Properties of polyunsaturated phosphatidylcholine membranes in the presence and absence of cholesterol (original) (raw)
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Biophysical Journal, 2006
To investigate the properties of a pure liquid ordered (L o ) phase in a model membrane system, a series of saturated phosphatidylcholines combined with cholesterol were examined by variable temperature multinuclear ( 1 H, 2 H, 13 C, 31 P) solidstate NMR spectroscopy and x-ray scattering. Compositions with cholesterol concentrations $40 mol %, well within the L o phase region, are shown to exhibit changes in properties as a function of temperature and cholesterol content. The 2 H-NMR data of both cholesterol and phospholipids were used to more accurately map the L o phase boundary. It has been established that the gel-L o phase coexistence extends to 60 mol % cholesterol and a modified phase diagram is presented. Combined 1 H-, 2 H-, 13 C-NMR, and x-ray scattering data indicate that there are large changes within the L o phase region, in particular, 1 H-magic angle spinning NMR and wide-angle x-ray scattering were used to examine the in-plane intermolecular spacing, which approaches that of a fluid L a phase at high temperature and high cholesterol concentrations. Although it is well known for cholesterol to broaden the gelto-fluid transition temperature, we have observed, from the 13 C magic angle spinning NMR data, that the glycerol region can still undergo a ''melting'', though this is broadened with increasing cholesterol content and changes with phospholipid chain length. Also from 2 H-NMR order parameter data it was observed that the effect of temperature on chain length became smaller with increasing cholesterol content. Finally, from the cholesterol order parameter, it has been previously suggested that it is possible to determine the degree to which cholesterol associates with different phospholipids. However, we have found that by taking into account the relative temperature above the phase boundary this relationship may not be correct. angle spinning; CSA, chemical shift anisotropy; Chol-d 1 , cholesterol deuterated at the C3 position or Cholesterol-3a-d 1 ; T m , temperature of the gel to fluid transition (chain melting); T*, temperature above the gel-L o to L o phase boundary.
Biophysical Journal, 2002
The behavior of cholesterol is different in mixtures with phosphatidylcholine as compared with phosphatidylserine. In 13 C cross polarization/magic angle spinning nuclear magnetic resonance spectra, resonance peaks of the vinylic carbons of cholesterol are a doublet in samples containing 0.3 or 0.5 mol fraction cholesterol with 1-palmitoyl-2-oleoyl phosphatidylserine (POPS) or in cholesterol monohydrate crystals, but a singlet with mixtures of cholesterol and 1-palmitoyl-2-oleoyl phosphatidylcholine (POPC). At these molar fractions of cholesterol with POPS, resonances of the C-18 of cholesterol appear at the same chemical shifts as in pure cholesterol monohydrate crystals. These resonances do not appear in samples of POPS with 0.2 mol fraction cholesterol or with POPC up to 0.5 mol fraction cholesterol. In addition, there is another resonance from the cholesterol C18 that appears in all of the mixtures of phospholipid and cholesterol but not in pure cholesterol monohydrate crystals. Using direct polarization, the fraction of cholesterol present as crystallites in POPS with 0.5 mol fraction cholesterol is found to be 80%, whereas with the same mol fraction of cholesterol and POPC none of the cholesterol is crystalline. After many hours of incubation, cholesterol monohydrate crystals in POPS undergo a change that results in an increase in the intensity of certain resonances of cholesterol monohydrate in 13 C cross polarization/magic angle spinning nuclear magnetic resonance, indicating a rigidification of the C and D rings of cholesterol but not other regions of the molecule.
Biochimica et Biophysica Acta (BBA) - Biomembranes, 1986
Dynamic properties of phosphatidylcholine-cholesterol membranes in the fluid phase and water accessibility to the membranes have been studied as a function of phospholipid alkyl chain length, saturation, mole fraction of cholesterol, and temperature by using spin and fluorescence labelling methods. The results are the following: (1) The effect of cholesterol on motional freedom of 5-doxyl stearic acid spin label (5-SASL) and 16-doxyl stearic acid spin label (16-SASL) in saturated phosphatidylcholine membrane is significantly larger than the effects of aikyl chain length and introduction of unsaturation in the aikyi chain. (2) Variation of alkyl chain length of saturated phospholipids does not alter the effects of cholesterol except in the case of dilauroylphosphatidylcholine, which possesses the shortest alkyl chains (12 carbons) used in this work. (3) Unsaturation of the alkyl chains greatly reduces the ordering effect of cholesterol at C-5 and C-16 positions although unsaturation alone gives only minor fluidizing effects. (4) Introduction of 30 mol% cholesterol to dimyristoylphosphatidylcholine membranes decreases the lateral diffusion constants of lipids by a factor of four, while it causes only a slight decrease of lateral diffusion in dioleoylphosphatidylcholine membranes. (5) If compared at the same temperature, 5-SASL mobilities plotted as a function of mole fraction of cholesterol in the fluid phases of dimyristoylphosphatidylcholine-, dipalmitoylphosphatidylcholine-and distearoylphosphatidylcholine-cholesterol membranes are similar in wide ranges of temperature (45-82°C) and cholesterol mole fraction (0-50%). (6) In isothermal experiments with saturated phosphatidylcholine membranes, 5-SASL is maximally immobilized at the phase boundary between Regions I and lII reported by other workers (Recktenwald, D.J. and McConnell, H.M. (1981) Biochemistry 20, 4505-4510) and becomes more mobile away from the boundary in Regions I and IIl. (7) 5-SASL in unsaturated phosphatidylcholine membranes showed a gradual monotonic immobilization with increase of cholesterol mole fraction without showing any maximum in the range of cholesterol fractions studied. (8) By rigorously determining rigid-limit
The Journal of Membrane Biology, 2011
A direct and quantitative analysis of the internal structure and dynamics of a polyunsaturated lipid bilayer composed of 1-stearoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine (18:0-22:6n3-PC) containing 29 mol% cholesterol was carried out by neutron diffraction, 2 H-NMR and 13 C-MAS NMR. Scattering length distribution functions of cholesterol segments as well as of the sn-1 and sn-2 hydrocarbon chains of 18:0-22:6n3-PC were obtained by conducting experiments with specifically deuterated cholesterol and lipids. Cholesterol orients parallel to the phospholipids, with the A-ring near the lipid glycerol and the terminal methyl groups 3 Å away from the bilayer center. Previously, we reported that the density of polyunsaturated docosahexaenoic acid (DHA, 22:6n3) chains was higher near the lipid-water interface. Addition of cholesterol partially redistributes DHA density from near the lipid-water interface to the center of the hydrocarbon region. Cholesterol raises chain-order parameters of both stearic acid and DHA chains. The fractional order increase for stearic acid methylene carbons C 8 -C 18 is larger, reflecting the redistribution of DHA chain density toward the bilayer center. The correlation times of DHA chain isomerization are short and mostly unperturbed by the presence of cholesterol. The uneven distribution of saturated and polyunsaturated chain densities and the cholesterol-induced balancing of chain distributions may have important implications for the function and integrity of membrane receptors, such as rhodopsin.
Dynamics of phosphatidylcholine-cholesterol mixed model membranes in the liquid crystalline state
Biophysical Journal, 1990
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 250C, whereas it shows only a 40% decrease at 50 mol % and 500C. 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 S2sL 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 RI, 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
Biochimica et Biophysica Acta (BBA) - Biomembranes, 1995
We report the results of a comparative study of the molecular order and dynamics of phosphatidylcholine (PC) bilayer membranes in the absence and presence of cholesterol, ergosterol and lanosterol, using deuterium (2 H) nuclear magnetic resonance (NMR) of deuterated phospholipid molecules, in addition to solid state m3C and 31P-NMR. Using dimyristoylphosphatidylcholines (DMPCs) specifically labeled at positions 2', 3', 4', 6', 8', 10' and 12' of the sn-2 chain together with the perdeuterated 2-[2HzT]DMPC derivative, the order profile for 9 of the 13 methylene groups of the sn-2 chain was established at 25°C for DMPC, DMPC/cholesterol, DMPC/ergosterol and DMPC/lanosterol membranes, at a fixed sterol/phospholipid mol ratio of 30%, and in the presence of excess water. The overall ordering effects were found to be ergosterol > cholesterol >> lanosterol. Transverse relaxation (T2c) studies of these systems indicated that while for DMPC, DMPC/cholesterol and DMPC/ergosterol the relative relaxation rates were in qualitative agreement with models which assume cooperative motions of the bilayer molecules as the main relaxation mechanism, those in DMPC/lanosterol were anomalously high, suggesting alterations of lipid packing. Using dipalmitoylphosphatidylcholine (DPPC) deuterated at the trimethylammonium group of the choline moiety, we found that the differential ordering and motional effects induced by the sterols in the acyl chains were also reflected in the headgroup, both in the gel (L~) and liquid-crystalline phases, t3C and ~H spin dynamics studies of these systems, including cross-polarization, rotating frame longitudinal relaxation and dipolar echo relaxation rates showed that the mobility of the different regions of the phospholipid molecules in the binary lipid systems were inversely correlated with the ordering effects induced by the sterols. A novel combination of C-D bond order parameters (obtained by :H-NMR) and 13C-IH cross polarization rates confirmed these results. The effects of the same sterols at the same molar proportion on the unsaturated lipid l-[2H3~]palmitoyl-2-oleoyl-snglycero-3-phosphatidylcholine (2H3:POPC) at 25 and 35°C were different from those observed on DMPC and showed ordering effects which are largest for cholesterol, while ergosterol and lanosterol produced significantly smaller effects. Transverse relaxation studies indicate that while cholesterol does not perturb cooperative motions in POPC, both ergosterol and lanosterol do. Again, high-resolution solid state J3C-NMR studies support the conclusions of the 2H-NMR experiments. Titration experiments using both 2H and13C-NMR show that ergosterol affects POPC bilayer structure up to 50 mol% but it increases the order of the phospholipid acyl chains only up to about 25 mol%. Beyond that level, it has a smaller ordering effect, possibly indicating aggregation or other more complex phase behavior. At > 30 mol% ergosterol, ~3C spectra reveal the presence of a second form of the sterol. However, 31p-NMR spectra show that all POPC/sterol systems retain a bilayer configuration up to 30 mol% sterol. The concentration of ergosterol which induces maximum order in the POPC membranes coincides with that present in the plasma membranes of the protozoan parasite To'panosoma cruzi. Taken together, our results indicate that the effects of sterols on PC bilayers are very complex, and depend on both sterol structure and on the fatty acids esterified to the phospholipid.
Interactions of cholesterol with the membrane lipid matrix. A solid state NMR approach
Biochimie, 1991
The effects of cholesterol on the structure and dynamics of dimyristoylphosphatidylcholine (DMPC) model membranes have been monitored as functions of temperature and cholesterol concentration in the membrane. The use of deuterium labels both on the cholesterol fused ring system and on the lipid chains in conjunction with solid state deuterium nuclear magnetic resonance (2H-NMR) afforded to monitor the degree of ordering of both molecules in a mixed system. The degree of ordering of the lipid head group was followed by phosphorus-31 (31p)-NMR. New findings on the effect of cholesterol on DMPC may be summarized as follows: i) cholesterol disorders the lipid chains below temperature of the DMPC gel-to-fluid transition (To) and orders them above; the effect is linear with cholesterol concentration at 0 and 60°C but for intermediate temperatures, a saturation effect is observed at 20-30 mol %; ii) the ordering-disordering effects are perceived similarly by all chain segments with, however, a greater sensitivity for positions near the bilayer center; iii) below To, the lipid head group is considerabily disordered by increasing amounts of cholesterol but slightly affected above; iv) the degree of ordering of cholesterol is quasi temperature independent for fractions greater than or equal to 30%; v) the average orientation of the cholesterol rigid body is perpendicular to the bilayer surface and exhibits little variations with temperature and cholesterol concentration. Variations in membrane dynamics are interpreted in terms of cholesterol-induced changes in bilayer thickness. The sterol is described as a regulator of membrane dynamics (for fractions greater than or equal to 30%) by providing the bilayer with quasi constant motional amplitudes over a large temperature scale.
Phosphatidylcholine structure determines cholesterol solubility and lipid polymorphism
Chemistry and Physics of Lipids, 2005
In the present work, we demonstrate that phosphatidylcholine with (16:1) 9 acyl chains undergoes polymorphic rearrangements in mixtures with 0.6-0.8 mol fraction cholesterol. Studies were performed using differential scanning calorimetry, X-ray diffraction, cryo-electron microscopy, 31 P NMR static powder patterns and 13 C MAS/NMR. Mixtures of phosphatidylcholine with (16:1) 9 acyl chains and 0.6 mol fraction cholesterol, after being heated to 100 • C, can form an ordered array with periodicity 14 nm which may be indicative of a cubic phase. Our results indicate that the formation of highly curved bilayer structures, such as those required for membrane fusion, can occur in mixtures of cholesterol with certain phosphatidylcholines that do not form non-lamellar structures in the absence of cholesterol. We also determine the polymorphic behavior of mixtures of symmetric phosphatidylcholines with cholesterol. Species of phosphatidylcholine with (20:1) 11 , (22:1) 13 or (24:1) 15 acyl chains in mixtures with 0.6-0.8 mol fraction cholesterol undergo a transition to the hexagonal phase at temperatures 70-80 • C. This is not the case for phosphatidylcholine with (18:1) 6 acyl chains which remains in the lamellar phase up to 100 • C when mixed with as much as 0.8 mol fraction cholesterol. Thus, the polymorphic behavior of mixtures of phosphatidylcholine and cholesterol is not uncommon and is dependent on the intrinsic curvature of the phospholipid. Crystals of cholesterol can be detected in mixtures of all of these phosphatidylcholines at sufficiently high cholesterol mole fraction. What is unusual about the formation of these crystals in several cases is that cholesterol crystals are present in the Abbreviations: PC, phosphatidylcholine; PE, phosphatidylethanolamine; Di(X:Y) Z PC, symmetric phosphatidylcholine with each acyl chain having X carbon atoms and Y double bonds at position Z in the acyl chain; CP, cross-polarization; DP, direct polarization; MAS, magic angle spinning; DSC, differential scanning calorimetry; cryo-TEM, cryo-transmission electron microscopy; ILA, interlamellar attachment site
1993
A method for obtaining the thermodynamic activity of each membrane component in phosphatidylcholine (PC)/ cholesterol mixtures, that is based upon ESR spin labeling is examined. The thermodynamic activity coefficients, Ypc and Ychol, for the PC and cholesterol, respectively, are obtained from the measured orientational order parameters, Spc and SChOI, as a function of cholesterol content for a spin-labeled PC and the sterol-type cholestane spin probe (CSL), respectively, and the effects of water concentration are also considered. At water content of 24 weight%, the thermodynamics of DMPC/cholesterol/water mixtures in the liquid-crystalline state may be treated as a two-component solution ignoring the water, but at lower water content the role of water is important, especially at lower cholesterol concentrations. At lower water content (17 wt%), Ychol decreases with increasing cholesterol content which implies aggregation. However, at higher water content (24 wt°/o), lYchol is found initially to increase as a function of cholesterol content before decreasing at higher cholesterol content. This implies a favorable accommodation for the cholesterol in the membrane at high water and low cholesterol content. Good thermodynamic consistency according to the Gibbs-Duhem equation was obtained for mypc and Wchol at 24 wt%/o water. The availability of-chol (and Ypc) as a function of cholesterol concentration permits the estimate of the boundary for phase separation. The rotational diffusion coefficients of the labeled PC and of CSL were also obtained from the ESR spectra. A previously proposed universal relation for the perpendicular component of the rotational diffusion tensor, R1, for CSL in PC/cholesterol mixtures (i.e., R1 = R°exp(-AS2hOIh9T)) is confirmed. Achange in composition of cholesterol or of waterfor DMPC/cholesterol/ water mixtures affects R1 only through the dependence of Schol on the composition. In particular, the amount of water affects the membrane fluidity, monitored by R1 for CSL, solely by the structural changes it induces in the membrane for the compositions studied. Rotational diffusion for the labeled PC is found to be more complex, most likely due to the combined action of the internal modes of motion of the flexible chain and of the overall molecular reorientation.