Dynamics of phosphatidylcholine-cholesterol mixed model membranes in the liquid crystalline state.

The effects of cholesterol on the dynamics of cholestane spin probe (CSL) in various phosphatidylcholine-cholesterol mixed model membranes are examined. The lateral diffusion, D of CSL in DMPC/POPC/cholesterol ternary mixtures, is measured utilizing an improved dynamic imaging electron spin resonance method. It shows a factor of two decrease at 10 mol % and 25 degrees C, whereas it shows only a 40% decrease at 50 mol % and 50 degrees C. A comparison with results in POPC/cholesterol mixtures, which show a stronger effect of cholesterol on D, indicates that acyl chain unsaturation leads to stronger self association of cholesterol in PC model membranes. An S2CSL dependence of the activation energy for D, has been confirmed for the DMPC/POPC/cholesterol mixtures. Here SCSL is the order parameter for CSL. A similar correlation of R perpendicular, the perpendicular component of the rotational diffusion coefficient, with SCSL, which is true for all three mixtures (DMPC/cholesterol, POPC/cholesterol, and DMPC/POPC/cholesterol) we have studied, is also found. These are associated with the effects of enhanced local ordering on the free volume needed for translation and reorientation. Such correlations of dynamic properties D and R perpendicular with the thermodynamic quantity S, as well as the consistent interpretations of the effect of acyl chain unsaturation on the dynamics in terms of the activity coefficients, strongly emphasize the interrelation between the dynamic structure and the thermodynamics of the PC/cholesterol mixtures.     

Dynamic imaging of lateral diffusion by electron spin resonance and study of rotational dynamics in model membranes. Effect of cholesterol.

The effects of cholesterol on the dynamics and the structural properties of two different spin probes, the sterol type CSL and the phospholipid type 16-PC, in POPC/cholesterol oriented multilayer model membranes were examined. Our results are consistent with a nonideal solution containing cholesterol-rich clusters created by the self association of cholesterol in POPC model membranes. The lateral diffusion coefficient D of the spin probes was measured over the temperature range of 15 to 60 degrees C and over the concentration range of 0 to 30 mol% of cholesterol in the model membrane by the electron spin resonance (ESR) imaging method. The rotational diffusion coefficients (including R perpendicular) and the order parameter S were determined utilizing a nonlinear least square ESR spectral simulation method. D, R perpendicular and S of CSL deviate considerably from linear dependence on mole percent cholesterol. The D of CSL was decreased by a factor of four at 15 degrees C and a factor of two at 60 degrees C for concentrations of cholesterol over 10 mol %, whereas those of 16-PC were hardly affected. Cholesterol decreased R perpendicular by a factor of 10 at 30 mol % of cholesterol, but it increased slightly that of 16-PC. A significant increase of S for CSL due to the presence of cholesterol was observed. It is shown how the difference in variation of S for CSL vs. 16-PC with composition may be interpreted in terms of their respective activity coefficients, and how a single universal linear relation is obtained for the S of both probes in terms of a scaled temperature. Simple but general correlations of D and of R perpendicular with S were also found, which aid in the interpretation of these diffusion coefficients.     

Dynamics and ordering in mixed model membranes of dimyristoylphosphatidylcholine and dimyristoylphosphatidylserine: a 250-GHz electron spin resonance study using cholestane.

We report here on a 250-GHz electron spin resonance (ESR) study of macroscopically aligned model membranes composed of mixtures of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylserine (DMPS), utilizing the nixtroxide-labeled cholesterol analog cholestane (CSL). Two clearly resolved spectral components, distinct in both their ordering and dynamics, are resolved. The major component in membranes composed mostly of DMPC shows typical characteristics, with the long axis of CSL parallel to the bilayer normal with slow (10(6) </= R </= 10(7) s-1) rotational diffusion rates, as expected for cholesterol. The second component grows in as the mole fraction of DMPS increases. A detailed analysis shows that CSL senses a local, strongly biaxial environment. Our results imply that the inefficient packing between cholesterol and DMPS occurs probably because of the strong interactions between the PS headgroups, which provide the local biaxiality. Such a packing of the headgroups has been predicted by molecular dynamics simulations but had not been observed experimentally. The analysis of these spectral components was greatly aided by the excellent orientational resolution provided by the 250-GHz spectra. This enabled the key qualitative features of this interpretation to be "read" off the spectra before the detailed analysis.     

Thermodynamics of phosphatidylcholine-cholesterol mixed model membranes in the liquid crystalline state studied by the orientational order parameter.

It is shown that good estimates of the activity of cholesterol in phosphatidylcholine-cholesterol mixed model membranes are obtained by examining the orientational order parameter S of cholestane spin probe (CSL) that is obtained from electron spin resonance by spectral simulation. By introducing thermodynamic stability conditions of liquid mixtures, the variation of activity (or S) as a function of cholesterol mole fraction is utilized to predict the concentration at which the phase separation occurs. These results for DMPC and cholesterol binary mixtures agree very well with those of Tempo-partitioning experiments. The comparison of activity coefficients and the phase boundary in DMPC/cholesterol mixtures with those of POPC/cholesterol mixtures suggests that acyl chain unsaturation leads to poorer mixing of cholesterol in phosphatidylcholine model membranes at higher temperatures (i.e., greater than 35 degrees C). In ternary solutions of DMPC, POPC, and cholesterol, it is found that cholesterol shows less deviation from ideality than in either of the two binary mixtures, and this implies that the phase separation occurs at higher cholesterol concentration than in either of the two binary mixtures. The present analysis suggests that there may not be a critical point in DMPC/cholesterol mixtures, even though phase separation does occur.     

Rotational diffusion of a steroid molecule in phosphatidylcholine membranes: effects of alkyl chain length, unsaturation, and cholesterol as studied by a spin-label method

Rotational diffusion of cholestane spin-label (CSL), a sterol analogue, in various phosphatidylcholine (PC)-cholesterol membranes was systematically studied by computer simulation of steady-state ESR spectra as a function of chain length and unsaturation of alkyl chains, cholesterol mole fraction, and temperature for better understanding of phospholipid-cholesterol and cholesterol-cholesterol interactions. CSL motion in the membrane was treated as Brownian rotational diffusion of a rigid rod within the confines of a cone imposed by the membrane environment. The wobbling rotational diffusion constant of the long axis, its activation energy, and the cone angle of the confines are obtained for various membranes in the liquid-crystalline phase. The wobbling diffusion constant decreases in the order dilauroyl-PC greater than dimyristoyl-PC greater than dioleoyl-PC approximately dipalmitoyl-PC greater than distearoyl-PC greater than dioleoyl-PC/cholesterol = 3/1 greater than dioleoyl-PC/cholesterol = 1/1 membranes. Activation energy for the wobbling diffusion of the long axis of CSL is strongly dependent on alkyl chain length, unsaturation, and cholesterol mole fraction. It decreases with decrease in alkyl chain length and by introduction of unsaturation in the alkyl chains. In dioleoylphosphatidylcholine membranes, activation energy decreases by a factor of approximately 3 in the presence of 50 mol % cholesterol. Activation energy for wobbling diffusion of CSL in phosphatidylcholine membranes is smaller than the activation energy for translational diffusion of a phospholipid. The former is more dependent on alkyl chain length and unsaturation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cholesterol and ergosterol superlattices in three-component liquid crystalline lipid bilayers as revealed by dehydroergosterol fluorescence.

We have examined the fractional sterol concentration dependence of dehydroergosterol (DHE) fluorescence in DHE/cholesterol/dimyristoyl-L-alpha-phosphatidylcholine (DMPC), DHE/ergosterol/DMPC and DHE/cholesterol/dipalmitoyl-L-alpha-phosphatidylcholine (DPPC) liquid-crystalline bilayers. Fluorescence intensity and lifetime exhibit local minima (dips) whenever the total sterol mole fraction, irrespective of the DHE content, is near the critical mole fractions predicted for sterols being regularly distributed in hexagonal superlattices. This result provides evidence that all three of these naturally occurring sterols (e.g., cholesterol, ergosterol, and DHE) can be regularly distributed in the membrane and that the bulky tetracyclic ring of the sterols is the cause of regular distribution. Moreover, at the critical sterol mole fractions, the steady-state anisotropy of DHE fluorescence and the calculated rotational relaxation times exhibit distinct peaks, suggesting that membrane free volume reaches a local minimum at critical sterol mole fractions. This, combined with the well-known sterol condensing effect on lipid acyl chains, provides a new understanding of how variations in membrane sterol content change membrane free volume. In addition to the fluorescence dips/peaks corresponding to hexagonal superlattices, we have observed intermediate fluorescence dips/peaks at concentrations predicted by the centered rectangular superlattice model. However, the 22.2 mol% dip for centered rectangular superlattices in DHE/ergosterol/DMPC mixtures becomes diminished after long incubation (4 weeks), whereas on the same time frame the 22.2 mol% dip in DHE/cholesterol/DMPC mixtures remains discernible, suggesting that although all three of these sterols can be regularly distributed, subtle differences in sterol structure cause changes in lateral sterol organization in the membrane.     

Electron spin resonance and electron-spin-echo study of oriented multilayers of L alpha-dipalmitoylphosphatidylcholine water systems.

A detailed electron spin resonance (ESR) study of spin-labeled-oriented multilayers of L alpha-dipalmitoylphosphatidylcholine (DPPC) water systems for low water content (2-10% by weight) is reported with the purpose of characterizing the dynamical and structural properties of model membrane systems. Emphasis is placed on the value of combining such experiments with detailed simulations based on current slow-motional theories. Information is obtained regarding ordering and anisotropic rotational diffusion rates via ESR lineshape analysis over the entire motional range, from the fast motional region through the moderately slow and slow to the rigid limit. This includes the low-temperature gel phase, the liquid crystalline L alpha (1) phase and what appears to be a third high-temperature phase above the L alpha phase. Cholestane (CSL) and spin-labeled DPPC (5-PC, 8-PC, and 16-PC) have been used to probe different depths of the bilayer. While CSL and 5-PC both reflect the high ordering of the bilayer close to the lipid-water interface, CSL appears to be located close enough to the water for the nitroxide to be involved in hydrogen bonding with water molecules. 16-PC reflects the relatively low ordering near the tail of the hydrocarbon chain in the bilayer. Quantitative estimates of ordering and motion are obtained for these cases. The results from CSL indicate that close to the lipid-water interface the DPPC molecule is oriented approximately perpendicular to the bilayer in these low water-content systems. However, all three labeled lipid probes indicate that the hydrocarbon chain of DPPC may be bent away from the bilayer normal by as much as 30 degrees and this evidence is stronger at low temperatures. When cholesterol is added to the DPPC-water system at a concentration greater than or equal to 2.5 mol %, the ordering is greatly increased although the rotational diffusion rate remains almost unaffected in the gel phase. Electron spin echoes (ESE) are observed for the first time from oriented lipid-water multilayers. Results obtained from cw ESR lineshape analysis are correlated with data from ESE experiments, which give a more direct measurement of relaxation times. These results indicate that for detection of very slow motions (close to the rigid limit) ESE experiments are more sensitive to dynamics than continuous wave ESR for which inhomogeneous broadening becomes a major problem.     

Electron spin resonance characterization of liquid ordered phase of detergent-resistant membranes from RBL-2H3 cells.

The dynamic structure of detergent-resistant membranes (DRMs) isolated from RBL-2H3 cells was characterized using two different acyl chain spin-labeled phospholipids (5PC and 16PC), a headgroup labeled sphingomyelin (SM) analog (SD-Tempo) and a spin-labeled cholestane (CSL). It was shown, by comparison to dispersions of SM, dipalmitoylphosphatidylcholine (DPPC), and DPPC/cholesterol of molar ratio 1, that DRM contains a substantial amount of liquid ordered phase: 1) The rotational diffusion rates (R( perpendicular)) of 16PC in DRM between -5 degrees C and 45 degrees C are nearly the same as those in molar ratio DPPC/Chol = 1 dispersions, and they are substantially greater than R( perpendicular) in pure DPPC dispersions in the gel phase studied above 20 degrees C; 2) The order parameters (S) of 16PC in DRM at temperatures above 4 degrees C are comparable to those in DPPC/Chol = 1 dispersions, but are greater than those in DPPC dispersions in both the gel and liquid crystalline phases. 3) Similarly, R( perpendicular) for 5PC and CSL in DRM is greater than in pure SM dispersions in the gel phase, and S for these labels in DRM is greater than in the SM dispersions in both the gel and liquid crystalline phases. 4) R( perpendicular) of SD-Tempo in DRM is greater than in dispersions of SM in both gel and liquid phases, consistent with the liquid-like mobility in the acyl chain region in DRM. However, S of SD-Tempo in DRM is substantially less than that of this spin label in SM in gel and liquid crystalline phases (in absolute values), indicating that the headgroup region in DRMs is less ordered than in pure SM. These results support the hypothesis that plasma membranes contain DRM domains with a liquid ordered phase that may coexist with a liquid crystalline phase. There also appears to be a coexisting region in DRMs in which the chain labels 16PC and 5PC are found to cluster. We suggest that other biological membranes containing high concentrations of cholesterol also contain a liquid ordered phase.     

Electron paramagnetic resonance studies of magnetically aligned phospholipid bilayers utilizing a phospholipid spin label: The effect of cholesterol

X-band EPR spectroscopy has been employed to study the dynamic properties of magnetically aligned phospholipid bilayers (bicelles) utilizing a variety of phosphocholine spin labels (n-PCSL) as a function of cholesterol content. The utilization of both perpendicular and parallel aligned bicelles in EPR spectroscopy provides a more detailed structural and orientational picture of the phospholipid bilayers. The magnetically aligned EPR spectra of the bicelles and the hyperfine splitting values reveal that the addition of cholesterol increases the phase transition temperature and alignment temperature of the DMPC/DHPC bicelles. The corresponding molecular order parameter, S_mol, of the DMPC/DHPC bicelles increased upon addition of cholesterol. Cholesterol also decreased the rotational motion and increased the degree of anisotropy in the interior region of the bicelles. This report reveals that the dynamic properties of DMPC/DHPC bicelles agree well with other model membrane systems and that the magnetically aligned bicelles are an excellent model membrane system.     

Electron paramagnetic resonance studies of magnetically aligned phospholipid bilayers utilizing a phospholipid spin label: the effect of cholesterol

X-band EPR spectroscopy has been employed to study the dynamic properties of magnetically aligned phospholipid bilayers (bicelles) utilizing a variety of phosphocholine spin labels (n-PCSL) as a function of cholesterol content. The utilization of both perpendicular and parallel aligned bicelles in EPR spectroscopy provides a more detailed structural and orientational picture of the phospholipid bilayers. The magnetically aligned EPR spectra of the bicelles and the hyperfine splitting values reveal that the addition of cholesterol increases the phase transition temperature and alignment temperature of the DMPC/DHPC bicelles. The corresponding molecular order parameter, Smol, of the DMPC/DHPC bicelles increased upon addition of cholesterol. Cholesterol also decreased the rotational motion and increased the degree of anisotropy in the interior region of the bicelles. This report reveals that the dynamic properties of DMPC/DHPC bicelles agree well with other model membrane systems and that the magnetically aligned bicelles are an excellent model membrane system.     

Effects of steroid molecules on the dynamical structure of dioleoylphosphatidylcholine and digalactosyldiacylglycerol bilayers

The ESR spectra of cholestane spin labels (CSL) in dioleoylphosphatidylcholine (DOPC) bilayers containing 20 wt% of cholesterol, 7-dehydrocholesterol, beta-sitosterol, stigmasterol and lanosterol exhibit a marked similarity, thus indicating that these steroids induced the same effects on the lipid bilayer over the temperature range 21-55 degrees C. The incorporation of these steroids into the DOPC bilayers enhances the orientational order of the CSL molecules at every temperature studied, but only induces a pronounced slow-down in their rotational motions at temperatures above 35 degrees C. Similar results were obtained in DOPC/ergosterol multilamellar liposomes, but the changes are now less pronounced than in the other five DOPC/steroid systems. In contrast, the addition of stigmasterol to digalactosyldiacylglycerol (DGDG) bilayers appears to increase the order parameter mean value of P2, without affecting the diffusion coefficients. Furthermore, the incorporation of 7-dehydrocholesterol to DGDG bilayers causes a large enhancement in the orientational order, but has only a small effect on D perpendicular of the CSL molecules. Importantly, this latter effect appears to be independent of temperature. The marked changes in the rates of the rotational motion brought about by the addition of steroids, contrasts with the lack of a significant effect of unsaturation on the bilayer dynamics reported by us previously (Korstanje et al. (1989), Biochim. Biophys. Acta 980, 225-233, and 982, 196-204).     

Rotational diffusion of a steroid molecule in phosphatidylcholine-cholesterol membranes: fluid-phase microimmiscibility in unsaturated phosphatidylcholine-cholesterol membranes

Rotational diffusion of androstane spin-label (ASL), a sterol analogue, in various phosphatidylcholine (PC)-cholesterol membranes was systematically studied by computer simulation of steady-state ESR spectra as a function of the chain length and unsaturation of the alkyl chains, cholesterol mole fraction, and temperature for a better understanding of phospholipid-cholesterol and cholesterol-cholesterol interactions. Special attention was paid to the differences in the cholesterol effects on ASL motion between saturated and unsaturated PC membranes. ASL motion in the membrane was treated as Brownian rotational diffusion of a rigid rod within the confines of a cone imposed by the membrane environment. The wobbling rotational diffusion constant of the long axis, its activation energy, and the cone angle of the confines were obtained for various PC-cholesterol membranes in the liquid-crystalline phase. Cholesterol decreases both the cone angle and the wobbling rotational diffusion constant for ASL in all PC membranes studied in this work. The cholesterol effects are the largest in DMPC membranes. An increase of cholesterol mole fraction from 0 to 30% decreases the rotational diffusion constant by a factor of 9-15 (depending on temperature) and the cone angle by a factor of about 2. In dioleoyl-PC membranes, addition of 30 mol % cholesterol reduces both the rotational diffusion constant and the cone angle of ASL by factors of approximately 2.5 and approximately 1.3, respectively, while it was previously found to cause only modest effects on the motional freedom of phospholipid analogue spin probes [Kusumi, A., Subczynski, W. K., Pasenkiewicz-Gierula, M., Hyde, J. S., & Merkle, H. (1986) Biochim. Biophys. Acta 854, 307-317]. It is proposed that fluid-phase microimmiscibility takes place in dioleoyl-PC-cholesterol membranes at physiological temperatures, which induces cholesterol-rich domains in the membrane, partially due to the steric nonconformability between the rigid fused-ring structure of cholesterol and the 30 degrees bend at the C9-C10 cis double bond of the alkyl chains of dioleoyl-PC. The mechanism by which cholesterol influences the lipid dynamics in the membrane is different between saturated and unsaturated PC membranes.     

Organization of water molecules by adhering to oriented layers of dipalmitoylphosphatidyl serine in the presence of varying concentrations of cholesterol

About seven water molecules adhere to one molecule of dipalmitoylphosphatidyl serine (DPPS) in an oriented surface layer as inferred from the increase of the dichroic ratio R of their OH stretching vibration band (3400 cm(-1)) from 2 in the random bulk state to about 2.8 when adhering to DPPS. In DPPS-cholesterol mixtures the number of water molecules adhering to the phospholipid molecules and oriented by them increases as cholesterol content increases. This increase is very steep between molar fractions of cholesterol X(chol)=0.2-0.4 and at X(chol)=0.6 about 13 water molecules adhere and are oriented by one DPPS molecule.     

An electron spin resonance study of interactions between phosphatidylcholine and phosphatidylserine in oriented membranes.

A detailed electron spin resonance (ESR) study of mixtures of 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) and phosphatidylserine (POPS) in oriented multilayers in the liquid crystalline phase is reported with the purpose of characterizing the effects of headgroup mixing on the structural and dynamical properties of the acyl chains. These studies were performed over a range of blends of POPC and POPS and temperatures, utilizing the spin-labeled lipids 16-phosphatidylcholine and 5-phosphatidylcholine as well as cholestane (CSL). The ESR spectra were analyzed by nonlinear least-squares fitting using detailed spectral simulations. Whereas CSL shows almost no variation in ordering and rotational dynamics versus mole fraction POPS, (i.e. XPS), and 5-PC shows small effects, the weakly ordered end-chain labeled 16-PC shows large relative effects, such that the orientational order parameter, S is at a minimum for XPS = 0.5 where it is about one-third the value observed for XPS = 0 and 1. This is directly reflected in the ESR spectrum as a substantial variation in the hyperfine splitting with XPS. The least-squares analysis also shows a reduction in rotational diffusion coefficient, R perpendicular by a fractor of 2 for XPS = 0.5 and permits the estimation of S2, the ordering parameter representing deviations from cylindrically symmetric alignment. These results are contrasted with 2H NMR studies which were insensitive to effects of mixing headgroups on the acyl chains. The ESR results are consistent with a somewhat increased disorder in the end-chain region as well as a small amount of chain tilting upon mixing POPC and POPS.(ABSTRACT TRUNCATED AT 250 WORDS)     

Bilayer polarity and its thermal dependency in the l(o) and l(d) phases of binary phosphatidylcholine/cholesterol mixtures

Diverse variations in membrane properties are observed in binary phosphatidylcholine/cholesterol mixtures. These mixtures are nonideal, displaying single or phase coexistence, depending on chemical composition and other thermodynamic parameters. When compared with pure phospholipid bilayers, there are changes in water permeability, bilayer thickness and thermomechanical properties, molecular packing and conformational freedom of phospholipid acyl chains, in internal dipolar potential and in lipid lateral diffusion. Based on the phase diagrams for DMPC/cholesterol and DPPC/cholesterol, we compare the equivalent polarity of pure bilayers with specific compositions of these mixtures, by using the Py empirical scale of polarity. Besides the contrast between pure and mixed lipid bilayers, we find that liquid-ordered (l(o)) and liquid-disordered (l(d)) phases display significantly different polarities. Moreover, in the l(o) phase, the polarities of bilayers and their thermal dependences vary with the chemical composition, showing noteworthy differences for cholesterol proportions at 35, 40, and 45 mol%. At 20 degrees C, for DMPC/cholesterol at 35 and 45 mol%, the equivalent dielectric constants are 21.8 and 23.8, respectively. Additionally, we illustrate potential implications of polarity in various membrane-based processes and reactions, proposing that for cholesterol containing bilayers, it may also go along with the occurrence of lateral heterogeneity in biological membranes.     

Lipid chain length effect on the phase behaviour of PCs/PEG:2000-PEs mixtures. A spin label electron spin resonance and spectrophotometric study

Spin-label electron spin resonance (ESR) spectroscopy and spectrophotometry at fixed wavelength are used to study fully hydrated aqueous dispersions of phosphatidylcholines (PCs) with poly(ethylene glycol:2000)-phosphatidylethanolamines (PEG:2000-PEs). PEG:2000-PE is a micelle-forming polymer-lipid that is extensively used for increasing the lifetime of PC liposomes in the blood circulation through a steric stabilisation effect. The PC lipids and the PEG:2000-PE polymer-lipids have the same acyl chain length of either dimiristoyl (DM) or distearoyl (DS) chains. DMPC/PEG:2000-DMPE and DSPC/PEG:2000-DSPE mixtures were investigated over the entire range of relative compositions (0-100 mol%). In both dispersions, the low-temperature conventional spin label ESR spectra and the temperature dependence of the absorbance at 400 nm give an indication of the conversion from lamellae to micelles with increasing PEG:2000-PEs content. The physical state of the lipid assemblies, lamellar or micellar, is dependent not only on PEG:2000-PEs content, but also on the length of hydrocarbon chain of the lipid matrix. Micellisation is attained more readily in dispersions with longer hydrocarbon chains (i.e. in DSPC/PEG:2000-DSPE mixtures) than in those with shorter acyl chains (i.e. in DMPC/PEG:2000-DMPE mixtures). Saturation transfer ESR (ST-ESR) and absorbance measurements reflect the disaggregation of the bilayers and a reduction in the size of the lipid aggregates by PEG:2000-PEs at low content.     

Effects of polar carotenoids on dimyristoylphosphatidylcholine membranes: a spin-label study.

Spin labeling methods were used to study the structure and dynamic properties of dimyristoylphosphatidylcholine (DMPC) membranes as a function of temperature and the mole fraction of polar carotenoids. The results in fluid phase membranes are as follows: (1) Dihydroxycarotenoids, zeaxanthin and violaxanthin, increase order, decrease motional freedom and decrease the flexibility gradient of alkyl chains of lipids, as was shown with stearic acid spin labels. The activation energy of rotational diffusion of the 16-doxylstearic acid spin label is about 35% less in the presence of 10 mol% of zeaxanthin. (2) Carotenoids increase the mobility of the polar headgroups of DMPC and increase water accessibility in that region of membrane, as was shown with tempocholine phosphatidic acid ester. (3) Rigid and highly anisotropic molecules dissolved in the DMPC membrane exhibit a bigger order of motion in the presence of polar carotenoids as was shown with cholestane spin label (CSL) and androstane spin label (ASL). Carotenoids decrease the rate of reorientational motion of CSL and do not influence the rate of ASL, probably due to the lack of the isooctyl side chain. The abrupt changes of spin label motion observed at the main phase transition of the DMPC bilayer are broadened and disappear at the presence of 10 mol% of carotenoids. In gel phase membranes, polar carotenoids increase motional freedom of most of the spin labels employed showing a regulatory effect of carotenoids on membrane fluidity. Our results support the hypothesis of Rohmer, M., Bouvier, P. and Ourisson, G. (1979) Proc. Natl. Acad. Sci. USA 76, 847-851, that carotenoids regulate the membrane fluidity in Procaryota as cholesterol does in Eucaryota. A model is proposed to explain these results in which intercalation of the rigid rod-like polar carotenoid molecules into the membrane enhances extended trans-conformation of the alkyl chains, decreases free space in the bilayer center, separate the phosphatidylcholine headgroups and decreases interaction between them.     

Bilayer polarity and its thermal dependency in the ?o and ?d phases of binary phosphatidylcholine/cholesterol mixtures

Diverse variations in membrane properties are observed in binary phosphatidylcholine/cholesterol mixtures. These mixtures are nonideal, displaying single or phase coexistence, depending on chemical composition and other thermodynamic parameters. When compared with pure phospholipid bilayers, there are changes in water permeability, bilayer thickness and thermomechanical properties, molecular packing and conformational freedom of phospholipid acyl chains, in internal dipolar potential and in lipid lateral diffusion. Based on the phase diagrams for DMPC/cholesterol and DPPC/cholesterol, we compare the equivalent polarity of pure bilayers with specific compositions of these mixtures, by using the Py empirical scale of polarity. Besides the contrast between pure and mixed lipid bilayers, we find that liquid-ordered (?_o) and liquid-disordered (?_d) phases display significantly different polarities. Moreover, in the ?_o phase, the polarities of bilayers and their thermal dependences vary with the chemical composition, showing noteworthy differences for cholesterol proportions at 35, 40, and 45 mol%. At 20 °C, for DMPC/cholesterol at 35 and 45 mol%, the equivalent dielectric constants are 21.8 and 23.8, respectively. Additionally, we illustrate potential implications of polarity in various membrane-based processes and reactions, proposing that for cholesterol containing bilayers, it may also go along with the occurrence of lateral heterogeneity in biological membranes.     

ESR studies of spin-labeled membranes aligned by isopotential spin-dry ultracentrifugation: lipid-protein interactions.

Electron spin resonance (ESR) studies have been performed on spin-labeled model membranes aligned using the isopotential spin-dry ultracentrifugation (ISDU) method of Clark and Rothschild. This method relies on sedimentation of the membrane fragments onto a gravitational isopotential surface with simultaneous evaporation of the solvent in a vacuum ultracentrifuge to promote alignment. The degree of alignment obtainable using ISDU, as monitored by ESR measurements of molecular ordering for both lipid (16-PC) and cholestane spin labels (CSL), in dipalmitoylphosphatidylcholine (DPPC) model membranes compares favorably with that obtainable by pressure-annealing. The much gentler conditions under which membranes may be aligned by ISDU greatly extends the range of macroscopically aligned membrane samples that may be investigated by ESR. We report the first ESR study of an integral membrane protein, bacteriorhodopsin (BR) in well-aligned multilayers. We have also examined ISDU-aligned DPPC multilayers incorporating a short peptide gramicidin A' (GA), with higher water content than previously studied. 0.24 mol% BR/DPPC membranes with CSL probe show two distinct components, primarily in the gel phase, which can be attributed to bulk and boundary regions of the bilayer. The boundary regions show sharply decreased molecular ordering and spectral effects comparable to those observed from 2 mol% GA/DPPC membranes. The boundary regions for both BR and GA also exhibit increased fluidity as monitored by the rotational diffusion rates. The high water content of the GA/DPPC membranes reduces the disordering effect as evidenced by the reduced populations of the disordered components. The ESR spectra obtained slightly below the main phase transition of DPPC from both the peptide- and protein-containing membranes reveals a new component with increased ordering of the lipids associated with the peptide or protein. This increase coincides with a broad endothermic peak in the DSC, suggesting a disaggregation of both the peptide and the protein before the main phase transition of the lipid. Detailed simulations of the multicomponent ESR spectra have been performed by the latest nonlinear least-squares methods, which have helped to clarify the spectral interpretations. It is found that the simulations of ESR spectra from CSL in the gel phase for all the lipid membranes studied could be significantly improved by utilizing a model with CSL molecules existing as both hydrogen-bonded to the bilayer interface and non-hydrogen-bonded within the bilayer.     

A 2H solid-state NMR spectroscopic investigation of biomimetic bicelles containing cholesterol and polyunsaturated phosphatidylcholine

Deuterium solid-state NMR spectroscopy was used to qualitatively study the effects of both 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphatidylcholine (PLiPC) and cholesterol on magnetically aligned phospholipid bilayers (bicelles) as a function of temperature utilizing the chain-perdeuterated probe 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine (DMPC-d54) in DMPC/dihexanoylPC (DHPC) phospholipid bilayers. The results demonstrate that polyunsaturated PC and cholesterol were successfully incorporated into DMPC/DHPC phospholipid bilayers, leading to a bicelle that will be useful for investigations of eukaryotic membrane protein-lipid interactions. The data indicate that polyunsaturated PC increases membrane fluidity and decreases the minimum magnetic alignment temperature for DMPC/DHPC bicelles. Conversely, the introduction of cholesterol into aligned DMPC/DHPC bilayers decreases fluidity in the membrane and increases the minimum temperature necessary to magnetically align the phospholipid bilayers. Finally, the addition of Tm3+ to magnetically aligned DMPC/DMPC-d54/PLiPC/DHPC bilayers doubles the quadrupolar splittings, indicating that this unique bicelle system can be aligned with the bilayer normal parallel to the static magnetic field.