Elasticity and phase behavior of DPPC membrane modulated by cholesterol, ergosterol, and ethanol

Giant vesicles formed of 1,2-dipalmitoylphosphatidylcholine (DPPC) and sterols (cholesterol or ergosterol) in water and water/ethanol solutions have been used to examine the effect of sterol composition and ethanol concentration on the area compressibility modulus (K(a)), overall mechanical behavior, vesicle morphology, and induction of lipid alkyl chain interdigitation. Our results from micropipette aspiration suggest that cholesterol and ergosterol impact the order and microstructure of the gel (L(beta)') phase DPPC membrane. At low concentration (10-15 mol%) these sterols disrupt the long-range lateral order and fluidize the membrane (K(a) approximately 300 mN/m). Then at 18 mol%, these sterols participate in the formation of a continuous cohesive liquid-ordered (L(o)) phase with a sterol-dependent membrane density (K(a) approximately 750 for DPPC/ergosterol and K(a) approximately 1100 mN/m for DPPC/cholesterol). Finally at approximately 40 mol% both cholesterol and ergosterol impart similar condensation to the membrane (K(a) approximately 1200 mN/m). Introduction of ethanol (5-25 vol%) results in drops in the magnitude of K(a), which can be substantial, and sometimes individual vesicles with lowered K(a) reveal two slopes of tension versus apparent area strain. We postulate that this behavior represents disruption of lipid-sterol intermolecular interactions and therefore the membrane becomes interdigitation prone. We find that for DPPC vesicles with sterol concentrations of 20-25 mol%, significantly more ethanol is required to induce interdigitation compared to pure DPPC vesicles; approximately 7 vol% more for ergosterol and approximately 10 vol% more for cholesterol. For lower sterol concentrations (10-15 mol%), interdigitation is offset, but by <5 vol%. These data support the idea that ergosterol and cholesterol do enhance survivability for cells exposed to high concentrations of ethanol and provide evidence that the appearance of the interdigitated (L(beta)I) phase bilayer is a major factor in the disruption of cellular activity, which typically occurs between approximately 12 and approximately 16 vol% ethanol in yeast fermentations. We summarize our findings by producing, for the first time, "elasticity/phase diagrams" over a wide range of sterol (cholesterol and ergosterol) and ethanol concentrations.     

Molecular dynamics simulation of the structure of dimyristoylphosphatidylcholine bilayers with cholesterol, ergosterol, and lanosterol

Five molecular dynamics computer simulations were performed on different phospholipid:sterol membrane systems in order to study the influence of sterol structure on membrane properties. Three of these simulated bilayer systems were composed of a 1:8 sterol:phospholipid ratio, each of which employed one of the sterol molecules: cholesterol, ergosterol, and lanosterol. The two other simulations were of a bilayer with a 1:1 sterol:phospholipid ratio. These simulations employed cholesterol and lanosterol, respectively, as their sterol components. The observed differences in simulations with cholesterol and lanosterol may have their implication on the form of the phospholipid/sterol phase diagram.     

Anisotropic motion and molecular dynamics of cholesterol,lanosterol, and ergosterol in lecithin bilayers studied by quasi-elastic neutron scattering

Quasi-elastic neutron scattering (QENS) was employed to study the molecular dynamics of three structurally related sterols, namely, cholesterol, lanosterol, and ergosterol. Oriented bilayers of dipalmitoylphosphatidylcholine (DPPC) were investigated at 40 mol % sterol content and at three temperatures (20, 36, and 50 degrees C) for two energy resolutions. Data analysis was concentrated on a direct comparison of the out-of-plane and the in-plane high-frequency motions of the three sterols in terms of their rates and amplitudes. The (spatially restricted) diffusive motion of the three sterols in the two directions was characterized by diffusion constants in the range of (5-30) x 10(-12) x m(2) x s(-1), with a significantly faster rate of diffusion along the membrane normal, resulting in a diffusional anisotropy, D(a). At low temperature (20 degrees C), cholesterol showed the highest value (D(a) = 4.5), while lanosterol gave the lowest one (D(a) = 2.0). At high temperature (50 degrees C), ergosterol diffusion had the highest diffusion anisotropy (D(a) = 2.0) compared to lanosterol (D(a) = 1.8) and cholesterol (D(a) = 1.6). Most interestingly, cholesterol showed at all three temperatures an amplitude of its out-of-plane-motion of 1.0-1.1 nm, more than a factor of 3 higher than measured for the other two sterols. This finding suggests that the short alkyl chain of the cholesterol molecule may cross at high frequency the bilayer midplane, while the other two sterols remain confined within the geometrical limits of each monolayer leaflet. The results provide an example of how slight structural alterations of sterols can affect their molecular dynamics in bilayers, which in turn may be relevant to the membrane micromechanical properties.     

Differences in the modulation of collective membrane motions by ergosterol, lanosterol, and cholesterol: a dynamic light scattering study

A dynamic light scattering setup was used to study the undulations of freely suspended planar lipid bilayers, the so-called black lipid membranes, over a previously inaccessible range of frequency and wave number. A pure synthetic lecithin bilayer, 1,2-dielaidoyl-sn-3-glycero-phoshatidylcholine (DEPC), and binary mixtures of DEPC with 40 mol % of cholesterol, ergosterol, or lanosterol were studied. By analyzing the dynamic light scattering data (oscillation and damping curves) in terms of transverse shear motion, we extracted the lateral tension and surface viscosity of the composite bilayers for each sterol. Cholesterol gave the strongest increase in lateral tension (approximately sixfold) with respect to the DEPC control, followed by lanosterol (approximately twofold), and ergosterol (1.7-fold). Most interestingly, only cholesterol simultaneously altered the surface viscosity of the bilayer by almost two orders of magnitude, whereas the other two sterols did not affect this parameter. We interpret this unique behavior of cholesterol as a result of its previously established out-of-plane motion which allows the molecule to cross the bilayer midplane, thereby effectively coupling the bilayer leaflets to form a highly flexible but more stable composite membrane.     

Phase behavior of lipid monolayers containing DPPC and cholesterol analogs

We investigate the miscibility phase behavior of lipid monolayers containing a wide variety of sterols. Six of the sterols satisfy a definition from an earlier study of "membrane-active sterols" in bilayers (cholesterol, epicholesterol, lathosterol, dihydrocholesterol, ergosterol, and desmosterol), and six do not (25-hydroxycholesterol, lanosterol, androstenolone, coprostanol, cholestane, and cholestenone). We find that monolayers containing dipalmitoyl phosphatidylcholine mixed with membrane-active sterols generally produce phase diagrams containing two distinct regions of immiscible liquid phases, whereas those with membrane-inactive sterols generally do not. This observation establishes a correlation between lipid monolayers and bilayers. It also demonstrates that the ability to form two regions of immiscibility in monolayers is not one of the biophysical attributes that explains cholesterol's predominance in animal cell membranes. Furthermore, we find unusual phase behavior for dipalmitoyl phosphatidylcholine monolayers containing 25-hydroxycholesterol, which produce both an upper and a lower miscibility transition. The lower transition correlates with a sharp change of slope in the pressure-area isotherm.     

Bilayer thickness and thermal response of dimyristoylphosphatidylcholine unilamellar vesicles containing cholesterol, ergosterol and lanosterol: a small-angle neutron scattering study

Small-angle neutron scattering (SANS) measurements are performed on pure dimyristoyl phosphatidylcholine (DMPC) unilamellar vesicles (ULV) and those containing either 20 or 47 mol% cholesterol, ergosterol or lanosterol. From the SANS data, we were able to determine the influence of these sterols on ULV bilayer thickness and vesicle area expansion coefficients. While these parameters have been determined previously for membranes containing cholesterol, to the best of our knowledge, this is the first time such results have been presented for membranes containing the structurally related sterols, ergosterol and lanosterol. At both molar concentrations and at temperatures ranging from 10 to 45 degrees C, the addition of the different sterols leads to increases in bilayer thickness, relative to pure DMPC. We observe large differences in the influence of these sterols on the membrane thermal area expansion coefficient. All three sterols, however, produce very similar changes to membrane thickness.     

Effects of trehalose on the phase behavior of DPPC–cholesterol unilamellar vesicles

A systematic study is presented of the effects of trehalose on the physical properties of extruded DPPC–cholesterol unilamellar vesicles. Particular emphasis is placed on examining how the interactions present in the hydrated state translate into those in the dehydrated state. Observations from HSDSC and DSC are used to examine the phase behavior of hydrated and dehydrated vesicles, respectively. The concentration of trehalose inside and outside the vesicles is manipulated, and is shown to affect the relative stability of the membranes. Our results show for the first time that a combination of high inner and low outer trehalose concentration is able to decrease the gel-to-liquid crystalline phase temperature (T_m), while any other combination will not. Upon dehydration, the T_m of all lipid mixtures increases. The extent of the increase depends on the trehalose distribution across the bilayer. The T_m changes in the same direction with trehalose concentration for both freeze-dried and fully hydrated samples, suggesting that the trehalose distribution across the vesicle membrane, as well as the trehalose–phospholipid interaction, is maintained upon lyophilization. The results presented in this work may aid in the formulation of systems to be used in the lyophilization of liposomes for drug delivery applications.     

FTIR spectroscopic study of molecular organization of the antibiotic amphotericin B in aqueous solution and in DPPC lipid monolayers containing the sterols cholesterol and ergosterol

Langmuir monolayers of amphotericin B (AmB) were investigated by recording π-A isotherms under different pH conditions. To gain a better insight into antibiotic-membrane interactions they were monitored by use of the ATR-FTIR spectroscopy. It was observed for AmB monolayers that the limiting molecular area was larger at high than at neutral pH. Analysis of FTIR spectra at different pH revealed substantial differences, depending on ionic state, for different orientations of AmB molecules. These results enable better understanding of the participation of functional groups in the interactions between AmB and sterol-containing DPPC membranes. AmB molecules incorporated into two-component lipid monolayers bind strongly to the ergosterol-rich membrane (maximum penetration surface pressures ca 35?mN/m). The FTIR spectra revealed that the ionic state of AmB and the presence of sterols led to changes in membrane fluidity and molecular packing of the AmB molecules in the lipid membranes. These investigations should be further investigated to discover the molecular mechanism responsible for the mode of action AmB in biological systems.     

New phases of DPPC/water mixtures

Hydration of DPPC at low temperatures yielded two new phases, a non-lamellar C1 phase and a lamellar C2 phase, as well as the normal gel phase, depending upon the initial physical state of the dry lipid. From the results of wide-angle diffraction and calorimetry the C2 phase appears very similar to the normal C phase, but the D spacing is considerably larger, suggesting that the C2 phase is a C phase with untilted chains.     

Structure formation in low-concentrated solutions of cholesterol and ergosterol

The conformational behavior of biologically important chiral molecules of cholesterol and ergosterol was studied via the method of molecular dynamics. The formation of strings and their absence was experimentally observed in methanol solutions of cholesterol and ergosterol, respectively. It was shown that the intermolecular dynamics of the molecule significantly affects the possibility of structure formation. We proposed an alternative explanation of the functional significance of cholesterol, which is apparently associated with the formation of commutation structures outside the membrane, and the biological feasibility of the presence of ergosterol in the non-commuting cells of fungi and cholesterol in the commuting cells of macroorganisms.     

Changes of the physical properties of the liquid-ordered phase with temperature in binary mixtures of DPPC with cholesterol: A 2H-NMR, FT-IR, DSC, and neutron scattering study

The structure of the so-called liquid-ordered (lo) phase of binary mixtures of DPPC-d₆₂ with cholesterol was studied between 20-50 mol% cholesterol using ²H-NMR, FT-IR, DSC, and neutron specular reflection. Different model systems such as multilamellar vesicles, spherical supported vesicles, and oriented multilayers were used. We observed significant changes of the lo phase structure in the physiological relevant temperature region between 30-45°C. ²H-NMR in combination with lineshape simulations provides evidence for a drastic effect of cholesterol on the shape of multilamellar vesicles due to magnetic field orientation. Moreover, the data indicates a significant change of the extent of this partial orientation for DPPC-d₆₂ multilamellar vesicles containing 25 mol% cholesterol between 36-42°C. The semiaxes ratio of the (due to magnetic field orientation) ellipsoidal multilamellar vesicles changes over this temperature range by ≈25%. ²H-NMR and FT-IR measurements indicate changes of the average orientational order at the bilayer center in the same temperature range and a significant increase of the number of end-gauche conformers while the majority of the methylene groups remain in an all-trans conformation. Additionally, specular reflection of neutrons shows a concomitant reduction of the bilayer thickness by 4 Å. Based on a model of the arrangement of DPPC and cholesterol in the lo phase, a molecular mechanism is proposed in which increasing the temperature between 30 and 45°C has the effect of driving cholesterol from the bilayer center towards the head group region.     

Hydration of DMPC and DPPC at 4 degrees C produces a novel subgel phase with convex-concave bilayer curvatures

Hydration of dimyristoyl- and dipalmitoylphosphatidylcholines at 4 degrees C results in the formation of a characteristic subgel phase designated Pcc. Examination of the phase by freeze-fracture electron microscopy shows convex-concave deformations of the planar bilayer which are of two types. A smaller type with a radius of curvature of about 20 nm predominates in DMPC, and a larger type with about 70 nm radii of curvatures dominates in DPPC. The Pcc phase can also be formed in samples hydrated at temperatures above the main phase transition if the dispersion is frozen slowly and subsequently incubated at 4 degrees C for several days. The subgel Pcc phase was distinguished from the subgel Lc phase by the temperature of transition, packing of the acyl chains on the basis of wide-angle X-ray diffraction, and 2H-NMR spectra characteristic of a 'solid-ordered' phase. Vibrational spectra of the carbonyl and phosphate regions are consistent with a partially reduced hydration state. The origin of the convex-concave bilayer deformation is believed to result from constraints imposed by limiting hydration of the headgroup and a frustration arising from the spontaneous curvature of both monolayers.     

Discrimination between cholesterol and ergosterol by yeast membranes

The typical sterol of animal membranes (cholesterol) failed to support normal growth of yeast under anaerobic conditions when compared to the growth induced by the organism's natural sterol (ergosterol). This pathology was evident in strongly reduced numbers of cells, failure of budded cells to separate, and premature death. This study demonstrates that one or all of the Δ^5,7-, Δ²²-, and 24β-methyl groupings present in ergosterol are functionally significant.     

Cholesterol, lanosterol, and ergosterol attenuate the membrane association of LL-37(W27F) and temporin L

Sterols impart significant changes to the biophysical properties of lipid bilayers. In this regard the impact of cholesterol on membrane organization and dynamics is particularly well documented and serves for comparison with other sterols. However, the factors underlying the molecular evolution of cholesterol remain enigmatic. To this end, cholesterol attenuates membrane perturbation by the so-called antimicrobial peptides (AMPs), produced ubiquitously by eukaryotic cells to combat bacterial infections by compromising the permeability barrier function of the microbial target membranes. In the present study, we addressed the effects of cholesterol, ergosterol, and lanosterol on the membrane association of two structurally and functionally diverse AMPs viz. LL-37(F27W) and temporin L (TemL) using fluorescence spectroscopy. Interestingly, sterol concentration dependent effects on the membrane association of these peptides were observed. At X(Sterol)=0.5 cholesterol was most effective in reducing the membrane intercalation of both LL-37(F27W) and TemL, the corresponding efficiencies of the three sterols decreasing as cholesterol>lanosterol> or =ergosterol, and cholesterol>lanosterol>ergosterol. It is conceivable that part of the selection pressure for the chemical evolution of cholesterol may have derived from the ability to protect the AMP-secreting host cell from the membrane damaging action of the antimicrobial peptides.