Small-scale lipid-membrane structure: simulation versus experiment.

Recently, it has become obvious that the conventional picture of the fluid lipid-bilayer component of biological membranes being a fairly structureless 'fluid mosaic' solvent is far from correct. The lipid bilayer displays distinct static and dynamic structural organization on a small scale, for example in terms of differentiated lipid domains, and evidence is accumulating that these structures are of importance for the functioning of biological membranes, including the activity of membrane-bound enzymes and receptors and morphological changes at the cell surface. Insight into the relationship between this small-scale structure and biological functioning holds promise for a more rational approach to modulate function via manipulation of the lipid microenvironment and the lipid/protein interface in particular. Computer simulation has proved to be a useful tool in investigating membrane structure on a small scale-specifically the nanometer scale (1-100 nm), which is in between the molecular scale accessible by various spectroscopic techniques and molecular dynamics calculations, and the micrometer scale accessible by scattering and microscopy techniques.

Studies on the lack of cooperativity in the melting of lipid bilayers.

The gel-to-fluid first-order melting transition of lipid bilayers is simulated by the use of a microscopic interaction model which includes a variable number of lipid-chain conformational states. The results suggest that the experimental observation of 'continuous melting' in pure wet lipid bilayers, rather than being ascribed to the presence of impurities, may be explained as a result of kinetically caused metastability of intermediate lipid-chain conformations.

Modelling the phase equilibria in two-component membranes of phospholipids with different acyl-chain lengths.

A phenomenological model is proposed to describe the membrane phase equilibria in binary mixtures of saturated phospholipids with different acyl-chain lengths. The model is formulated in terms of thermodynamic and thermomechanic properties of the pure lipid bilayers, specifically the chain-melting transition temperature and enthalpy, the hydrophobic bilayer thickness, and the lateral area compressibility modulus. The model is studied using a regular solution theory made up of a set of interaction parameters which directly identify that part of the lipid-lipid interaction which is due to hydrophobic mismatch of saturated chains of different lengths. It is then found that there is effectively a single universal interaction parameter which, in the full composition range, describes the phase equilibria in mixtures of DMPC/DPPC, DPPC/DSPC, DMPC/DSPC, and DLPC/DSPC, in excellent agreement with experimental measurements. The model is used to predict the variation with temperature and composition of the specific heat, as well as of the average membrane thickness and area in each of the phases. Given the value of the universal interaction parameter, the model is then used to predict the phase diagrams of binary mixtures of phospholipids with different polar head groups, e.g., DPPC/DPPE, DMPC/DPPE and DMPE/DSPC. By comparison with experimental results for these mixtures, it is shown that difference in acyl-chain lengths gives the major contribution to deviation from ideal mixing. Application of the model to mixtures with non-saturated lipids is also discussed.

Passive ion permeability of lipid membranes modelled via lipid-domain interfacial area.

A microscopic interaction model of the gel-to-fluid chain-melting phase transition of fully hydrated lipid bilayer membranes is used as a basis for modelling the temperature dependence of passive transmembrane permeability of small ions, e.g. Na+. Computer simulation of the model shows that the phase transition is accompanied by strong lateral density fluctuations which manifest themselves in the formation of inhomogeneous equilibrium structures of coexisting gel and fluid domains. The interfaces of these domains are found to be dominated by intermediate lipid-chain conformations. The interfacial area is shown to have a pronounced peak at the phase transition. By imposing a simple model for ion diffusion through membranes which assigns a high relative permeation rate to the domain interfaces, the interfacial area is then identified as a membrane property which has the proper temperature variation to account for the peculiar experimental observation of a strongly enhanced passive ion permeability at the phase transition. The excellent agreement with the experimental data for Na+-permeation, taken together with recent experimental results for the phase transition kinetics, provides new insight into the microphysical mechanisms of reversible electric breakdown. This insight indicates that there is no need for aqueous pore-formation to explain the experimental observation of a dramatic increase in ion conductance subsequent to electric pulses.

Lindane suppresses the lipid-bilayer permeability in the main transition region.

The effects of a small molecule, the insecticide lindane, on unilamellar DMPC bilayers in the phase transition region, have been studied by means of differential scanning calorimetry and fluorescence spectroscopy. The calorimetric data show that increasing concentrations of lindane broaden the transition and lower the transition temperature, without changing the transition enthalpy significantly. Lindane therefore enhances the thermal fluctuations of the bilayer. The calorimetric data furthermore suggest that the bilayer structure is intact and not disrupted by even high concentrations (32 mol%) of lindane. Fluorescence spectroscopy was used to measure the passive permeability of unilamellar DMPC bilayers to Co2+ ions. The data show that lindane seals the bilayer for Co2+ penetration and that this effect increases with increasing lindane concentration. The results are discussed in relation to the effects on the permeability of other small molecules, e.g., anesthetics.

Calorimetric and theoretical studies of the effects of lindane on lipid bilayers of different acyl chain length.

The effects of the insecticide lindane on the phase transition in multilamellar bilayers of saturated diacylphosphatidylcholines of different acyl chain length (DC14PC, DC16PC, and DC18PC) have been studied by means of differential scanning calorimetry (DSC), as well as computer-simulation calculations on a molecular interaction model. The calorimetric data show that increasing concentrations of lindane lower the transition temperature and lead to a broadening of the specific heat in a systematic way depending on the lipid acyl chain length. Kinetic effects in the observed calorimetric traces indicate that the incorporation of lindane into multilamellar lipid bilayers is slow, but faster for the shorter lipid species. Large unilamellar vesicles do not show such kinetic effects. The transition enthalpy is for all three lipid species found to be independent of the lindane concentration which implies that the entropy of mixing is vanishingly small. This lends support to a microscopic molecular interaction model which assigns the absorbed lindane molecules to interstitial sites in the bilayer. Computer-simulation calculations on this model, which assumes a specific interaction between lindane and certain excited acyl chain configurations, lead to predictions of the lipid-water partition coefficient in qualitative agreement with experimental measurements (Antunes-Madeira and Madeira (1985) Biochim. Biophys. Acta 820, 165-172). The partition coefficient has a peak near the phase transition which is a consequence of enhanced interfacial adsorption of lindane at lipid-domain interfaces.

Phospholipase A(2) activity towards vesicles of DPPC and DMPC-DSPC containing small amounts of SMPC.

Phospholipase A(2) (PLA(2)) is an interfacially active enzyme whose hydrolytic activity is known to be enhanced in one-component phospholipid bilayer substrates exhibiting dynamic micro-heterogeneity. In this study the activity of PLA(2) towards large unilamellar vesicles composed of DPPC:SMPC and DMPC:DSPC:SMPC is investigated using fluorescence and HPLC techniques. Phase diagrams of the mixtures are established by differential scanning calorimetry and the PLA(2) activity, monitored by the lag time, is correlated with the phase behavior of the mixtures. In addition, the degree of lipid hydrolysis in the DMPC:DSPC:SMPC lipid mixtures is detected by HPLC. The PLA(2) activity is found to be significantly increased in the temperature range of the coexistence region where the lipid mixtures exhibit lateral gel-fluid phase separation. Furthermore, in the entire temperature range it is demonstrated that PLA(2) preferentially hydrolyzes the short chain DMPC lipid. This discriminative effect becomes less pronounced when the asymmetric lipid SMPC is present in the lipid substrate. Inclusion of SMPC into either DPPC or DMPC:DSPC vesicles prolongs the lag time. The results clearly show that the PLA(2) activity is significantly enhanced by lipid bilayer micro-heterogeneity in both one-component and multi-component lipid bilayer substrates. The PLA(2) activity measurements are discussed in terms of dynamic gel-fluid lipid domain formation due to density fluctuations and static lipid domain formation due to gel-fluid phase separation.

Phase behavior and permeability properties of phospholipid bilayers containing a short-chain phospholipid permeability enhancer.

The thermodynamic phase behavior and trans-bilayer permeability properties of multilamellar phospholipid vesicles containing a short-chain DC10PC phospholipid permeability enhancer have been studied by means of differential scanning calorimetry and fluorescence spectroscopy. The calorimetric scans of DC14PC lipid bilayer vesicles incorporated with high concentrations of DC10PC demonstrate a distinct influence on the lipid bilayer thermodynamics manifested as a pronounced freezing-point depression and a narrow phase coexistence region. Increasing amounts of DC10PC lead to a progressive lowering of the melting enthalpy, implying a mixing behavior of the DC10PC in the bilayer matrix similar to that of a substitutional impurity. The phase behavior of the DC10PC-DC14PC mixture is supported by fluorescence polarization measurements which, furthermore, in the low-temperature gel phase reveal a non-monotonic concentration-dependent influence on the structural bilayer properties; small concentrations of DC10PC induce a disordering of the acyl chains, whereas higher concentrations lead to an ordering. Irreversible fluorescence quench measurements demonstrate a substantial increase in the trans-bilayer permeability over broad temperature and composition ranges. At temperatures corresponding to the peak positions of the heat capacity, a maximum in the trans-bilayer permeability is observed. The influence of DC10PC on the lipid bilayer thermodynamics and the associated permeability properties is discussed in terms of microscopic effects on the lateral lipid organization and heterogeneity of the bilayer.

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.

Intrinsic molecules in lipid membranes change the lipid-domain interfacial area: cholesterol at domain interfaces.

A theoretical analysis of the effects of intrinsic molecules on the lateral density fluctuations in lipid bilayer membranes is carried out by means of computer simulations on a microscopic interaction model of the gel-to-fluid chain-melting phase transition. The inhomogeneous equilibrium structures of gel and fluid domains, which in previous work (Cruzeiro-Hansson, L. and Mouritsen, O.G. (1988) Biochim. Biophys. Acta 944, 63-72) were shown to characterize the transition region of pure lipid membranes, are here shown to be enhanced by intrinsic molecules such as cholesterol. Cholesterol is found to increase the interfacial area and to accumulate in the interfaces. The interfacial area, the average cluster size, the lateral compressibility, and the membrane area are calculated as functions of temperature and cholesterol concentration. It is shown that the enhancement by cholesterol of the lateral density fluctuations and the lipid-domain interfacial area is most pronounced away from the transition temperature. The implications of the results are discussed in relation to passive ion permeability and function of interfacially active enzymes such as phospholipase.