dynamics. At concentrations with a majority of the longer lipid, we find phase separation into a gel and a liquid state in a small temperature window. This leads to a strong dynamic heterogeneity. Experimental phase transition temperatures are reproduced semiquantitatively. We see a clear shift in the phase transition to higher temperatures with increasing concentration of the longer lipid.     

The hydrophobic acyl-chain effect in the lipid domains appearance through phospholipid bilayers

An intermolecular interaction model for selective association processes of double-chain phospholipids in bilayer lipid membranes has been proposed, analysed and solved numerically. A large variety of binary mixtures of asymmetrical double-chain phospholipids with the cross-sectional areas of the polar headgroups a₁ = 40 Ų (the first component) and a₂ = 60 Ų (the second component) have been investigated. Changing the hydrophobic acyl-chain lengths of both mixture components, we found in all cases that the self-association probability (the association of like-pairs of phospholipids) of the first component in parallel alignment of the electric dipole moments of the polar headgroups is higher than the cross-association probability (the association of cross-pairs of phospholipids) and the self-association probability of the second component. This result is in good agreement with the experimental evidence that where the cross-sectional area of the polar headgroups matches the hydrocarbon chain-packing cross-sectional area (a 2Ξ 40 Ų), lipids possess a high tendency to aggregate into well packed bilayer structures with the acyl-chains oriented perpendicularly to the bilayer plane. Our theoretical data confirm that the double-chain phospholipids may associate themselves into anti-parallel alignment of the polar headgroups (P′₂₂) as well. The hydrophobic acyl-chain effect of phospholipids may modulate the distribution of lipid domains within bilayers that have a large variety of functional roles in cellular metabolism.     

Giant phospholipid/block copolymer hybrid vesicles: mixing behavior and domain formation

Lipids and block copolymers can be individually assembled into unsupported, spherical membranes (liposomes or polymersomes), each having their own particular benefits and limitations. Here we demonstrate the preparation of microscale, hybrid "lipopolymersomes" composed of the common lipid POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine) and the commercially available copolymer PBd-b-PEO (polybutadiene-b-poly(ethylene oxide)) with the goal of incorporating the advantageous qualities of the unitary systems into mixed-membrane capsules. We investigate the lipopolymersomes using confocal fluorescence microscopy and demonstrate that these hybrid membranes are well mixed on nanoscopic length scales within the permittable compositional windows for hybrid vesicle formation. We measure the intramembrane dynamics and mechanical properties of these hybrid membranes by fluorescence recovery after photobleaching (FRAP) and micropipet aspiration, respectively. For the first time, we demonstrate the demixing of lipid-rich and polymer-rich membrane domains within the same vesicle membrane. This is achieved by the biotinylation of one of the constituent species and cross linking with the protein NeutrAvidin. The resultant domain patterning is dependent upon which component carries the biotin functionality: cross linking of the copolymer species results in domains that ripen into a single, large, copolymer-rich island, and cross linking of the lipids yields many small, "spot-like", lipid-rich domains within a copolymer-rich matrix. We discuss these morphological differences in terms of the fluidity and mechanical properties of the membrane phases and the possible resultant interdomain interactions within the membrane. These heterogeneous hybrid lipopolymersomes could find applications in fields such as targeted delivery, controlled release, and environmental detection assays where these capsules possess the characteristics of biocompatible lipid membranes combined with enhanced mechanical strength and stability from the copolymer matrix.     

Effect of dipolar moments in domain sizes of lipid bilayers and monolayers

Lipid domains are found in systems such as multicomponent bilayer membranes and single component monolayers at the air-water interface. It was shown by Keller et al. [J. Phys. Chem. 91, 6417 (1987)] that in monolayers, the size of the domains results from balancing the line tension, which favors the formation of a large single circular domain, against the electrostatic cost of assembling the dipolar moments of the lipids. In this paper, we present an exact analytical expression for the electric potential, ion distribution, and electrostatic free energy for different problems consisting of three different slabs with different dielectric constants and Debye lengths, with a circular homogeneous dipolar density in the middle slab. From these solutions, we extend the calculation of domain sizes for monolayers to include the effects of finite ionic strength, dielectric discontinuities (or image charges), and the polarizability of the dipoles and further generalize the calculations to account for domains in lipid bilayers. In monolayers, the size of the domains is dependent on the different dielectric constants but independent of ionic strength. In asymmetric bilayers, where the inner and outer leaflets have different dipolar densities, domains show a strong size dependence with ionic strength, with molecular-sized domains that grow to macroscopic phase separation with increasing ionic strength. We discuss the implications of the results for experiments and briefly consider their relation to other two dimensional systems such as Wigner crystals or heteroepitaxial growth.     

Pore formation in phospholipid bilayers by branched-chain pyrogallol[4]arenes

Pyrogallolarenes are tetrameric macrocycles that form from 1,2,3-trihydroxybenzene and aldehydes under acidic conditions. When 2-ethylbutanal or 2-propylpentanal was so treated, the branched-chain pyrogallolarenes crystallized as nanotubes or bilayers, respectively. When the behavior of each compound was assessed by using the planar bilayer conductance method, pore formation was observed. The properties of the pores were significantly different from each other, probably reflecting different types of pore organization within the membrane.     

Domain formation in lipid bilayers probed by two-dimensional infrared spectroscopy

Two-dimensional infrared (2D-IR) spectroscopy has been used to probe structure and dynamics in binary sphingomyelin/phospholipid liposomes. The liposomes consist of 1-palmitoyl-2-linoleyl phosphatidylcholine (PLPC) and sphingomyelin (SPM) in the ratio 1:1. The diagonal part of the 2D-IR spectra shows two bands which are due to amide I of SPM and to the carbonyl moieties of PLPC. The diagonal components of the 2D-IR spectra reveal a difference in the molecular dynamics. The presence of off-diagonal cross-peaks indicates the occurrence of intermolecular structural correlation. The intensity of the cross-peaks is consistent with segregation of two lipid components into PLPC and SPM molecular domains.     

Phase and mixing behavior in two-component lipid bilayers: a molecular dynamics study in DLPC/DSPC mixtures

Phase and mixing behavior of dilauroylphosphatidylcholine (DLPC)/distearoylphosphatidylcholine (DSPC) lipid mixtures are studied by molecular dynamics simulations with use of a coarse-grained model over a wide range of concentrations. The results reveal that phase transformations from the fluid to the gel state can be followed over a microsecond time scale. The changes in structure suggest regions of phase coexistence allowing us to outline the entire phase diagram for this lipid mixture using a molecular based model. We show that simulations yield good agreement with the experimental phase diagram. We also address the effect of macroscopic phase separation on the determination of the transition temperature, different leaflet composition, and finite size effects. This study may have implications on lateral membrane organization and the associated processes dependent on these membrane regions on different time and length scales.     

Simulation studies of pore and domain formation in a phospholipid monolayer

Despite extensive study the phase behavior of phospholipid monolayers at an air-water interface is still not fully understood. In particular recent vibrational sum-frequency generation (VSFG) spectra of DPPC monolayers as a function of area density show a sharp transition in the order of the lipid chains at 1.10 nm2/molecule. This is in a region where the lateral pressure as a function of area is effectively constant. We have investigated the nature of this transition by studying the phase behavior of DPPC monolayers as a function of area density using molecular-dynamics simulations. The changes in order within the monolayer as a function of area density correlate well with the experimental signal. At 0.58 nm2/molecule we observe the onset of lateral separation of highly ordered and disordered lipids, indicating the coexistence of a gel-like liquid condensed and a fluidlike liquid expanded phase. At 0.97 nm2/molecule the monolayer ruptures, marking the onset of the liquid-gas (G) coexistence region. This is much earlier than suggested by fluorescence microscopy results and implies that at the point of rupture, the initial pores have an equilibrium size smaller than approximately 500 nm in diameter. The rupture of the monolayer leads to a sharp increase in the overall lipid order that explains the sharp transition observed in the VSFG measurements. VSFG measurements thus may represent a sensitive means to determine the onset of the liquid-gas (G) coexistence region for such systems.     

Air-exposure technique for the formation of artificial lipid bilayers in microsystems

To develop a reliable method for on-chip bilayer lipid membrane (BLM) formation, which could be employed for use in a biosensor array platform, a polymer microfluidic device has been constructed, and the formation of suspended BLMs within it has been investigated. A simple, yet reproducible BLM formation protocol has been developed, in which a brief air-exposure period is employed to induce the rapid thinning of an initially thick lipid-solvent layer. The technique is rapid, reproducible, and amenable to the simple injection of proteins or analytes, as well as to buffer exchange on both sides of the membrane. Scaling up the technique for use in an array platform is also straightforward, the simultaneous formation of three individually addressable BLMs being demonstrated.     

Spontaneous formation of asymmetric lipid bilayers by adsorption of vesicles

We have investigated the adsorption of phospholipid mixtures using neutron reflection. Small sonicated unilamellar vesicles (SUV) composed of DOPC and d(62)-DPPC were incubated at 50 degrees C in contact with a silica surface using a method commonly employed to form supported model membranes. The composition of the mixed supported bilayer was found to be substantially different from that of the bulk vesicles in a direction indicating a higher affinity of DPPC for the silica surface. Formation of an asymmetric bilayer arrangement was also discovered in all the cases studied. DPPC tended to dominate the composition of the leaflet next to silica, while the outer leaflet was generally closer to the bulk composition. The supported bilayers also exhibited increasing interfacial roughness in the outer membrane leaflet in the region of the DOPC-DPPC gel-liquid immiscibility region. To our knowledge, this is the first time that both the structure and the absolute composition of a mixed-lipid supported bilayer have been resolved, and the results raise a number of questions regarding the adsorption of vesicles and the properties of supported bilayers, which are discussed in terms of the bulk phase diagram of DOPC and DPPC.     

Effect of acidity on the formation of solvent-free lipid bilayers

Glasses with hydrophobized surface are used to form “dry” bilayer lipid membranes (BLM). The contact angle on the silaned glass plates reaches 125°. Partitions of this kind, as opposed to polymer films, make a system more resistant to heat oscillations and mechanical perturbations generated when studying membranes properties. The specific capacitance of BLM (0.86 ± 0.04 μF cm^?2) testifies to the solvent absence in the bilayer. The dramatic drop of the capacitance (area) of the dry membranes with increasing pH is caused by a greater adsorption of lipid molecules on the hydrophobic substrate, which is probably due to changing conformation of their polar “head.”     

Simulation of pore formation in lipid bilayers by mechanical stress and electric fields

We describe computer simulations of pore formation and membrane rupture of phospholipid bilayers under mechanical and electrical stress. On the nanosecond simulation time scale, pores are induced by a lateral pressure exceeding -200 bar or by an applied electric field of 0.5 V/nm.     

Penetration and saturation of lysozyme in phospholipid bilayers

We show that the combination of X-ray reflectivity, tryptophan fluorescence spectrum, and fluorescence quenching by bromine provides a useful tool to probe the location of lysozyme in lipid bilayers. To this end, we prepare lamellar complexes composed of phospholipids and lysozyme on solid surfaces using a solution-casting method. The proteins lie spontaneously between adjacent bilayers in the complexes. The results indicate that lysozyme may penetrate into the lipid bilayers. But the penetration depth is very shallow, and the tryptophan residues do not penetrate beyond the interface between the hydrocardon core and the headgroup region of the lipid bilayer. The penetration becomes saturated when more proteins are incorporated into the lamellar complex. The excess proteins stay in the interlamellar aqueous layers.     

Probing the hydration of lipid bilayers using a solvent isotope effect on phospholipid mixing

Nearest-neighbor recognition experiments, which have been carried out using exchangeable dimers derived from 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine,and 1-palmitoyl-2-oleoyl-sn-glycerophosphoethanolamine, indicate that replacement of H2O by D2O can significantly influence phospholipid mixing, but only in bilayers that are saturated and devoid of cholesterol. These findings, together with those of previous electron spin resonance spin-labeling studies,indicate that mammalian membranes, which are rich in cholesterol and unsaturated phospholipids, are ideal hydrophobic barriers.