The influence of cholesterol on molecular motion in egg lecithin bilayers--a variable-frequency electron spin resonance study of a cholestane spin probe

Electron spin resonance spectra at 9.5, 24. and 35 GHz were obtained for a cholestane spin probe in oriented multibilayers of egg lecithin of varying cholesterol content. In agreement with earlier studies, cholesterol induced a higher degree of spectral anisotropy in the multibilayers—the variation of the hyperfine separations with cholesterol content was in agreement with the model of Lapper et al. (Can. J. Biochem.50, 969 (1972)) where the amplitude of anisotropic probe motion decreased with increasing cholesterol content. Analysis of the electron spin resonance line shapes was done using the relatively simple modified Bloch equation approach, and correlation times for anisotropic probe motion were extracted from the spectra at three frequencies. The data demonstrate that increasing cholesterol content results in a decreased rate of anisotropic motion of the probe, providing further insight into the molecular mechanism of the condensing effect of cholesterol.     

Thermal effects on motion and orientation of the cholestane spin label in planar multibilayers of dimyristoylphosphatidylcholine and dipalmitoylphosphatidylcholine

A detailed picture of the orientation and restricted motion of the cholestane spin label (3-spiro-doxyl-5α-cholestane) in planar multibilayers of dipalmitoylphosphatidylcholine and dimyristoylphosphatidylcholine has been recorded by simultaneous simulation of ESR spectra obtained with the magnetic field parallel and perpendicular to the bilayers (Shimoyama, Y., Eriksson, L.E.G. and Ehrenberg, A. (1978) Biochim. Biophys. Acta 508, 213–235). The analysis has been made over the temperature range ?30°C to 60°C on samples containing 20 to 22% water. At low temperatures the cholestane spin label is tilted with respect to the lipid bilayer normal by an angle of approx. 30° which disappears at the pretransition. In this low temperature range the restricted twisting motion has an activation energy of 5.5 kJ·mol^?1. Above the main transition the twisting motion is unrestricted and has the activation energy 20 kJ·mol^?1. From below the pretransition to above the main transition the velocity of the twisting motion increases by an order of magnitude. The amplitude of the wobbling motion increases abruptly from 0° to 35° at the main transition.     

An EPR spin probe study of liposomes from sunflower and soybean phospholipids

Comparative properties of lecithin-based liposomes prepared from the mixed phospholipids of sunflower seeds, soybean and egg yolk were investigated by electron paramagnetic resonance (EPR) spectroscopy. For these investigations, stable nitroxide radicals, 1-oxyl-2,2,6,6-tetramethylpiperidin-4-yl 5,7-dimethyladamantane-1-carboxylate (DMAC-TEMPO), 5-doxylstearic acid (5-DSA) and 16-doxylstearic acid (16-DSA) were used as spin probes. Binding of the spin probes to the liposome membranes resulted in a substantial increase of the apparent rotational diffusion correlation times. The EPR spectra of the incorporated nitroxides underwent temperature-dependent changes. For every spin probe, values of apparent enthalpy and entropy of activation were calculated from the temperature dependence of rotational diffusion correlation times via Arrhenius equation. In case of DMAC-TEMPO, the data point to differences between the phospholipid bilayer of liposomes derived from sunflower and soy lecithin, and some similarity between the sunflower and egg yolk liposomes. Anisotropic hyperfine interaction constants of DMAC-TEMPO and 16-DSA included in the liposomes have been analyzed and attributed to different micropolarity of the surroundings of the spin probes. The kinetics of EPR signal decay of DMAC-TEMPO in the presence of 2,2′-azobis(2-amidinopropane) suggest the better stability of the sunflower liposomes to lipid peroxidation as compared to the liposomes prepared from soy lecithin.     

An ESR Study of the mobility of the cholestane spin label in oriented lecithin-cholesterol multibilayers.

The motion of the cholestane spin label in oriented lecithin-cholesterol multibilayers is described in terms of a rotational diffusion about the long molecular axis with diffusion coefficient D parrell and a restricted random librational motion about axes perpendicular to the long axis with diffusion coefficient D1. The diffusion coefficients have been determined from the angular dependence of the ESR line shape at various temperatures and cholesterol contents. The temperature dependence of D parrell and D1 clearly shows the transition from the gel to liquid crystalline phase. Increasing amounts of cholesterol reduce the transition temperature. A strong reduction is found from o to 10 mole % cholesterol. At 50 mole % no longer a sharp transition is observed. In the temperature range from 40 to 80 degrees C the range of D is about 10 times larger than the range of D parrell, indicating a high activation energy for the librational motion arising from a strong hindrance by interaction with surrounding molecules. Cholesterol contents up to 10-20 mole % give an increase of D parrell and D1, arising from strong decrease of the transition temperature in this range. Above 10-20 mole % a reduction of D parrell and D1 is found. However, the effect of cholesterol is much stronger on D1 than on D parrell. In the liquid crystalline phase at about 60 degrees C the effect of cholesterol on D parrell is even negligible, while D1 strongly changes. This indicates that in the liquid crystalline phase only the librational motion is influenced by cholesterol, due to a denser packing of the molecules in the bilayer.     

Interaction of cholesterol with photoactivable phospholipids in sonicated vesicles

Photoactivable phospholipids containing either α-diazo-β-trifluoropropionyloxy or m-diazirinophenoxyl groups in the ω-positions of sn-2 fatty acyl chains were synthesized and incorporated into sonicated vesicles containing 33 mol% of cholesterol. Photolysis of the vesicles at 350 nm produced covalent cross-links between the synthetic phospholipids and cholesterol. The cross-linked products obtained using [¹⁴C]cholesterol were characterized by their chromatographic behavior, cleavage on phospholipase A₂ treatment, base-catalyzed transesterification and mass spectral measurements. The cross-linking was shown not to involve the 3-β-hydroxyl group of cholesterol, and it was concluded that the reactive carbene intermediates formed from the photolabels inserted into the hydrocarbon skeleton of cholesterol in the bilayer. The extent of cross-linking obtained was comparable to that observed previously using phospholipids alone, indicating that no lateral phase separation occurred. The present approach is promising for further precise studies of the molecular interactions between cholesterol and phospholipids in biological membranes.     

The effect of cholesterol on the lateral diffusion of phospholipids in oriented bilayers

Pulsed field gradient NMR was utilized to directly determine the lipid lateral diffusion coefficient for the following macroscopically aligned bilayers: dimyristoylphosphatidylcholine (DMPC), sphingomyelin (SM), palmitoyloleoylphosphatidylcholine (POPC), and dioleoylphosphatidylcholine (DOPC) with addition of cholesterol (CHOL) up to approximately 40 mol %. The observed effect of cholesterol on the lipid lateral diffusion is interpreted in terms of the different diffusion coefficients obtained in the liquid ordered (l(o)) and the liquid disordered (l(d)) phases occurring in the phase diagrams. Generally, the lipid lateral diffusion coefficient decreases linearly with increasing CHOL concentration in the l(d) phase for the PC-systems, while it is almost independent of CHOL for the SM-system. In this region the temperature dependence of the diffusion was always of the Arrhenius type with apparent activation energies (E(A)) in the range of 28-40 kJ/mol. The l(o) phase was characterized by smaller diffusion coefficients and weak or no dependence on the CHOL content. The E(A) for this phase was significantly larger (55-65 kJ/mol) than for the l(d) phase. The diffusion coefficients in the two-phase regions were compatible with a fast exchange between the l(d) and l(o) regions in the bilayer on the timescale of the NMR experiment (100 ms). Thus, strong evidence has been obtained that fluid domains (with size of micro m or less) with high molecular ordering are formed within a single lipid bilayer. These domains may play an important role for proteins involved in membrane functioning frequently discussed in the recent literature. The phase diagrams obtained from the analysis of the diffusion data are in qualitative agreement with earlier published ones for the SM/CHOL and DMPC/CHOL systems. For the DOPC/CHOL and the POPC/CHOL systems no two-phase behavior were observed, and the obtained E(A):s indicate that these systems are in the l(d) phase at all CHOL contents for temperatures above 25 degrees C.     

Interaction of cholesterol with conformationally restricted phospholipids in vesicles.

The interaction of cholesterol with conformationally restricted analogs of dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidylglycerol (DPPG) in the liquid-crystalline phase has been studied in vesicles. These analogs contain one of three cyclopentane triols in place of the glycerol moiety found in natural phospholipids and make possible an analysis of whether a limitation of the conformational mobility in the glycerol backbone region affects the interaction with cholesterol. When cholesterol was incorporated into vesicles from cyclopentanoid phospholipids in which the acyl group vicinal to the head group is trans, the first-order rate constant for Cl- efflux is decreased similarly to that in vesicles from 'natural' DPPC or DPPG (about 50%). However, when the head group is in the unnatural 2 position, cholesterol has a much smaller effect on the rate of Cl- efflux (a decrease of about 20%). Cholesterol decreased the rate constants for valinomycin-mediated 86Rb+ efflux from vesicles of the cyclopentanoid PC analogs and of DPPC to a similar extent. The half-time values for spontaneous intervesicle cholesterol exchange were not markedly different using vesicles prepared with the natural glycerophospholipids and with the cyclopentano-phospholipids, suggesting that the geometrical orientation of the acyl chains or the head group has little influence on cholesterol desorption from the lipid/water interface.     

Cholestane spin label study of filipin action on lipid planar multibilayers.

EPR spectra of a cholestane probe dissolved in egg yolk lecithin and lecithin-cholesterol planar multibilayers were observed as a function of the filipin dose. The probe is structurally similar to cholesterol; its normal position when dissolved is with the long axis approximately along the bilayer normal. Both cholesterol-containing and cholesterol-free samples showed spectral components characteristic of bilayer fragmentation (tilted domains) which increased with dose. Furthermore, the cholesterol-free spectra indicated that some of the probe was frozen with the long molecular axis perpendicular to the slide normal. The frozen spectral component increased with dose. Spectra from a fatty acid probe did not have this feature. We interpret this as due to probe complexed with filipin (in place of cholesterol) in accordance with the filipin-cholesterol aggregate model of deKruijff and Demel. An ultraviolet study of filipin-probe interaction indicates that the probe is capable of complexing in just such a manner but has less affinity for the drug than cholesterol. Spectra from the cholesttane probe in liposomes were also observed.     

Effect of cholesterol on viscoelastic properties of dipalmitoylphosphatidylcholine multibilayers as measured by a laser-induced ultrasonic probe

Using a novel laser-induced ultrasonic probe, we have examined the bulk viscoelastic properties of fully hydrated dipalmitoylphosphatidylcholine (DPPC) aligned multibilayers in terms of the anisotropic in-plane elastic stiffness (C11) and viscosity (eta 11). Our measurements of C11 are in accord with those reported on Brillouin light scattering on a similar system. Our measurements on viscosity are the first of their kind and are, on the average, a factor of 10 lower than microviscosities estimated by spectroscopic techniques. We report the first comprehensive study of the effects of cholesterol on the bulk mechanical properties of DPPC multibilayers. At temperatures above the phase transition temperature of DPPC (Tc), an increase in both C11 and eta 11 is noticed when cholesterol is incorporated in the multibilayers. However, at temperatures below Tc, no measurable changes are detected in either C11 or eta 11. These results, reflecting changes in the bulk viscoelastic properties of the multibilayers, differ from the changes reported by local fluidity parameters in that the latter indicate a decrease in the bilayer fluidity in the presence of cholesterol above Tc and an increase below Tc ("dual effect" of cholesterol). Our data suggest that the "dual effect" of cholesterol is noticeable only on a molecular scale. Increasing cholesterol concentrations higher than 20 mol % cease to further affect C11 or eta 11 of the DPPC multibilayers. This agrees with various results reported in the literature, by techniques measuring the local effects of cholesterol, and supports the changes in molecular organization postulated to occur when cholesterol concentration reaches 20 mol % in the lipid bilayers.     

Effects of cholesterol on the divalent cation-mediated interactions of vesicles containing amino and choline phospholipids

We have used assays of lipid probe mixing, contents mixing and contents leakage to monitor the divalent cation-mediated interactions between lipid vesicles containing phosphatidylserine (PS) as a minority component together with mixtures of phosphatidylethanolamine (PE), phosphatidylcholine (PC) or sphingomyelin, and cholesterol in varying proportions. The initial rates of calcium- and magnesium-induced lipid probe quenching between vesicles, which reflect primarily the rates of vesicle aggregation, are strongly reduced as progressively higher proportions of PC or sphingomyelin are incorporated into PE/PS vesicles. The initial rates of divalent cation-induced contents mixing and contents leakage for PE/PS vesicles are also strongly reduced when choline phospholipids are incorporated into the vesicles in even low molar proportions. Sphingomyelin has a more potent inhibitory effect on these processes than does PC at an equal level in the vesicle membranes. The inclusion of cholesterol in these vesicles, at levels up to 1:2 moles sterol/mole phospholipid, has little effect on the rates of calcium- or magnesium-induced vesicle aggregation. However, cholesterol significantly enhances the initial rates of vesicle contents mixing and contents leakage in the presence of divalent cations when the vesicles contain choline as well as amino phospholipids. This effect is substantial only when the level of cholesterol exceeds the level of choline phospholipids in the vesicles. These results may have significance for the fusion of certain cellular membranes in mammalian cells, whose cytoplasmic faces have lipid compositions very similar to those of the vesicles examined in this study.     

Effect of subtransition in 1,2-dipalmitoylphosphatidylcholine model membrane on the spin probe motion

The effect of subgel----gel phase transition (subtransition) on conventional electron spin resonance spectra of cholestane, fatty acid and alkylammonium-type spin probes has been studied in aqueous 1,2-dipalmitoyl-sn-phosphatidylcholine dispersions. The cooperative onset of the cholestane spin probe rotation about its long axis, with an effective correlation time of 2-3 ns, has been detected at a temperature coinciding with the calorimetric substransition, indicating onset of the host lipid rotational motion. The lipid rotation results in dissolution of the spin probe clusters in the host lipid. In the gel phase, the lateral distribution of impurity molecules is more isotropic than in the subgel phase.     

Separation of liquid phases in giant vesicles of ternary mixtures of phospholipids and cholesterol

We use fluorescence microscopy to directly observe liquid phases in giant unilamellar vesicles. We find that a long list of ternary mixtures of high melting temperature (saturated) lipids, low melting temperature (usually unsaturated) lipids, and cholesterol produce liquid domains. For one model mixture in particular, DPPC/DOPC/Chol, we have mapped phase boundaries for the full ternary system. For this mixture we observe two coexisting liquid phases over a wide range of lipid composition and temperature, with one phase rich in the unsaturated lipid and the other rich in the saturated lipid and cholesterol. We find a simple relationship between chain melting temperature and miscibility transition temperature that holds for both phosphatidylcholine and sphingomyelin lipids. We experimentally cross miscibility boundaries both by changing temperature and by the depletion of cholesterol with beta-cyclodextrin. Liquid domains in vesicles exhibit interesting behavior: they collide and coalesce, can finger into stripes, and can bulge out of the vesicle. To date, we have not observed macroscopic separation of liquid phases in only binary lipid mixtures.     

Photochemical study of the interaction of phenothiazine derivatives with spin labelled lecithin multibilayers.

Nitroxide free radicals are destroyed by ultraviolet irradiation in the presence of phenothiazine derivatives. This property has been used in order to determine the type of interaction of these drugs with spin-labelled lecithin multibilayers. Kinetic measurements of the spin label signal decay under irradiation have shown that chlorpromazine and perphenazine are preferentially located in the polar part of the bilayer, whereas promethazine and oxidized derivatives of chlorpromazine are found principally in the hydrophobic part.     

A laser-induced ultrasonic probe of the mechanical properties of aligned lipid multibilayers.

The recently developed laser-induced phonon spectroscopy (LIPS) technique is applied to the determination of dynamic mechanical properties of aligned dilauroylphosphatidylcholine (DLPC) multibilayer arrays containing 2 and 20% water by weight. Sample excitation by two crossed 100-ps laser pulses generates a longitudinal ultrasonic wave whose wavelength depends on the crossing angle. In these experiments, the acoustic wave propagates parallel to the bilayer planes. The ultrasonic velocity and attenuation are monitored through the diffraction of a variably delayed probe pulse by the acoustic grating. The velocity measures the lateral area compressibility of the bilayers, while the attenuation is related to the viscosity. Velocities obtained in the gel and liquid crystal phases are compared with those found previously using Brillouin scattering. The acoustic attenuation is shown to be an order of magnitude more sensitive to the gel-liquid crystal phase transition than the velocity. The lipid area compressibility and viscosity of DLPC-20% water multilayers with and without 100 mM CaCl2 are found to be identical within our experimental error.