Calculation of intermolecular interaction strengths in the P beta' phase in lipid bilayers. Implications for theoretical models.

The existence of the P beta' phase in certain lipid bilayers is evidence that molecular interactions between lipids are capable of producing unusual large-scale structures at or near biological conditions. The problem of identifying the specific intermolecular interactions responsible for the structures requires construction of theoretical models capable of clear predictions of the observable consequences of postulated intermolecular interactions. To this end we have carried out a twofold modeling effort aimed at understanding the ripple phase. First, we have performed detailed numerical calculations of potential energies of interaction between pairs and triplets of lipid molecules having different chain tilt angles and relative vertical alignments. The calculations support the notion that chain tilting in the gel phase is a result of successive 3-5-A displacements of neighboring molecules perpendicular to the bilayer plane rather than long-range cooperative chain tilting. Secondly, we have used these results as a guide to formulate a new lattice model for lipid bilayer condensed phases. The new model is less complex than our earlier model and it includes interactions which are, based on the energy calculations, more likely to be responsible for the ripple phase. In a certain limit the model maps onto the chiral clock model, a model of much interest in condensed matter theory. In this limit the model exhibits a chain-tilted ordered phase followed by (as temperature increases) a modulated phase followed by a disordered phase. Within this limit we discuss the properties of the model and compare structures of the modulated phase exhibited by the model with experimental data for the P beta' phase in lipid bilayers.     

Effect of membrane characteristics on phase separation and domain formation in cholesterol-lipid mixtures

We examine, using an analytical mean-field model, the distribution of cholesterol in a lipid bilayer. The model accounts for the perturbation of lipid packing induced by the embedded cholesterol, in a manner similar to that of transmembrane proteins. We find that the membrane-induced interactions between embedded cholesterol molecules vary as a function of the cholesterol content. Thus, the effective lipid-cholesterol interaction is concentration-dependent. Moreover, it transitions from repulsive to attractive to repulsive as the cholesterol content increases. As the concentration of cholesterol in the bilayer exceeds a critical value, phase separation occurs. The coexistence between cholesterol-rich and cholesterol-poor domains is universal for any bilayer parameters, although the composition of the cholesterol-rich phase varies as a function of the lipid properties. Although we do not assume specific cholesterol-lipid interactions or the formation of a lipid-cholesterol cluster, we find that the composition of the cholesterol-rich domains is constant, independent of the cholesterol content in the bilayer.     

Temperature-controlled structure and kinetics of ripple phases in one- and two-component supported lipid bilayers

Temperature-controlled atomic force microscopy (AFM) has been used to visualize and study the structure and kinetics of ripple phases in one-component dipalmitoylphosphatidylcholine (DPPC) and two-component dimyristoylphosphatidylcholine-distearoylphosphatidylcholine (DMPC-DSPC) lipid bilayers. The lipid bilayers are mica-supported double bilayers in which ripple-phase formation occurs in the top bilayer. In one-component DPPC lipid bilayers, the stable and metastable ripple phases were observed. In addition, a third ripple structure with approximately twice the wavelength of the metastable ripples was seen. From height profiles of the AFM images, estimates of the amplitudes of the different ripple phases are reported. To elucidate the processes of ripple formation and disappearance, a ripple-phase DPPC lipid bilayer was taken through the pretransition in the cooling and the heating direction and the disappearance and formation of ripples was visualized. It was found that both the disappearance and formation of ripples take place virtually one ripple at a time, thereby demonstrating the highly anisotropic nature of the ripple phase. Furthermore, when a two-component DMPC-DSPC mixture was heated from the ripple phase and into the ripple-phase/fluid-phase coexistence temperature region, the AFM images revealed that several dynamic properties of the ripple phase are important for the melting behavior of the lipid mixture. Onset of melting is observed at grain boundaries between different ripple types and different ripple orientations, and the longer-wavelength metastable ripple phase melts before the shorter-wavelength stable ripple phase. Moreover, it was observed that the ripple phase favors domain growth along the ripple direction and is responsible for creating straight-edged domains with 60 degrees and 120 degrees angles, as reported previously.     

Lateral organization in lipid-cholesterol mixed bilayers

Interactions between lipid and cholesterol molecules in membranes play an important role in the structural and functional properties of cell membranes. Although structural properties of lipid-cholesterol mixtures have been extensively studied, an understanding of the role of cholesterol in the lateral organization of bilayers has been elusive. In this article, we propose a simple yet powerful model, based on self-consistent mean-field theory and molecular dynamics simulations, for lipid bilayers containing cholesterol. Properties predicted by our model are shown to be in excellent agreement with experimental data. Our model predicts that cholesterol induces structural changes in the bilayer through the formation of regions of ordered lipids surrounding each cholesterol molecule. We find that the "smooth" and "rough" sides of cholesterol play crucial roles in formation and distribution of the ordered regions. Our model is predictive in that input parameters are obtained from independent atomistic molecular dynamics simulations. The model and method are general enough to describe other heterogeneous lipid bilayers, including lipid rafts.     

Macro-ripple phase formation in bilayers composed of galactosylceramide and phosphatidylcholine.

As determined by freeze fracture electron microscopy, increasing levels of bovine brain galactosylceramide (GalCer) altered the surface structure of 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) bilayers by inducing a striking "macro-ripple" phase in the larger, multilamellar lipid vesicles at GalCer mole fractions between 0.4 and 0.8. The term "macro-ripple" phase was used to distinguish it from the P beta' ripple phase observed in saturated, symmetric-chain length phosphatidylcholines. Whereas the P beta' ripple phase displays two types of corrugations, one with a wavelength of 12-15 nm and the other with a wavelength of 25-35 nm, the macro-ripple phase occurring in GalCer/POPC dispersions was of one type with a wavelength of 100-110 nm. Also, in contrast to the extended linear arrays of adjacent ripples observed in the P beta' ripple phase, the macro-ripple phase of GalCer/POPC dispersions was interrupted frequently by packing defects resulting from double dislocations and various disclinations and, thus, appeared to be continuously twisting and turning. Control experiments verified that the macro-ripple phase was not an artifact of incomplete lipid mixing or demixing during preparation. Three different methods of lipid mixing were compared: a spray method of rapid solvent evaporation, a sublimation method of solvent removal, and solvent removal using a rotary evaporation apparatus. Control experiments also revealed that the macro-ripple phase was observed regardless of whether lipid specimens were prepared by either ultra-rapid or manual plunge freezing methods as well as either in the presence or absence of the cryo-protectant glycerol. The macro-ripple phase was always observed in mixtures that were fully annealed by incubation above the main thermal transition of both POPC and bovine brain GalCer before rapid freezing. If the GalCer mixed with POPC contained only nonhydroxy acyl chains or only 2-hydroxy acyl chains, then the occurrence of macro-ripple phase decreased dramatically.     

The permeability and the effect of acyl-chain length for phospholipid bilayers containing cholesterol: theory and experiment.

The model of Cruzeiro-Hansson et al. (Biochim. Biophys. Acta (1989) 979, 166-1176) for lipid-cholesterol bilayers at low cholesterol concentrations is used to predict the thermodynamic properties and the passive ion permeability of lipid bilayers as a function of acyl-chain length and cholesterol concentration. Numerical simulations based on the Monte Carlo method are used to determine the equilibrium state of the system near the main gel-fluid phase transition. The permeability is calculated using an ansatz which relates the passive permeability to the amount of interfaces formed in the bilayer when cholesterol is present. The model predicts at low cholesterol contents an increase in the membrane permeability in the transition region both for increasing cholesterol concentration and for decreasing chain length at a given value of the reduced temperature. This is in contrast to the case of lipid bilayers containing high cholesterol concentrations where the cholesterol strongly suppresses the permeability. Experimental results for the Na+ permeability of C15PC and DPPC (C16PC) bilayers containing cholesterol are presented which confirm the theoretical predictions at low cholesterol concentrations.     

Theory of thermal anomalies in the specific heat of lipid bilayers containing cholesterol.

A theoretical explanation of the experimentally observed characteristic thermal anomalies in the specific heat of lipid bilayers containing cholesterol is provided in terms of the phase equilibria in the phosphatidylcholine-cholesterol system. The phase equilibria are calculated via a microscopic interaction model that takes proper account of both the conformational and the crystalline degrees of freedom of the lipid acyl chains. It is shown that the characteristic double-peaked specific heat, with a narrow and a broad component, is a natural consequence of the topology of the phase diagram. Some results for the enthalpy of the mixed system are also reported. It is suggested that there is no need for invoking special mechanisms such as lipid-cholesterol complexing or formation of special interfacial regions in the bilayer in order to explain the specific-heat anomalies.     

Lipid-cholesterol interactions. Monte Carlo simulations and theory.

Results of Monte Carlo calculations of order parameter profiles of lipid chains interacting with cholesterol are presented. Cholesterol concentrations in the simulations are sufficiently large that it is possible to analyze profiles for chains which are near neighbors of two or more cholesterol molecules, chains which are neighbors to a single cholesterol, and chains which are not near any cholesterol molecules. The profiles, show that cholesterol acts to significantly decrease the ability of neighboring chains to undergo trans-gauche isomeric rotations, although these chains are not all forced into all-trans conformations. The effect is significantly greater for chains which are neighbors to more than one cholesterol. The Monte Carlo results are next used as a guide to develop a theoretical model for lipid-cholesterol mixtures. The properties of this model and the phase diagram which it predicts are described. The phase diagram is then compared with experimentally determined phase diagrams. The model calculations and the computer simulations upon which they are based yield a molecular mechanism for several of the observed phases exhibited by lipid-cholesterol mixtures. The theoretical model predicts that at low temperatures the system should exhibit solid phase immiscibility.     

Ripples and the formation of anisotropic lipid domains: imaging two-component supported double bilayers by atomic force microscopy

Direct visualization of the fluid-phase/ordered-phase domain structure in mica-supported bilayers composed of 1,2-dimyristoyl-sn-glycero-3-phosphocholine/1,2-distearoyl-sn-glycero-3-phosphocholine mixtures is performed with atomic force microscopy. The system studied is a double bilayer supported on a mica surface in which the top bilayer (which is not in direct contact with the mica) is visualized as a function of temperature. Because the top bilayer is not as restricted by the interactions with the surface as single supported bilayers, its behavior is more similar to a free-standing bilayer. Intriguing straight-edged anisotropic fluid-phase domains were observed in the fluid-phase/ordered-phase coexistence temperature range, which resemble the fluid-phase/ordered-phase domain patterns observed in giant unilamellar vesicles composed of such phospholipid mixtures. With the high resolution provided by atomic force microscopy, we investigated the origin of these anisotropic lipid domain patterns, and found that ripple phase formation is directly responsible for the anisotropic nature of these domains. The nucleation and growth of fluid-phase domains are found to be directed by the presence of ripples. In particular, the fluid-phase domains elongate parallel to the ripples. The results show that ripple phase formation may have implications for domain formation in biological systems.     

Evolution of a rippled membrane during phospholipase A2 hydrolysis studied by time-resolved AFM

The sensitivity of phospholipase A(2) (PLA(2)) for lipid membrane curvature is explored by monitoring, through time-resolved atomic force microscopy, the hydrolysis of supported double bilayers in the ripple phase. The ripple phase presents a corrugated morphology. PLA(2) is shown to have higher activity toward the ripple phase compared to the gel phase in 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) membranes, indicating its preference for this highly curved membrane morphology. Hydrolysis of the stable and metastable ripple structures is monitored for equimolar DMPC/1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC)-supported double bilayers. As shown by high-performance liquid chromatography results, DSPC is resistant to hydrolysis at this temperature, resulting in a more gradual hydrolysis of the surface that leads to a change in membrane morphology without loss of membrane integrity. This is reflected in an increase in ripple spacing, followed by a sudden flattening of the lipid membrane during hydrolysis. Hydrolysis of the ripple phase results in anisotropic holes running parallel to the ripples, suggesting that the ripple phase has strip regions of higher sensitivity to enzymatic attack. Bulk high-performance liquid chromatography measurements indicate that PLA(2) preferentially hydrolyzes DMPC in the DMPC/DSPC ripples. We suggest that this leads to the formation of a flat gel-phase lipid membrane due to enrichment in DSPC. The results point to the ability of PLA(2) for inducing a compositional phase transition in multicomponent membranes through preferential hydrolysis while preserving membrane integrity.     

Effects of cholesterol concentration on the interaction of cytarabine with lipid membranes: a molecular dynamics simulation study

Liposomal cytarabine, DepoCyt, is a chemotherapy agent which is used in cancer treatment. This form of cytarabine has more efficacy and fewer side effects relative to the other forms. Since DepoCyt contains the cytarabine encapsulated within phosphatidylcholine and the sterol molecules, we modeled dioleoylphosphatidylcholine (DOPC)/cholesterol bilayer membrane as a carrier for cytarabine to study drug–bilayer interactions. For this purpose, we performed a series of united-atom molecular dynamics (MD) simulations for 25?ns to investigate the interactions between cytarabine and cholesterol-containing DOPC lipid bilayers. Only the uncharged form of cytarabine molecule was investigated. In this study, different levels of the cholesterol content (0, 20, and 40%) were used. MD simulations allowed us to determine dynamical and structural properties of the bilayer membrane and to estimate the preferred location and orientation of the cytarabine molecule inside the bilayer membrane. Properties such as membrane thickness, area per lipid, diffusion coefficient, mass density, bilayer packing, order parameters, and intermolecular interactions were examined. The results show that by increasing the cholesterol concentration in the lipid bilayers, the bilayer thickness increases and area per lipid decreases. Moreover, in accordance with the experiments, our calculations show that cholesterol molecules have ordering effect on the hydrocarbon acyl chains. Furthermore, the cytarabine molecule preferentially occupies the polar region of the lipid head groups to form specific interactions (hydrogen bonds). Our results fully support the experimental data. Our finding about drug–bilayer interaction is crucial for the liposomal drug design.     

Real-time imaging of drug-membrane interactions by atomic force microscopy

Understanding drug-biomembrane interactions at high resolution is a key issue in current biophysical and pharmaceutical research. Here we used real-time atomic force microscopy (AFM) imaging to visualize the interaction of the antibiotic azithromycin with lipid domains in model biomembranes. Various supported lipid bilayers were prepared by fusion of unilamellar vesicles on mica and imaged in buffer solution. Phase-separation was observed in the form of domains made of dipalmitoylphosphatidylcholine (DPPC), sphingomyelin (SM), or SM/cholesterol (SM/Chl) surrounded by a fluid matrix of dioleoylphosphatidylcholine (DOPC). Time-lapse images collected following addition of 1 mM azithromycin revealed progressive erosion and disappearance of DPPC gel domains within 60 min. We attribute this effect to the disruption of the tight molecular packing of the DPPC molecules by the drug, in agreement with earlier biophysical experiments. By contrast, SM and SM-Chl domains were not modified by azithromycin. We suggest that the higher membrane stability of SM-containing domains results from stronger intermolecular interactions between SM molecules. This work provides direct evidence that the perturbation of lipid domains by azithromycin strongly depends on the lipid nature and opens the door for developing new applications in membrane biophysics and pharmacology.     

Real-time imaging of drug-membrane interactions by atomic force microscopy

Understanding drug-biomembrane interactions at high resolution is a key issue in current biophysical and pharmaceutical research. Here we used real-time atomic force microscopy (AFM) imaging to visualize the interaction of the antibiotic azithromycin with lipid domains in model biomembranes. Various supported lipid bilayers were prepared by fusion of unilamellar vesicles on mica and imaged in buffer solution. Phase-separation was observed in the form of domains made of dipalmitoylphosphatidylcholine (DPPC), sphingomyelin (SM), or SM/cholesterol (SM/Chl) surrounded by a fluid matrix of dioleoylphosphatidylcholine (DOPC). Time-lapse images collected following addition of 1 mM azithromycin revealed progressive erosion and disappearance of DPPC gel domains within 60 min. We attribute this effect to the disruption of the tight molecular packing of the DPPC molecules by the drug, in agreement with earlier biophysical experiments. By contrast, SM and SM-Chl domains were not modified by azithromycin. We suggest that the higher membrane stability of SM-containing domains results from stronger intermolecular interactions between SM molecules. This work provides direct evidence that the perturbation of lipid domains by azithromycin strongly depends on the lipid nature and opens the door for developing new applications in membrane biophysics and pharmacology.     

Computation of mixed phosphatidylcholine-cholesterol bilayer structures by energy minimization.

The energetically preferred structures of dimyristoylphosphatidylcholine (DMPC)-cholesterol bilayers were determined at a 1:1 mole ratio. Crystallographic symmetry operations were used to generate planar bilayers of cholesterol and DMPC. Energy minimization was carried out with respect to bond rotations, rigid body motions, and the two-dimensional lattice constants. The lowest energy structures had a hydrogen bond between the cholesterol hydroxyl and the carbonyl oxygen of the sn-2 acyl chain, but the largest contribution to the intermolecular energy was from the nonbonded interactions between the flat alpha surface of cholesterol and the acyl chains of DMPC. Two modes of packing in the bilayer were found; in structure A (the global minimum), unlike molecules are nearest neighbors, whereas in structure B (second lowest energy) like-like intermolecular interactions predominate. Crystallographic close packing of the molecules in the bilayer was achieved, as judged from the molecular areas and the bilayer thickness. These energy-minimized structures are consistent with the available experimental data on mixed bilayers of lecithin and cholesterol, and may be used as starting points for molecular dynamics or other calculations on bilayers.     

Phase diagrams for impure lipid systems. Application to lipid/anaesthetic mixtures

A physical model is presented to describe theoretically the temperature-dependent interactions of lipid bilayers with small molecules such as anaesthetics. Based on an earlier model, a triangular lattice in which each site is occupied by a single lipid chain is constructed and the small (anaesthetic) molecules are assumed to occupy interstitial sites in the centre of each lattice triangle. The phase characteristics of such lipid/anaesthetic mixtures are described in terms of the interaction parameters between lipid-lipid, lipid-anaesthetic and anaesthetic-anaesthetic molecules. Depending on the chemical nature of the interacting species the following three models are formulated: Model I. An interstitial model in which the only perturbation is in the head-group region of the bilayer and direct interactions between neighbouring anaesthetic molecules are taken into account. Model II. Here, only hydrophobic interactions between anaesthetics and lipids are considered. Model III. Both van der Waals' and coulombic interactions are taken into account. Phase diagrams for the three models are obtained by numerical calculation over a wide range of interaction parameters. It is shown that in all three models, lateral phase separation takes place due to the presence of anaesthetics. The heat of transition, however, is found to be virtually independent of the anaesthetic concentration.     

Role of cholesterol in lipid raft formation: lessons from lipid model systems

Biochemical and cell-biological experiments have identified cholesterol as an important component of lipid 'rafts' and related structures (e.g., caveolae) in mammalian cell membranes, and membrane cholesterol levels as a key factor in determining raft stability and organization. Studies using cholesterol-containing bilayers as model systems have provided important insights into the roles that cholesterol plays in determining lipid raft behavior. This review will discuss recent progress in understanding two aspects of lipid-cholesterol interactions that are particularly relevant to understanding the formation and properties of lipid rafts. First, we will consider evidence that cholesterol interacts differentially with different membrane lipids, associating particularly strongly with saturated, high-melting phospho- and sphingolipids and particularly weakly with highly unsaturated lipid species. Second, we will review recent progress in reconstituting and directly observing segregated raft-like (liquid-ordered) domains in model membranes that mimic the lipid compositions of natural membranes incorporating raft domains.     

A theoretical model for lipid mixtures, phase transitions, and phase diagrams.

We present a new model for the thermodynamic properties of lipid bilayers. The model consists of a system of hard cylinders of varying radii that correspond to the different molecular radii of lipids having different numbers of gauche rotations in their chains. Scaled particle theory is used to provide an accurate estimate of the entropy of packing of the cylinders. To apply the model to bilayers we introduce a semiempirical attractive potential energy. Once the form of this potential is chosen, we adjust one parameter, the interaction strength, so that the model fits the transition temperatures and entropies for various phospholipids. The model then agrees quite well with other published data for these systems. We also directly generalize our model to lipid mixtures, and we obtain phase diagrams that we compare to existing data for these systems. We use the model to describe lipid protein interactions in bilayers as well.     

Role of cholesterol in lipid raft formation: lessons from lipid model systems

Biochemical and cell-biological experiments have identified cholesterol as an important component of lipid ‘rafts’ and related structures (e.g., caveolae) in mammalian cell membranes, and membrane cholesterol levels as a key factor in determining raft stability and organization. Studies using cholesterol-containing bilayers as model systems have provided important insights into the roles that cholesterol plays in determining lipid raft behavior. This review will discuss recent progress in understanding two aspects of lipid-cholesterol interactions that are particularly relevant to understanding the formation and properties of lipid rafts. First, we will consider evidence that cholesterol interacts differentially with different membrane lipids, associating particularly strongly with saturated, high-melting phospho- and sphingolipids and particularly weakly with highly unsaturated lipid species. Second, we will review recent progress in reconstituting and directly observing segregated raft-like (liquid-ordered) domains in model membranes that mimic the lipid compositions of natural membranes incorporating raft domains.     

Molecular dynamics simulation of lipid packing and mobility in bilayer membranes

A model of lipid bilayer membrane in water has been developed. Parameters have been selected that allow molecular dynamics simulation of lipid bilayers in the all-atom approximation. The calculated indices of packing and mobility of lipid molecules for the liquid crystalline state of the bilayer agree well with the experimental data. Based on the model of the liquid crystalline state of the membrane, a system in the gel-like state has been constructed. The gel-state model reproduces well the packing of lipids in real bilayers, whereas the mobility of molecules proves to be overestimated.