In situ determination of structure and fluctuations of coexisting fluid membrane domains

Abstract

Biophysical understanding of membrane domains requires accurate knowledge of their structural details and elasticity. We report on a global small angle x-ray scattering data analysis technique for coexisting liquid-ordered (Lo) and liquid-disordered (Ld) domains in fully hydrated multilamellar vesicles. This enabled their detailed analysis for differences in membrane thickness, area per lipid, hydrocarbon chain length, and bending fluctuation as demonstrated for two ternary mixtures (DOPC/DSPC/CHOL and DOPC/DPPC/CHOL) at different cholesterol concentrations. Lo domains were found to be ~10 Å thicker, and laterally up to 20 Å(2)/lipid more condensed than Ld domains. Their bending fluctuations were also reduced by ~65%. Increase of cholesterol concentration caused significant changes in structural properties of Ld, while its influence on Lo properties was marginal. We further observed that temperature-induced melting of Lo domains is associated with a diffusion of cholesterol to Ld domains and controlled by Lo/Ld thickness differences.

Figure 1

Overview of samples studied in this work. (Solid circles) Their location is shown in previously reported compositional phase diagrams of (A) DOPC/DSPC/CHOL (50) and (B) DOPC/DPPC/CHOL (49). In fluid-fluid phase coexistence regions (dark-shaded areas), demixing into L_o and L_d domains occurs along tielines. Two tielines for each system (A_t1, A_t2 and B_t1, B_t2) with three different compositions at the L_d, L_o endpoints and the tieline center were studied (see Table S1 for detailed lipid composition). Note that tieline endpoints for L_o on A_t1 and L_d on B_t2 (star) are outside previously reported phase boundaries. This is either due to updates in phase boundaries for isolated tielines (A_t1 (35)) or experiments performed in this study (B_t2, see Results).

Figure 2

Parsing scheme of ternary lipid mixtures based on MD simulations of an L_o phase of DOPC/DPPC/CHOL (31). (A) Snapshot of the equilibrated system. DPPC lipids, blue; DOPC, red; cholesterol, yellow. (B) Calculated electron density profile. (C) Electron densities of molecular groups, calculated using SIMTOEXP (70). (Left) Individual contributions of DPPC (solid lines) and DOPC (dashed lines) for the CholCH₃, PCN, CG, CH₂, and CH₃ groups. (Yellow line) Contribution of cholesterol. (Right) Condensed parsing scheme after merging individual contributions as detailed in the main text. To see this figure in color, go online.

Figure 3

Validation of the global analysis for two coexisting phases for the A_t1 tieline (T = 22°C). (A and B) Fits to L_d and L_o endpoint data, respectively. (Insets, both panels) Derived volume probability distributions. (C) Best fit to SAXS data at the A_t1 tieline midpoint. Bragg reflections of L_o (dashes) and L_d (crosses) domains. (Inset) ED profiles for L_o and L_d phases. To see this figure in color, go online.

Figure 4

Temperature behavior of DOPC/DSPC/CHOL (tieline t2) as revealed by SAXS. (A) Contour plot of the second-order Bragg reflections indicated as L_o and L_d. Note that the smooth appearance of data is due to an interpolation procedure between the individual frames. The critical temperature T_C is between 45 and 50°C. At >T_C, only a single lamellar lattice is observed. (B) Measured scattering at 22°C with the indicated Bragg reflections for L_o (dashes) and L_d (crosses) domains. (C) The same, for 50°C. (Solid lines) Best fits. (Insets, both panels) Resulting ED profiles for L_o and L_d phases. To see this figure in color, go online.

Figure 5

Temperature dependence of structural parameters of DOPC/DSPC/CHOL (squares) and DOPC/DPPC/CHOL (triangles), starting from the t1 tieline midpoints (Fig. 1). (A) Bilayer thickness, (B) area per lipid, and (C) water layer thickness, for L_d (solid symbols) and L_o (open symbols) domains as a function of temperature.

Figure 6

Temperature dependence of bending fluctuations of coexisting DOPC/DSPC/CHOL (squares) and DOPC/DPPC/CHOL (triangles) domains, for t1 tieline compositions (Fig. 1 and Table S1). (Shaded symbols) Results for L_d domain; (open symbols) results for L_o domain.

Figure 7

Variation of L_d phase fraction with temperature. (Left) Results for DOPC/DSPC/CHOL. (Right) Results for DOPC/DPPC/CHOL. (Squares) Results for t1 tielines; (triangles) results for t2 tielines.