Free-radical label--new approach to the study of dynamics of lipid systems

A new method of EPR-spectroscopy--the recombination of free radicals appearing as a result of indirect radiolysis of biological molecules after a low temperature irradiation--is applied to the study of molecular dynamics of phosphatidylcholine dimyristoyl in mass and in the structure of liposomes above and below the transition temperature. It was shown, that the mobility of lipid molecules in crystalline liposomes is higher than in the structure of liquid-crystalline liposomes. The addition of cholesterol in liposome membranes decreases the lateral molecular motion of lipids in crystalline and liquid-crystalline state, in the latter case the effect of cholesterol addition is more pronounced. The activation energy for the displacement of the fragments of lipid molecules and the lipid molecule as a whole was estimated, and it was shown, that lipid matrix possesses a high degree of heterogeneity.     

Free radical label: new approach to the study of super-slow molecular dynamics of lipid systems

A new method of EPR-spectroscopy, the recombination of free radicals appearing as a result of indirect radiolysis of biological molecules after a low temperature irradiation, was applied to the study of molecular dynamics of dimyristoyl phosphatidylcholine in mass and in the structure of liposomes above and below the transition temperature. It was shown that the mobility of lipid molecules in crystalline liposomes was lower than in the structure of liquid-crystalline liposomes. The addition of cholesterol in liposome membranes decreased the lateral molecular motion of lipids in crystalline and liquid-crystalline states; in the latter case, the effect of cholesterol addition was more pronounced. The activation energy for the displacement of the fragments of lipid molecules and the lipid molecule as a whole was estimated, and it was shown that the lipid matrix possesses a high degree of heterogeneity. The solubility of oxygen in the lipid bilayer and the mechanism of lipid diffusion are discussed.     

Pulmonary Surfactant Protein SP-C Counteracts the Deleterious Effects of Cholesterol on the Activity of Surfactant Films under Physiologically Relevant Compression-Expansion Dynamics

The presence of cholesterol is critical in defining a dynamic lateral structure in pulmonary surfactant membranes. However, an excess of cholesterol has been associated with impaired surface activity of surfactant. It has also been reported that surfactant protein SP-C interacts with cholesterol in lipid/protein interfacial films. In this study, we analyzed the effect of SP-C on the thermodynamic properties of phospholipid membranes containing cholesterol, and the ability of lipid/protein complexes containing cholesterol to form and respread interfacial films capable of producing very low surface tensions upon repetitive compression-expansion cycling. SP-C modulates the effect of cholesterol to reduce the enthalpy associated with the gel-to-liquid-crystalline melting transition in dipalmitoylphosphatidylcholine (DPPC) bilayers, as analyzed by differential scanning calorimetry. The presence of SP-C affects more subtly the effects of cholesterol on the thermotropic properties of ternary membranes, mimicking more closely the lipid composition of native surfactant, where SP-C facilitates the miscibility of the sterol. Incorporation of 1% or 2% SP-C (protein/phospholipid by weight) promotes almost instantaneous adsorption of suspensions of DPPC/palmitoyloleoylphospatidylcholine (POPC)/palmitoyloleoyl-phosphatidylglycerol (POPG) (50:25:15, w/w/w) into the air-liquid interface of a captive bubble, in both the absence and presence of cholesterol. However, cholesterol impairs the ability of SP-C-containing films to achieve very low surface tensions in bubbles subjected to compression-expansion cycling. Cholesterol also substantially impairs the ability of DPPC/POPC/POPG films containing 1% surfactant protein SP-B to mimic the interfacial behavior of native surfactant films, which are characterized by very low minimum surface tensions with only limited area change during compression and practically no compression-expansion hysteresis. However, the simultaneous presence of 2% SP-C practically restores the compression-expansion dynamics of cholesterol- and SP-B-containing films to the efficient behavior shown in the absence of cholesterol. This suggests that cooperation between the two proteins is required for lipid-protein films containing cholesterol to achieve optimal performance under physiologically relevant compression-expansion dynamics.     

Properties of bilayer membranes in the phase transition or phase separation region

The increase in passive permeability of bilayer membranes near the phase transition temperature is usually explained as caused by either the increase in the amount of ‘boundary lipid’ present in the membrane, or by the increase in lateral compressibility of the membrane. Since both the amount of ‘boundary lipid’ and the lateral compressibility show a similar anomaly near the transition temperature, it is difficult to distinguish experimentally between the two proposed mechanisms.We have examined some details of both of the proposed pictures. The fluid-solid boundary energy, neglected in previous work, has been computed as a function of the domain size. For a single component uncharged lipid bilayer, the results rule out the existence of even loosely defined solid domains in a fluid phase, or vice versa. Thermodynamic fluctuations, which are responsible for anomalous behaviour near the phase transition temperature, are not intense enough to approximate the formation of a domain of the opposite phase.Turning next to lateral compressibility of bilayer membranes we have considered two-component mixtures in the phase separation region. We present the first calculation of lateral compressibility for such systems. The behaviour shows interesting anomalies, which should correlate with existing and future data on transport across membranes.     

From small fluctuations to large-scale phase separation: lateral organization in model membranes containing cholesterol

Macroscopic coexisting liquid phases are readily observed in certain model membranes containing cholesterol and at least two other lipid components. Recent fluorescence microscopy and deuterium NMR work indicates that submicron fluctuations are also found, in vesicles with near-critical lipid compositions. In principle, the magnitude of critical fluctuations can be controlled by changing temperature, or through other means of shifting the phase boundary such as including impurities or cross-linking components. Critical fluctuations are dynamic submicron domains in model membranes, and provide a plausible physical mechanism to produce putative 'raft' domains in cholesterol-rich biomembranes.     

Chain ordering in liquid crystals: II. Structure of bilayer membranes

The ordering of the hydrocarbon chain interior of bilayer membranes has been calculated using the molecular field approximation developed in previous work on liquid crystals. Different statistical averages are evaluated by exact summation over all conformations of a single chain in the field due to neighboring molecules. The internal energy of each conformation, as well as contributions arising from interaction with the molecular field and from a lateral pressure on the chain have been included.The results describe properties of both lipid monolayers and bilayers. For monolayers, the calculated pressure-area relationships are in good agreement with experimental observations. The order parameter for hydrocarbon chains in bilayers (or monolayers) as a function of temperature, lateral pressure and position along the chain, is shown and compared with the available NMR data. Combining the results of calculation and NMR measurements we obtain the value for intrinsic lateral pressure within bilayer membranes, in excellent agreement with direct measurements on surface monolayers.The calculation also gives average length of hydrocarbon chains, thermal expansion coefficient and fraction of bonds in gauche conformations. The effect of cholesterol and proteins within the bilayer is qualitatively described, and the contribution of the bilayer interior to membrane elasticity is determined.     

A model for the packing of lipids in bilayer membranes

A number of known structural properties of mixed lipid bilayer membranes and monolayers are accounted for by a model in which lipids pack into bilayers and monolayers like building blocks, each characterized by a surface head group area and characteristic solid angle. In phospholipids above the melting transition the head group area (at a given temperature and degree of hydration) is fairly invariant while the hydrocarbon region may be liquid-like so long as the molecule is not compressed beyond its characteristic solid angle.Phosphotidylcholine and phosphotidylserine are tapered lipids, i.e. their surface head group areas are greater than their non-polar end areas; cholesterol is frayed, i.e. its polar end area is less than its non-polar end area; while phosphotidylethanolamine is almost cylindrical. The “condensing” effect of cholesterol in mixed phospholipid-cholesterol films is seen as a taper-fray accomodation. The lipid distribution in erythrocyte membranes is shown to be conductive to a stable strain-free membrane.     

Softening of lipid bilayers

The softening of wet lipid bilayer membranes during their gel-to-fluid first-order phase transition is studied by computer simulation of a family of two-dimensional microscopic interaction models. The models include a variable number, q, of lipid chain conformational states, where 2q10. Results are presented as functions of q and temperature for a number of bulk properties, such as internal energy, specific heat, and lateral compressibility. A quantitative account is given of the statistics of the lipid clusters which are found to form in the neighborhood of the transition. The occurrence of these clusters is related to the softening and the strong thermal density fluctuations which dominate the specific heat and the lateral compressibility for the high-q models. The cluster distributions and the fluctuations behave in a manner reminiscent of critical phenomena and percolation. The findings of long-lived metastable states and extremely slow relaxational behavior in the transition region are shown to be caused by the presence of intermediate lipid chain conformational states which kinetically stabilize the cluster distribution and the effective phase coexistence. This has as its macroscopic consequence that the first-order transition apperas as a continuous transition, as invariably observed in all experiments on uncharged lecithin bilayer membranes. The results also suggest an explanation of the non-horizontal isotherms of lipid monolayers. Possible implications of lipid bilayer softening and enhanced passive permeability for the functioning of biological membranes are discussed.Abbreviations PC phosphatidvlcholine - DMPC dimyristoyl PC - DPPC dipalmitoyl PC - ac alternating current - DSC differential scanning calorimetry - T _m lipid gel-to-fluid phase transition temperature - TEMPO 2,2,6,6-tetramethylpiperidine-N-oxyl Supported by the Danish Natural Science Research Council and A/S De Danske Spritfabrikkers JubilæumslegatSupported in part by the NSERC of Canada and Le FCAC du Quebec     

Molecular dynamics simulations of phospholipid bilayers with cholesterol

To investigate the microscopic interactions between cholesterol and lipids in biological membranes, we have performed a series of molecular dynamics simulations of large membranes with different levels of cholesterol content. The simulations extend to 10 ns, and were performed with hydrated dipalmitoylphosphatidylcholine (DPPC) bilayers. The bilayers contain 1024 lipids of which 0-40% were cholesterol and the rest DPPC. The effects of cholesterol on the structure and mesoscopic dynamics of the bilayer were monitored as a function of cholesterol concentration. The main effects observed are a significant ordering of the DPPC chains (as monitored by NMR type order parameters), a reduced fraction of gauche bonds, a reduced surface area per lipid, less undulations--corresponding to an increased bending modulus for the membrane, smaller area fluctuations, and a reduced lateral diffusion of DPPC-lipids as well as cholesterols.     

Cholesterol Induces Specific Spatial and Orientational Order in Cholesterol/Phospholipid Membranes

Background

In lipid bilayers, cholesterol facilitates the formation of the liquid-ordered phase and enables the formation of laterally ordered structures such as lipid rafts. While these domains have an important role in a variety of cellular processes, the precise atomic-level mechanisms responsible for cholesterol''s specific ordering and packing capability have remained unresolved.

Methodology/Principal Findings

Our atomic-scale molecular dynamics simulations reveal that this ordering and the associated packing effects in membranes largely result from cholesterol''s molecular structure, which differentiates cholesterol from other sterols. We find that cholesterol molecules prefer to be located in the second coordination shell, avoiding direct cholesterol-cholesterol contacts, and form a three-fold symmetric arrangement with proximal cholesterol molecules. At larger distances, the lateral three-fold organization is broken by thermal fluctuations. For other sterols having less structural asymmetry, the three-fold arrangement is considerably lost.

Conclusions/Significance

We conclude that cholesterol molecules act collectively in lipid membranes. This is the main reason why the liquid-ordered phase only emerges for Chol concentrations well above 10 mol% where the collective self-organization of Chol molecules emerges spontaneously. The collective ordering process requires specific molecular-scale features that explain why different sterols have very different membrane ordering properties: the three-fold symmetry in the Chol-Chol organization arises from the cholesterol off-plane methyl groups allowing the identification of raft-promoting sterols from those that do not promote rafts.     

Interactions of proteins and cholesterol with lipids in bilayer membranes.

Mixtures of lipids and protein, the ATPase from rabbit sarcoplasmic reticulum, were studied by freeze-fracture electron microscopy and by measurement of the amount of fluid lipid with the spin label 2,2,6,6-tetramethylpiperidine-1-oxyl (TEM-PO). In dimyristoyl phosphatidylcholine vesicles the protein molecules were randomly distributed above the transition temperature, Tt, of the lipid and aggregated below Tt. For mixtures of dimyristoyl and dipalmitoyl phosphatidylcholine the existence of fluid and solid domains were shown in the temperature interval predicted from earlier TEMPO measurements. When protein was incorporated into this lipid mixture, freeze-fracture particles were randomly distributed in fluid lipids, or aggregated when only solid lipids were present. In mixtures of dimyristoyl phosphatidylcholine with cholesterol the protein was distributed randomly above the transition temperature of the phosphatidylcholine. Below that transition temperature the protein was excluded from a banded phase of solid lipid in the case of 10 mol% cholesterol. In mixtures containing 20 mol% cholesterol, protein molecules formed linear arrays, 50-200 nm in length, around smooth patches of lipid. Phase diagrams for lipid/cholesterol and lipid/protein systems are proposed which account for many of the available data. A model for increasing solidification of lipid around protein molecules or cholesterol above the transition temperature of the lipid is discussed.     

Preferential orientation of an immunoglobulin in a glycolipid monolayer controlled by the disintegration kinetics of proteo-lipidic vesicles spread at an air-buffer interface

The insertion of immunoglobulin (IgG) in a glycolipid monolayer was achieved by using the ability of new proteo-glycolipid vesicles to disintegrate into a mixed IgG-glycolipid interfacial film after spreading at an air-buffer interface. The interfacial disintegration kinetics was shown to be directly dependent on the initial vesicle surface density and on the buffer ionic strength. The presence of the immunoglobulin in the glycolipid film was displayed by an increase of the lateral compressibility (Cs) during monolayer compression. Cs magnitude modifications, due to the antibody effect on the monolayer packing, decreases as the spread vesicle density increases. At interfacial saturation, the lateral compressibility profile becomes similar to that of a control monolayer without antibody. However, the careful analysis of the mixed monolayer after transfer by Langmuir-Blodgett technique (ATR-FTIR characterisation, enzyme immunoassociation) clearly demonstrated that the antibody was still present in such conditions and was not completely squeezed out from the interface as compressibility changes could have meant. At nonsaturating vesicle surface density, IgG molecules initially lying in the lipid matrix with the Y-shape plane parallel to the interface move to a standing-up position during the compression, leading to lateral compressibility modifications. For a saturating vesicle surface density, the glycolipid molecules force the IgG molecules to directly adopt a more vertical position in the interfacial film and, consequently, no lateral compressibility modification was recorded during the compression.     

A simple theoretical model for the effects of cholesterol and polypeptides on lipid membranes

A simple theoretical model for the effects of impurities on biomembranes is proposed. The model accounts for the cholesterol-induced decrease of membrane phase transition temperature, membrane condensation above the gel to liquid crystalline phase transition, and increase in lateral compressibility. The model also predicts that addition of molecules such as cholesterol and polypeptides to membranes results in unmasking of a continuous phase transition. This results in a second broad peak in the calorimetric curves for melting of lipid-cholesterol mixtures, and the appearance of a second melting transition in membranes modified by the incorporation of polypeptides. The theory assumes that the membrane may be adequately described by a kink model, and that impurities are randomly distributed in the membrane. The difference in size and shape of impurity molecules, compared to membrane lipids, results in a spatial disordering in the membrane which in turn causes increased chain disorder and membrane condensation, as well as a decrease in the cooperativity of melting. The second transition results from a second expansion of the condensed, partially disordered membrane, which takes place over a several degree temperature range. This transition, although unmasked by boundary effects of non-lipid molecules, does not correspond to melting of a boundary annulus or phase separation.     

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.     

Lateral diffusion of gramicidin S, M-13 coat protein and glycophorin in bilayers of saturated phospholipids. Mean field and Monte Carlo studies

We have developed a general model that relates the lateral diffusion coefficient of one isolated large intrinsic molecule (mol. wt. greater than or approximately 1000) in a phosphatidylcholine bilayer to the static lipid hydrocarbon chain order. We have studied how protein lateral diffusion can depend upon protein-lipid interactions but have not investigated possible non-specific contributions from gel-state lattice defects. The model has been used in Monte Carlo simulations or in mean-field approximations to study the lateral diffusion coefficients of Gramicidin S, the M-13 coat protein and glycophorin in dimyristoyl- and dipalmitoylphosphatidylcholine (DMPC and DPPC) bilayers as functions of temperature. Our calculated lateral diffusion coefficients for Gramicidin S and the M-13 coat protein are in good agreement with what has been observed and suggest that Gramicidin S is in a dimeric form in DMPC bilayers. In the case of glycophorin we find that the 'ice breaker' effect can be understood as a consequence of perturbation of the lipid polar region around the protein. In order to understand this effect is necessary that the protein hydrophilic section perturb the polar regions of at least approx. 24 lipid molecules, in good agreement with the numbers of 29-30 measured using 31P-NMR. Because of lipid-lipid interactions this effect extends itself out to four or five lipid layers away from the protein so that the hydrocarbon chains of between approx. 74 and approx. 108 lipid molecules are more disordered in the gel phase, so contributing less to the transition enthalpy, in agreement with the numbers of 80-100 deduced from differential scanning calorimetry (DSC). An understanding of the abrupt change in the diffusion coefficient at a temperature below the main bilayer transition temperature requires an additional mechanism. We propose that this change may be a consequence of a 'coupling-uncoupling' transition involving the protein hydrophilic section and the lipid polar regions, which may be triggered by the lipid bilayer pretransition. Our calculation of the average number of gauche bonds per lipid chain as a function of temperature and distance away from an isolated polypeptide or integral protein shows the extent of statically disordered lipid around such molecules. The range of this disorder depends upon temperature, particularly near the main transition.     

A model for the packing of lipids in bilayer membranes.

A number of known structural properties of mixed lipid bilayer membranes and monolayers are accounted for by a model in which lipids pack into bilayers and monolayers like building blocks, each characterized by a surface head group area and characteristic solid angle. In phospholipids above the melting transition the head group area (at a given temperature and degree of hydration) is fairly invariant while the hydrocarbon region may be liquid-like so long as the molecule is not compressed beyond its characteristic solid angle. Phosphatidylcholine and phosphatidylserine are tapered lipids, i.e. their surface head group areas are greater than their non-polar end areas; cholesterol is frayed, i.e. its polar end area is less than its non-polar end area; while phosphatidylethanolamine is almost cylindrical. The "condensing" effect of cholesterol in mixed phospholipid-cholesterol films is seen as a taper-fray accommodation. The lipid distribution in erythrocyte membrane is shown to be conducive to a stable strain-free membrane.     

Critical point for membrane bilayer formation

Unilamellar liposomes often are employed in investigations of lipid-protein interactions and the delivery of drugs in therapies for disease. Also, related lipid-containing nanoparticles have been developed as elements of a new class of mRNA vaccines. We show that only unilamellar films form in equilibrium lipid dispersions, at temperature values {T*} that depend on the identities of the lipids (e.g., T* ≈ 29 °C for DMPC). Thermodynamic analysis confirms that films at air-water surfaces can be used to monitor the properties of the lipid vesicles that form in the dispersion. When T > T*, critical exponents describing film properties as T approaches T* are μ ≈ 1.4 and ν ≈ 0.7, which are close to values for the interfacial tension and the correlation length of density fluctuations at fluid interfaces. These results, and observations that within the bilayer the lateral diffusion of fluorescent lipid probes demonstrates increases at T*, suggest that unilamellar vesicles at T* are a transition state between two different multilamellar structures. We generalize the thermodynamic arguments to explain the linkage between lipid structures in the surface and bulk dispersion within more complex samples, showing that dispersions containing total lipid extracts of cell membranes have properties similar to those in dispersions containing single lipids. Information from various independent studies indicates that T* noted for bilayer membranes of a population of cells is identical to the temperature at which the growth or gestation of the cells occurs in vivo. Examples include whole-cell lipid extracts obtained from bacteria, and poikilothermic and homeothermic animals.     

Phase-State Dependent Current Fluctuations in Pure Lipid Membranes

Current fluctuations in pure lipid membranes have been shown to occur under the influence of transmembrane electric fields (electroporation) as well as a result from structural rearrangements of the lipid bilayer during phase transition (soft perforation). We demonstrate that the ion permeability during lipid phase transition exhibits the same qualitative temperature dependence as the macroscopic heat capacity of a D15PC/DOPC vesicle suspension. Microscopic current fluctuations show distinct characteristics for each individual phase state. Although current fluctuations in the fluid phase show spikelike behavior of short timescales (∼2 ms) with a narrow amplitude distribution, the current fluctuations during lipid phase transition appear in distinct steps with timescales of ∼20 ms. We propose a theoretical explanation for the origin of timescales and permeability based on a linear relationship between lipid membrane susceptibilities and relaxation times near the phase transition.     

Evidence for phase boundary lipid. Permeability of Tempo-choline into dimyristoylphosphatidylcholine vesicles at the phase transition.

The existence of distinct regions of mismatch in molecular packing at the interfaces of the fluid and ordered domains during the phase transition of dimyristoylphosphatidylcholine vesicles has been demonstrated by measuring the temperature dependence of the permeability to a spin-label cation and comparing this with a statistical mechanical calculation of the fraction of interfacial lipid. The kinetics of uptake and release of the 2,2,6,6-tetramethylpiperidinyl-1-oxycholine (Tempo-choline) spin label by single-bilayer dimyristoylphosphatidylcholine vesicles were measured using electron spin resonance spectroscopy to quantitate the amount of spin label present within the vesicles after removal of the external spin-label by ascorbate at 0 degrees C. Both the uptake and release experiments show that the Tempo-choline permeability peaks to a sharp maximum at the lipid-phase transition, the vesicles being almost impermeable to Tempo-choline below the transition and having a much reduced permeability above. The temperature profile of the permeability is in reasonable quantitative agreement with calculations of the fraction of interfacial boundary lipid from the Zimm and Bragg theory of cooperative transitions, which use independent spin-label measurements of the degree of transition to determine the cooperativity parameter. The relatively high intrinsic permeability of the interfacial regions (P approximately 0.2-1.0 X 10(-8) cm/s) is attributed to the mismatch in molecular packing of the lipid molecules at the ordered-fluid boundaries, which could have important implications not only for permeability in natural membranes (e.g., in transmitter release), but also for the function of membrane-bound enzymes and transport proteins.