Liquid–liquid immiscibility under non-equilibrium conditions in a model membrane: An X-ray synchrotron study (original) (raw)
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Physical Review E, 2009
The influence of cholesterol on lipid bilayer structure is significant and the effect of cholesterol on lipid sorting and phase separation in lipid-raft-forming model membrane systems has been well investigated by microscopy methods on giant vesicles. An important consideration however is the influence of fluorescence illumination on the phase state of these lipids and this effect must be carefully minimized. In this paper, we show that synchrotron x-ray scattering on solution lipid mixtures is an effective alternative technique for the identification and characterization of the l o ͑liquid ordered͒ and l d ͑liquid disordered͒ phases. The high intensity of synchrotron x rays allows the observation of up to 5 orders of diffraction from the l o phase, whereas only two are clearly visible when the l d phase alone is present. This data can be collected in ϳ1 min/ sample, allowing rapid generation of phase data. In this paper, we measure the lamellar spacing in both the liquidordered and liquid-disordered phases simultaneously, as a function of cholesterol concentration in two different ternary mixtures. We also observe evidence of a third gel-phaselike population at 10-12 mol % cholesterol and determine the thickness of the bilayer for this phase. Importantly we are able to look at phase coexistence in the membrane independent of photoeffects.
Phase behaviour of lipid–cholesterol membranes
Solid State Communications, 2006
The proposed existence of cholesterol-rich lipid rafts in eucaryotic plasma membranes has led to a large number of studies on the influence of cholesterol on the structure and phase behaviour of model membranes. In this article we first give a brief overview of the phase behaviour of membranes made up of binary and ternary lipid-cholesterol mixtures determined from these experiments, and then present the results of our recent x-ray diffraction and fluorescence microscopy studies on these systems. Our observations suggest a possible resolution of the reported discrepancy in the phase behaviour of these systems obtained using different experimental techniques.
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.
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.
Evidence for Lipid/Cholesterol Ordering in Model Lipid Membranes
Biophysical Journal, 2006
It has been postulated that for a binary mixture of phospholipid and cholesterol, phospholipid/cholesterol complexes are formed. Using grazing incidence x-ray diffraction, we have obtained evidence for lipid/cholesterol ordering in model membranes. Scattering features consistent with the existence of lipid/cholesterol complexes persist to high surface pressures even though fluorescence microscopy suggests a homogeneously fluid phase. Contrary to pure phospholipid and cholesterol systems, the resulting lattice spacing, integrated scattering intensity, and coherence lengths of these complexes are almost independent of surface pressure. Furthermore, the single peak observed in these mixed systems is very broad, suggesting that the extent of order for a single scattering structure only persists over a few molecules. This observation is consistent with these complexes being dynamic structures.
Order Parameters and Areas in Fluid-Phase Oriented Lipid Membranes Using Wide Angle X-Ray Scattering
Biophysical Journal, 2008
We used wide angle x-ray scattering (WAXS) from stacks of oriented lipid bilayers to measure chain orientational order parameters and lipid areas in model membranes consisting of mixtures of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)/cholesterol and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC)/cholesterol in fluid phases. The addition of 40% cholesterol to either DOPC or DPPC changes the WAXS pattern due to an increase in acyl chain orientational order, which is one of the main properties distinguishing the cholesterol-rich liquid-ordered (Lo) phase from the liquid-disordered (Ld) phase. In contrast, powder x-ray data from multilamellar vesicles does not yield information about orientational order, and the scattering from the Lo and Ld phases looks similar. An analytical model to describe the relationship between the chain orientational distribution and WAXS data was used to obtain an average orientational order parameter, S x-ray . When 40% cholesterol is added to either DOPC or DPPC, S x-ray more than doubles, consistent with previous NMR order parameter measurements. By combining information about the average chain orientation with the chain-chain correlation spacing, we extended a commonly used method for calculating areas for gel-phase lipids to fluid-phase lipids and obtained agreement to within 5% of literature values.
Functional lipid pairs as building blocks of phase-separated membranes
Proceedings of the National Academy of Sciences
Biological membranes exhibit a great deal of compositional and phase heterogeneity due to hundreds of chemically distinct components. As a result, phase separation processes in cell membranes are extremely difficult to study, especially at the molecular level. It is currently believed that the lateral membrane heterogeneity and the formation of domains, or rafts, are driven by lipid–lipid and lipid–protein interactions. Nevertheless, the underlying mechanisms regulating membrane heterogeneity remain poorly understood. In the present work, we combine inelastic X-ray scattering with molecular dynamics simulations to provide direct evidence for the existence of strongly coupled transient lipid pairs. These lipid pairs manifest themselves experimentally through optical vibrational (a.k.a. phononic) modes observed in binary (1,2-dipalmitoyl-sn-glycero-3-phosphocholine [DPPC]–cholesterol) and ternary (DPPC–1,2-dioleoyl-sn-glycero-3-phosphocholine/1-palmitoyl-2-oleoyl-glycero-3-phosphochol...
Biophysical Journal, 2003
In recent years, the implication of sphingomyelin in lipid raft formation has intensified the long sustained interest in this membrane lipid. Accumulating evidences show that cholesterol preferentially interacts with sphingomyelin, conferring specific physicochemical properties to the bilayer membrane. The molecular packing created by cholesterol and sphingomyelin, which presumably is one of the driving forces for lipid raft formation, is known in general to differ from that of cholesterol and phosphatidylcholine membranes. However, in many studies, saturated phosphatidylcholines are still considered as a model for sphingolipids. Here, we investigate the effect of cholesterol on mixtures of dioleoyl-phosphatidylcholine (DOPC) and dipalmitoyl-phosphatidylcholine (DPPC) or distearoyl-phosphatidylcholine (DSPC) and compare it to that on mixtures of DOPC and sphingomyelin analyzed in previous studies. Giant unilamellar vesicles prepared from ternary mixtures of various lipid compositions were imaged by confocal fluorescence microscopy and, within a certain range of sterol content, domain formation was observed. The assignment of distinct lipid phases and the molecular mobility in the membrane bilayer was investigated by fluorescence correlation spectroscopy. Cholesterol was shown to affect lipid dynamics in a similar way for DPPC and DSPC when the two phospholipids were combined with cholesterol in binary mixtures. However, the corresponding ternary mixtures exhibited different spatial lipid organization and dynamics. Finally, evidences of a weaker interaction of cholesterol with saturated phosphatidylcholines than with sphingomyelin (with matched chain length) are discussed.
Phase separation of saturated and mono-unsaturated lipids as determined from a microscopic model
The Journal of Chemical Physics, 2005
A molecular model is proposed of a bilayer consisting of fully saturated dipalmitoylphosphatidylcholine ͑DPPC͒ and mono-unsaturated dioleoylphosphatidylcholine ͑DOPC͒. The model not only encompasses the constant density within the hydrophobic core of the bilayer, but also the tendency of chain segments to align. It is solved within self-consistent field theory. A model bilayer of DPPC undergoes a main-chain transition to a gel phase, while a bilayer of DOPC does not do so above zero degrees centigrade because of the double bond which disrupts order. We examine structural and thermodynamic properties of these membranes and find our results in reasonable accord with experiment. In particular, order-parameter profiles are in good agreement with NMR experiments. A phase diagram is obtained for mixtures of these lipids in a membrane at zero tension. The system undergoes phase separation below the main-chain transition temperature of the saturated lipid. Extensions to the ternary DPPC, DOPC, and cholesterol system are outlined.