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Phase diagram and tie-line determination for the ternary mixture DOPC/eSM/cholesterol.

ABSTRACT: We propose a novel, to our knowledge, method for the determination of tie lines in a phase diagram of ternary lipid mixtures. The method was applied to a system consisting of dioleoylphosphatidylcholine (DOPC), egg sphingomyelin (eSM), and cholesterol (Chol). The approach is based on electrofusion of single- or two-component homogeneous giant vesicles in the fluid phase and analyses of the domain areas of the fused vesicle. The electrofusion approach enables us to create three-component vesicles with precisely controlled composition, in contrast to conventional methods for giant vesicle formation. The tie lines determined in the two-liquid-phase coexistence region are found to be not parallel, suggesting that the dominant mechanism of lipid phase separation in this region changes with the membrane composition. We provide a phase diagram of the DOPC/eSM/Chol mixture and predict the location of the critical point. Finally, we evaluate the Gibbs free energy of transfer of individual lipid components from one phase to the other.

SUBMITTER: Bezlyepkina N

PROVIDER: S-EPMC3617424 | biostudies-literature | 2013 Apr

REPOSITORIES: biostudies-literature

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Phase diagram and tie-line determination for the ternary mixture DOPC/eSM/cholesterol.

Bezlyepkina N N Gracià R S RS Shchelokovskyy P P Lipowsky R R Dimova R R

Biophysical journal 20130401 7

We propose a novel, to our knowledge, method for the determination of tie lines in a phase diagram of ternary lipid mixtures. The method was applied to a system consisting of dioleoylphosphatidylcholine (DOPC), egg sphingomyelin (eSM), and cholesterol (Chol). The approach is based on electrofusion of single- or two-component homogeneous giant vesicles in the fluid phase and analyses of the domain areas of the fused vesicle. The electrofusion approach enables us to create three-component vesicles ...[more]

PMID: 23561522

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Project description:We investigate the possibility of using light scattering data in the single-phase regions of a ternary liquid mixture phase diagram to infer ternary mixture coexistence curves, and to infer tie lines joining the compositions of isotropic liquid phases in thermodynamic equilibrium. Previous analyses of a nonlinear light scattering partial differential equation (LSPDE) show that it provides for reconstruction of ternary [D. Ross, G. Thurston, and C. Lutzer, J. Chem. Phys. 129, 064106 (2008); C. Wahle, D. Ross, and G. Thurston, J. Chem. Phys. 137, 034201 (2012)] and quaternary [C. Wahle, D. Ross, and G. Thurston, J. Chem. Phys. 137, 034202 (2012)] mixing free energies from light scattering data, and that if the coexistence curves are already known, it can also yield ternary tie lines and triangles [D. Ross, G. Thurston, and C. Lutzer, J. Chem. Phys. 129, 064106 (2008)]. Here, we show that the LSPDE can be used more generally, to infer phase boundaries and tie lines from light scattering data in the single-phase region, without prior knowledge of the coexistence curve, if the single-phase region is connected. The method extends the fact that the reciprocal light scattering intensity approaches zero at the thermodynamic spinodal. Expressing the free energy as the sum of ideal and excess parts leads to a natural family of Padé approximants for the reciprocal Rayleigh ratio. To test the method, we evaluate the single-phase reciprocal Rayleigh ratio resulting from the mean-field, regular solution model on a fine grid. We then use a low-order approximant to extrapolate the reciprocal Rayleigh ratio into metastable and unstable regions. In the metastable zone, the extrapolation estimates light scattering prior to nucleation and growth of a new phase. In the unstable zone, the extrapolation produces a negative function that in the present context is a computational convenience. The original and extrapolated reciprocal light scattering are jointly used as input to solving the LSPDE to deduce the mixing free energy and its convex hull. When projected onto the composition triangle, the boundary of the convexified part of the free energy is the phase boundary, and lines on the convexified region along which the second directional derivative is zero are the tie lines. We find that the tie lines and phase boundaries so deduced agree well with their exact values. This work is a step toward developing methods for inferring phase boundaries from real light scattering intensities measured with noise, from mixtures having compositions on a coarser grid.

Project description:Unsaturated and saturated phospholipids tend to laterally segregate, especially in the presence of cholesterol. Small molecules such as neurotransmitters, toxins, drugs etc. possibly modulate this lateral segregation. The small aromatic neurotransmitter serotonin (5-HT) has been found to bind to membranes. We studied the lipid structure and packing of a ternary membrane mixture consisting of palmitoyl-oleoyl-phosphatidylcholine, palmitoyl-sphingomyelin, and cholesterol at a molar ratio of 4/4/2 in the absence and in the presence of 5-HT, using a combination of solid-state 2H NMR, atomic force microscopy, and atomistic molecular dynamics (MD) simulations. Both NMR and MD report formation of a liquid ordered (L o ) and a liquid disordered (L d ) phase coexistence with small domains. Lipid exchange between the domains was fast such that single component 2H NMR spectra are detected over a wide temperature range. A drastic restructuring of the domains was induced when 5-HT is added to the membranes at a 9 mol% concentration relative to the lipids. 2H NMR spectra of all components of the mixture showed two prominent contributions indicative of molecules of the same kind residing both in the disordered and the ordered phase. Compared to the data in the absence of 5-HT, the lipid chain order in the disordered phase was further decreased in the presence of 5-HT. Likewise, addition of serotonin increased lipid chain order within the ordered phase. These characteristic lipid chain order changes were confirmed by MD simulations. The 5-HT-induced larger difference in lipid chain order results in more pronounced differences in the hydrophobic thickness of the individual membrane domains. The correspondingly enlarged hydrophobic mismatch between ordered and disordered phases is assumed to increase the line tension at the domain boundary, which drives the system into formation of larger size domains. These results not only demonstrate that small membrane binding molecules such as neurotransmitters have a profound impact on essential membrane properties. It also suggests a mechanism by which the interaction of small molecules with membranes can influence the function of membrane proteins and non-cognate receptors. Altered membrane properties may modify lateral sorting of membrane protein, membrane protein conformation, and thus influence their function as suspected for neurotransmitters, local anesthetics, and other small drug molecules.

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Phase diagram and tie-line determination for the ternary mixture DOPC/eSM/cholesterol.

Publications

Phase diagram and tie-line determination for the ternary mixture DOPC/eSM/cholesterol.

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