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.     

Interactions of proteins and cholesterol with lipids in bilayer membranes

Mixtures of lipids and proteins, 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 (TEMPO). In dimyristoyl phosphatidylcholine vesicles the protein molecules were randomly distributed above the transition temperature, $\text{T}{}_{\mathrm{<mtext>t</mtext>}}$, of the lipid and aggregated below $\text{T}{}_{\mathrm{<mtext>t</mtext>}}$. For mixtures af dimyristoyl and dipalmitoyl phosphatidylcholine the existence of fluid and solid domains was 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 temperarture of the lipid is discussed.     

Constant-pressure molecular dynamics investigation of cholesterol effects in a dipalmitoylphosphatidylcholine bilayer.

We report a 1.4-ns constant-pressure molecular dynamics simulation of cholesterol at 12.5 mol% in a dipalmitoylphosphatidylcholine (DPPC) bilayer at 50 degrees C and compare the results to our previous simulation of a pure DPPC bilayer. The interlamellar spacing was increased by 2.5 A in the cholesterol-containing bilayer, consistent with x-ray diffraction results, whereas the bilayer thickness was increased by only 1 A. The bilayer/water interface was more abrupt because the lipid headgroups lie flatter to fill spaces left by the cholesterol molecules. This leads to less compensation by the lipid headgroups of the oriented water contribution to the membrane dipole potential and could explain the experimentally observed increase in the magnitude of the dipole potential by cholesterol. Our calculations suggested that 12.5 mol% cholesterol does not significantly affect the conformations and packing of the hydrocarbon chains and produces only a slight reduction in the empty free volume. However, cholesterol has a significant influence on the subnanosecond time scale lipid dynamics: the diffusion constant for the center-of-mass "rattling" motion was reduced by a factor of 3, and the reorientational motion of the methylene groups was slowed along the entire length of the hydrocarbon chains.     

Effects of butanol isomers on dipalmitoylphosphatidylcholine bilayer membranes

Differential scanning calorimetry and (31)P-NMR were used to study the effects of butanol isomers on the thermotropic phase behavior of dipalmitoylphosphatidylcholine (DPPC) bilayers. The threshold concentration for the onset of interdigitation for each isomer was determined by the disappearance of the pretransition and the onset of a large hysteresis between the heating and cooling scans of the gel-to-liquid main transition. The threshold concentration was found to correlate with increased solubility of the isomers in the aqueous phase, led by tert-butanol. However, as the solution concentration of tert-butanol increased, there was an abrupt shrinking of the hysteresis, initially with well-resolved shoulder peaks indicating mixed phases. The eventual disappearance of the shoulder peaks was correlated with a breakdown of the multilamellar structure identified using (31)P-NMR.     

Interaction of cholesterol with sphingomyelin in bilayer membranes: evidence that the hydroxy group of sphingomyelin does not modulate the rate of cholesterol exchange between vesicles

Cholesterol undergoes exchange between membranes containing sphingomyelin at a much slower rate than between membranes lacking sphingomyelin. To investigate the role of the hydroxy group at the 3-position of sphingomyelin in the interaction between sphingomyelin and cholesterol, we have measured the rates of [4-14C]cholesterol exchange between unilamellar vesicles prepared with N-stearoylsphingomyelin or with synthetic analogues in which the hydroxy group is replaced with an O-alkyl group or with hydrogen. Vesicles prepared from 3-deoxy- and 3-O-methyl-N-stearoylsphingomyelin had the same rate of [14C]-cholesterol desorption. The half-times for exchange from vesicles prepared with 3-O-methyl- and 3-deoxy-N-stearoylsphingomyelins and 10 mol % of cholesterol were only slightly faster (a factor of only 1.5) than that found from vesicles prepared from N-stearoylsphingomyelin and 10 mol % cholesterol. The rate of cholesterol desorption from vesicles could be accelerated by preparing vesicles from bulky 3-O-alkyl analogues of sphingomyelin. Vesicles containing 3-O-ethyl-N-stearoylsphingomyelin and 3-O-tetrahydropyranyl egg sphingomyelin gave rate enhancements of approximately 14 and 35, compared with the rates observed in vesicles made from N-stearoyl- and egg sphingomyelin, respectively. These data indicate that insertion of sterically bulky groups at the 3-position of sphingomyelin (such as ethoxy and tetrahydropyranyloxy) in place of hydroxy interferes markedly with the molecular packing of cholesterol and sphingomyelin in bilayer membranes; however, the hydroxy group of sphingomyelin is not critical for the strong interaction of cholesterol with sphingomyelin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Miscibility of acyl-chain defined phosphatidylcholines with N-palmitoyl sphingomyelin in bilayer membranes

In this study we have used differential scanning calorimetry (DSC) to study the miscibility of different saturated phosphatidylcholines (PCs) with D-erythro-N-palmitoyl-sphingomyelin (16:0-SM). Information about the miscibility was obtained by observing the thermotropic phase behavior of binary mixtures of saturated PCs and 16:0-SM. The results obtained showed that PC miscibility in 16:0-SM was markedly affected by the PC acyl-chain composition. According to phase diagrams prepared from DSC data and the mid-transition temperatures of the main phase transition, the PC which formed the most ideal mixture with 16:0-SM was di-14:0-PC. However, the cooperativity of the main transition in PC/16:0-SM bilayers increased until the average acyl-chain length in the PC reached 15 carbons. Based on the criteria of the most ideal miscibility or the highest cooperativity of the main transition, we conclude that di-14:0-PC, 15:0/15:0-PC, and 14:0/16:0-PC interacted most favorably with 16:0-SM in bilayer membranes. Di-16:0-PC, to which 16:0-SM is often compared in biophysical studies, showed much less ideal miscibility.     

Interactions of liposomes with planar bilayer membranes

Summary The adhesion to horizontal, planar lipid membranes of lipid vesicles containing calcein in the aqueous compartment or fluorescent phospholipids in the membranes has been examined by phase contrast, differential interference contrast and fluorescence microscopy. With water-immersion lenses, it was possible to study the interactions of vesicles with planar bilayers at magnifications up to the useful limit of light microscopy. In the presence of 15 mM calcium chloride, vesicles composed of phosphatidylserine and either phosphatidylethanolamine or soybean lipids adhere to the torus, bilayer and lenses of planar bilayers of the same composition. Lenses of solvent appear, at the site where vesicles attach to decane-based bilayers and lipid fluorophores move from the vesicles to the lenses. Because the calcein contained in such vesicles is not released, we interpret this as indicating fusion of only the outer monolayer (hemifusion) of the vesicles with the decane lenses. In the case of squalene-based black lipid membranes (BLMs), in contrast, vesicles do not nucleate lenses but they apparently do fuse with the torus at the bilayer boundary. Interactions leading to hemifusions between vesicles and planar membranes thus occur predominantly in regions where hydrocarbon solvent is present. Osmotic water flow, induced by addition of urea to the compartment containing vesicles, causes coalescence of lenses in decane-based, BLMs as well as coalescence of the aqueous spaces of the vesicles that have undergone hemifusion with the lenses. We did not observe transfer of the aqueous phase of vesicles to therans side of either decane-or squalene-based planar membranes; however, we cannot rule out the possibility particularly in the latter case, that rupture of the planar membrane may have been an immediate result of vesicle fusion and thus precluded its detection.     

Interactions between cholesterol and lipids in bilayer membranes. Role of lipid headgroup and hydrocarbon chain-backbone linkage

We have employed four lipids in the present study, of which two are cationic and two bear phosphatidylcholine (PC) headgroups. Unlike dipalmitoylphosphatidylcholine, the other lipids employed herein do not have any ester linkage between the hydrocarbon chains and the respective lipid backbones. Small unilamellar vesicles formed from each of the PC and cationic lipids with or without varying amounts of cholesterol have been examined using the steady-state fluorescence anisotropy method as a function of temperature. The anisotropy data clearly indicate that the order in the lipid bilayer packing is strongly affected upon inclusion of cholesterol. This effect is similar irrespective of the electrostatic character of the lipid employed. The influence of cholesterol inclusion on multi-lamellar lipid dispersions has also been examined by 1H-nuclear magnetic resonance spectroscopy above the phase transition temperatures. With all the lipids, the line widths of (CH2)n protons of hydrocarbon chains in the NMR spectra respond to the addition of cholesterol to membranes. The influence on the bilayer widths of various lipids upon inclusion of cholesterol was determined from X-ray diffraction studies of the cast films of the lipid-cholesterol coaggregates in water. The effect of cholesterol on the efflux rates of entrapped carboxyfluorescein (CF) from the phospholipid vesicles was determined. Upon incremental incorporation of cholesterol into the phospholipid vesicles, the CF leakage rates were progressively reduced. Independent experiments measuring transmembrane OH- ion permeation rates from cholesterol-doped cationic lipid vesicles using entrapped dye riboflavin also demonstrated that the addition of cholesterol into the cationic lipid vesicles reduced the leakage rates irrespective of lipid molecular structure. It was found that the cholesterol induced changes on the membrane properties such as lipid order, linewidth broadening, efflux rates, bilayer widths, etc., did not depend on the ability of the lipids to participate in the hydrogen bonding interactions with the 3beta-OH of cholesterol. These findings emphasize the importance of hydrophobic interaction between lipid and cholesterol and demonstrate that it is not necessary to explain the observed cholesterol induced effects on the basis of the presence of hydrogen bonding between the 3beta-OH of cholesterol and the lipid chain-backbone linkage region or headgroup region.     

Structure of dipalmitoylphosphatidylcholine/cholesterol bilayer at low and high cholesterol concentrations: molecular dynamics simulation.

By using molecular dynamics simulation technique we studied the changes occurring in membranes constructed of dipalmitoylphosphatidylcholine (DPPC) and cholesterol at 8:1 and 1:1 ratios. We tested two different initial arrangements of cholesterol molecules for a 1:1 ratio. The main difference between two initial structures is the average number of nearest-neighbor DPPC molecules around the cholesterol molecule. Our simulations were performed at constant temperature (T = 50 degrees C) and pressure (P = 0 atm). Durations of the runs were 2 ns. The structure of the DPPC/cholesterol membrane was characterized by calculating the order parameter profiles for the hydrocarbon chains, atom distributions, average number of gauche defects, and membrane dipole potentials. We found that adding cholesterol to membranes results in a condensing effect: the average area of membrane becomes smaller, hydrocarbon chains of DPPC have higher order, and the probability of gauche defects in DPPC tails is lower. Our results are in agreement with the data available from experiments.     

Interactions between digitonin and bilayer membranes

The morphology of interactions between digitonin and cholesterol has been investigated. When precipitated from ethanolic solutions, digitonin-cholesterol complexes form in flat lamellar sheets. In contrast, when the complex is formed in a bilayer membrane, the membrane is deformed into corrugations of hemitubules. The polarity of the deformations formed in bilayer membranes is highly correlated with the direction of entry of digitonin into the membrane. We suggest that the morphology of digitonin/cholesterol hemitubules is dependent upon the complex being formed within a bilayer and, in addition, is not correlated with asymmetry of cholesterol concentration across the membrane.     

Interaction of cholesterol with galactocerebroside and galactocerebroside-phosphatidylcholine bilayer membranes

The interaction of the galactocerebroside, N-palmitoylgalactosylsphingosine (NPGS), with cholesterol has been studied by differential scanning calorimetry (DSC) and x-ray diffraction. Thermal and structural studies demonstrate complex behavior characterized by two endothermic transitions: transition I (TI approximately equal to 50-60 degrees C) corresponding to an NPGS-cholesterol bilayer gel----bilayer liquid crystal transition II (TII where TI less than TII less than TNPGS) corresponding to an NPGS bilayer crystal (stable E form)----bilayer liquid crystal transition. For mixtures containing from 6 to 80 mol % cholesterol, x-ray diffraction studies at 22 degrees C (T less than TI) indicate two separate lamellar phases; an NPGS crystal bilayer phase and a cholesterol monohydrate phase. For cholesterol concentrations less than 50 mol % at TI less than T less than TII, NPGS-cholesterol liquid crystal bilayer and excess NPGS crystal bilayer phases are observed. For greater than 50 mol % cholesterol concentrations at these temperatures, an excess cholesterol monohydrate phase coexists with the NPGS-cholesterol liquid crystal bilayers. At T greater than TII, complete NPGS-cholesterol miscibility is only observed for less than 50 mol % cholesterol concentrations, whereas at greater than 50 mol % cholesterol an excess cholesterol phase is present. The solid phase immiscibility of cerebroside and cholesterol at low temperatures is suggested to result from preferential NPGS-NPGS associations via hydrogen bonding. The unique thermal and structural behavior of NPGS-cholesterol dispersions is contrasted with the behavior of cholesterol-phosphatidycholine and cholesterol-sphingomyelin bilayers. Thermal and structural studies of NPGS in dipalmitoylphosphatidylcholine (DPPC)/cholesterol (1:1, molar ratio) bilayers have been performed. For dispersions containing less than 20 mol % NPGS at 22 degrees C there are no observable calorimetric transitions and x-ray diffraction studies indicate complete lipid miscibility. At greater than 20 mol % NPGS, a high temperature transition is observed that is shown by x-ray diffraction studies to be due to an excess NPGS crystal bilayer----liquid crystal bilayer transition. Complete miscibility of NPGS in DPPC/cholesterol bilayers is observed at T greater than TNPGS. The properties of NPGS/DPPC/cholesterol bilayers are discussed in terms of the lipid composition of the myelin sheath.     

Interactions of divalent cations with phosphatidylserine bilayer membranes

The interaction of divalent cations with a homologous series of diacylphosphatidylserines (diacyl-PS) has been studied by differential scanning calorimetry and X-ray diffraction. Hydrated di-C14-PS (DMPS) exhibits a gel leads to liquid-crystal bilayer transition at 39 degrees C (delta H = 7.2 kcal/mol of DMPS). With increasing MgCl2 concentration, progressive conversion to a phase exhibiting a high melting (98 degrees C), high enthalpy (delta H congruent to 11.0 kcal/mol of DMPS) transition is observed. Similar behavior is observed for DMPS with increasing CaCl2 concentration. In this case, the high-temperature transition of the Ca2+-DMPS complex occurs at approximately 155 degrees C and is immediately followed by an exothermic transition probably associated with PS decomposition. For di-C12-, di-C14-, di-C16- (DPPS), and di-C18-PS, the transition temperatures of the Ca2+-PS complexes are in the range 151-155 degrees C; only di-C10-PS exhibits a significantly lower value, 142 degrees C. A different pattern of behavior is exhibited by DPPS in the presence of Sr2+ or Ba2+, with transitions in the range 70-80 degrees C being observed. X-ray diffraction of the Ca2+-PS complexes at 20 degrees C provides evidence of structural homology. All Ca2+-PS complexes exhibit bilayer structures, the bilayer periodicity increasing linearly from 35.0 A for di-C10-PS to 52.5 A for di-C18-PS. Wide-angle X-ray diffraction data indicate that hydrocarbon chain "crystallization" occurs on Ca2+-PS complex formation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Miscibility of acyl-chain defined phosphatidylcholines with N-palmitoyl sphingomyelin in bilayer membranes

In this study we have used differential scanning calorimetry (DSC) to study the miscibility of different saturated phosphatidylcholines (PCs) with d-erythro-N-palmitoyl-sphingomyelin (16:0-SM). Information about the miscibility was obtained by observing the thermotropic phase behavior of binary mixtures of saturated PCs and 16:0-SM. The results obtained showed that PC miscibility in 16:0-SM was markedly affected by the PC acyl-chain composition. According to phase diagrams prepared from DSC data and the mid-transition temperatures of the main phase transition, the PC which formed the most ideal mixture with 16:0-SM was di-14:0-PC. However, the cooperativity of the main transition in PC/16:0-SM bilayers increased until the average acyl-chain length in the PC reached 15 carbons. Based on the criteria of the most ideal miscibility or the highest cooperativity of the main transition, we conclude that di-14:0-PC, 15:0/15:0-PC, and 14:0/16:0-PC interacted most favorably with 16:0-SM in bilayer membranes. Di-16:0-PC, to which 16:0-SM is often compared in biophysical studies, showed much less ideal miscibility.     

Interaction of enantiomeric alkylacyl and diacylglycerophosphocholines with cholesterol in bilayer membranes

The sn-1 and sn-3 isomers of dioleoylglycerophosphocholine form vesicles of the same size as the racemic lipid. Identical permeability coefficients were found for the diffusion of glucose and chloride across bilayer membranes of vesicles consisting of these lipids. Vesicles made of mixtures of enantiomeric or racemic dioleoyllecithin with 30 mol% cholesterol have identical radii. Cholesterol reduces the permeability of bilayers for glucose and chloride irrespective of the steric configuration of the constituent phospholipid. Increasing concentrations of cholesterol (17, 33 and 50 mol%, respectively) broaden the (CH₂)_n signal in the ¹H-NMR-spectra (90 MHz) of unilamellar vesicles containing sn-1, sn-3 or rac alkyloleoylglycerophosphocholine to the same extent. These results indicate that the steric configuration of phospholipids has no gross effect on the arrangement of phospholipids and cholesterol in bilayer membranes.     

Interactions between metabolism and intracellular distribution of cholesterol and sphingomyelin

There is ample evidence from experimental models and human metabolic disorders indicating that cholesterol and sphingomyelin (SM) levels are coordinately regulated. Generally it has been observed that altering the cellular content of sphingomyelin or cholesterol results in corresponding changes in mass and/or synthesis of the other lipid. In the case of cholesterol synthesis and trafficking, SM regulates the capacity of membranes to absorb cholesterol and thereby controls sterol flux between the plasma membrane and regulatory pathways in the endoplasmic reticulum. This relationship exemplifies the importance of cholesterol/sphingolipid-rich domains in cholesterol homeostasis, as well as other aspects of cell signaling and transport. Evidence for regulation of sphingomyelin metabolism by cholesterol is less convincing and dependent on the model system under study. Sphingomyelin biosynthetic rates are not dramatically affected by alterations in cholesterol balance suggesting that sphingomyelin or its metabolites serve other indispensable functions in the cell. A notable exception is the robust and specific regulation of both SM and cholesterol synthesis by 25-hydroxycholesterol. This finding is reviewed in the context of the role of oxysterol binding protein and its putative role in cholesterol and SM trafficking between the plasma membrane and Golgi apparatus.     

Ceramide acyl chain length markedly influences miscibility with palmitoyl sphingomyelin in bilayer membranes

Ceramides are precursors of major sphingolipids and can be important cellular effectors. The biological effects of ceramides have been suggested to stem from their biophysical effects on membrane structure affecting the lateral and transbilayer organization of other membrane components. In this study we investigated the effect of acyl chain composition in ceramides (C4-C24:1) on their miscibility with N-palmitoyl-sphingomyelin (PSM) using differential scanning calorimetry. We found that short-chain (C4 and C8) ceramides induced phase separation and lowered the T _m and enthalpy of the PSM endotherm. We conclude that short-chain ceramides were more miscible in the fluid-phase than in the gel-phase PSM bilayers. Long-chain ceramides induced apparent heterogeneity in the bilayers. The main PSM endotherm decreased in cooperativity and enthalpy with increasing ceramide concentration. New ceramide-enriched components could be seen in the thermograms at all ceramide concentrations above X _Cer = 0.05. These broad components had higher T _m values than pure PSM. C24:1 ceramide exhibited complex behavior in the PSM bilayers. The miscibility of C24:1 ceramide with PSM at low (X _Cer = 0.05–0.10) concentrations was exceptionally good according to the cooperativity of the transition. At higher concentrations, multiple components were detected, which might have arisen from interdigitated gel-phases formed by this very asymmetric ceramide. The results of this study indicate that short-chain and long-chain ceramides have very different effects on the sphingomyelin bilayers. There also seems to be a correlation between their miscibility in binary systems and the effect of ceramides of different hydrophobic length on sphingomyelin-rich domains in multicomponent membranes.     

Comparative calorimetric and spectroscopic studies of the effects of cholesterol and epicholesterol on the thermotropic phase behaviour of dipalmitoylphosphatidylcholine bilayer membranes

We carried out comparative differential scanning calorimetric and Fourier transform infrared spectroscopic studies of the effects of cholesterol (Chol) and epicholesterol (EChol) on the thermotropic phase behaviour and organization of dipalmitoylphosphatidylcholine (DPPC) bilayers. EChol is an epimer of Chol in which the axially oriented hydroxyl group of C3 of Chol is replaced by an equatorially oriented hydroxyl group, resulting in a different orientation of the hydroxyl group relative to sterol fused ring system. Our calorimetric studies indicate that the incorporation of EChol is more effective than Chol is in reducing the enthalpy of the pretransition of DPPC. EChol is also initially more effective than Chol in reducing the enthalpies of both the sharp and broad components of the main phase transition of DPPC. However, at higher EChol concentrations (~ 30-50 mol%), EChol becomes less effective than Chol in reducing the enthalpy and cooperativity of the main phase transition, such that at sterol concentrations of 50 mol%, EChol does not completely abolish the cooperative hydrocarbon chain-melting phase transition of DPPC, while Chol does. However, EChol does not appear to form a calorimetrically detectable crystallite phase at higher sterol concentrations, suggesting that EChol, unlike Chol, may form dimers or lower order aggregates at higher sterol concentrations. Our spectroscopic studies demonstrate that EChol incorporation produces more ordered gel and comparably ordered liquid-crystalline bilayers compared to Chol, which are characterized by increased hydrogen bonding in the glycerol backbone region of the DPPC bilayer. These and other results indicate that monomeric EChol is less miscible in DPPC bilayers than is Chol at higher sterol concentrations, but perturbs their organization to a greater extent at lower sterol concentrations, probably due primarily to the larger effective cross-sectional area of the EChol molecule. Nevertheless, EChol does appear to produce a lamellar liquid-ordered phase in DPPC bilayers.