Key molecular requirements for raft formation in lipid/cholesterol membranes

Abstract

The lipid mixture of DPPC (saturated lipid)/DUPC (unsaturated lipid)/CHOL (cholesterol) is studied with respect to its ability to form liquid-ordered and liquid-disordered phases. We employ coarse-grained simulations with MARTINI force field. All three components are systematically modified in order to explore the relevant molecular properties, leading to phase separation. Specifically, we show that the DPPC/DUPC/CHOL system unmixes due to enthalpic DPPC-DPPC and DPPC-CHOL interactions. The phase separation remains unchanged, except for the formation of a gel phase at long times after decreasing the conformational degrees of freedom of the unsaturated DUPC. In contrast, the phase separation can be suppressed by softening the DPPC chains. In an attempt to mimic the ordering and unmixing effect of CHOL the latter is replaced by a stiff and shortened DPPC-like lipid. One still observes phase separation, suggesting that it is mainly the rigid and planar structure of CHOL which is important for raft formation. Addition of an extra bead to the head of CHOL has no notable impact on the phase separation of the system, supporting the irrelevance of the Umbrella model for the phase separation. Reduction of the conformational entropy of CHOL by stiffening its last bead results in a significant increase of the order of the DPPC/CHOL domain. This suggests that the conformational entropy of CHOL is important to prohibit the gelation process. The interleaflet interactions as mediated by the terminal molecular groups seem to have a strong impact on the possibility of a subsequent gelation process after phase separation.

Figure 1. Martini CG models of DPPC, DUPC and CHOL.

All the beads are colored according to their types and numerated. Types of all the beads are written within the beads.

Figure 2. Time-dependent interaction energies and order parameters of DPPC, DUPC and CHOL.

The interaction energy gains (A) present the differences between the absolute energies at a given time and the energies at time 1 ns. The average order parameters of DPPC, DUPC and CHOL are shown in (B). The small error bars present the standard errors around the values which are the averages of five independent runs.

Figure 3. DPPC, DUPC and the modified DUPC lipids.

The modifications relate to the equilibrium angle bonds and/or angle force constants (stiffness) as well as the bead types of the chains of DUPC. The types of the two middle beads of the lipid chains are set to C1 type (cyan) according to the corresponding DPPC bead type for the modified DUb2, DUb3 and DUb23 lipids. The black circles on top of the lipids indicate assimilations of the angles and/or angle force constants of the DUPC to the ones of the DPPC lipid while the red color indicates no change with respect to the original DUPC. The final configurations and phases of DPPC/DUPC/CHOL, DPPC/DUas/CHOL and DPPC/DUb23/CHOL systems after 12 µs of simulation time are shown at the bottom. These are characteristic configurations also for the other modified DUPC types as indicated by the arrows. In the snapshots, the DPPC is shown in green, the DUPC and its modifications in blue and the CHOL in red.

Figure 4. Time-dependent average MSD for DUPC, DUas and DUb23 over five independent runs.

The dashed line has slope one, corresponding to pure diffusion.

Figure 5. Time-dependent interaction energies and order parameters of DPPC/DUPC/CHOL, DPPC/DUas/CHOL and DPPC/DUb23/CHOL systems.

The interaction energy gains (A) for each system present the differences between the absolute energies at a given time and the energies at time 1 ns. The average order parameters of the components for the three systems are shown in (B). The small error bars present the standard errors around the values which are the averages of five independent runs.

Figure 6. Snapshot, interaction energy gains and order parameters of DPPC_soft/DUPC/CHOL system.

The snapshot (top) presents one of the leaflets after 12 µs. The DPPC_soft, DUPC and CHOL are colored in green, blue and red, respectively. The energies at time 0 are taken for references (middle). For the sake of readability the energy gain of 0 at time 0 is mapped to 10 ps. The order parameters (bottom) of the systems DPPC_soft/DUPC/CHOL and DPPC/DUPC/CHOL are colored in red and black, respectively. The energy and order parameter data is averaged over three independent runs with standard error bars.

Figure 7. Snapshot and order parameters of DPPC/DUPC/DPPC_3b system.

The snapshot (top) presents one of the leaflets after 12 µs. The DPPC, DUPC and DPPC_3b are colored in the snapshot in green, blue and red, respectively. The order parameters (bottom) of the systems DPPC/DUPC/DPPC_3b and DPPC/DUPC/CHOL are colored in red and black, respectively. The data of DPPC_3b system is averaged over three independent runs with standard error bars smaller than the marker symbols.

Figure 8. Order parameters of DPPC/DUPC/CHOL₃₀₀ and DPPC/DUPC/CHOL systems.

The order parameters of DPPC/DUPC/CHOL₃₀₀ and DPPC/DUPC/CHOL systems are colored in red and black, respectively. The data of CHOL₃₀₀ system is averaged over three independent runs with standard error bars smaller than the marker symbols.

Figure 9. Interleaflet domain correlation coefficient and temporal z coordinates of lipid/CHOL heads and tails.

Interleaflet DPPC-domain correlation coefficient (A) and temporal z coordinates of heads and tails of lipids and CHOL relative to the bilayer center for the DPPC/DUPC/CHOL system (B). The relative coordinates of heads and tails in (B) are shown in solid and dashed lines, respectively. The horizontal gray line at 0.0 shows the bilayer center. The error bars present the standard errors from averaging over five independent runs.

Figure 10. Order parameters of the symmetric and asymmetric cases of the DPPC/DUPC/CHOL system.

Figure 11. Diagram of the gel formation stresses the importance of interleaflet interaction.

Figure 12. Order parameters and Umbrella model for CHOL and CHOL_+1HB.

The order parameters of DPPC/DUPC/CHOL_+1HB (red) and DPPC/DUPC/CHOL (black) systems (A) and the temporal ratios of all those CHOL or CHOL_+1HB molecules which are covered by one or two lipid heads i.e. belonging to the Umbrella model to the total number of sterols in the systems (B). The data is averaged over five and three independent runs for the systems with CHOL and CHOL_+1HB, respectively The error bars show the standard error.