Molecular Dynamics (MD) simulations

The bilayer properties of the different lipid types used for exploring NLPs stability were characterized using MD simulations. All the simulations were generated using the CHARMM-GUI Membrane Builder,^45,46 and performed using the GROMACS molecular dynamics suite version 5.1.x.^47,48 Each bilayer system contained 144 lipids (72 in each leaflet), 60 water molecules per lipid, and 150 mM NaCl. The CHARMM36 lipid force field⁴⁹ was used with the TIP3P water model.^50,51 The initial energy minimization and six-step equilibration was done according to the CHARMM-GUI GROMACS equilibration scripts. Production runs were 200 ns long at 310 K and 1 bar. Temperature was controlled with a Nosé-Hoover thermostat⁵² (with a time constant of 1 ps) and pressure with a semi-isotropic Parrinello-Rahman barostat (with a time constant of 5 ps and a compressibility of 4.5×10⁻⁵ bar⁻¹).⁵³ The simulations were run under periodic boundary conditions (PBC), time step was 2 fs, the LINCS algorithm⁵⁴ was used to restrain h-bonds, Lennard–Jones interactions were cutoff using a force-switch over 1.0 to 1.2 nm, electrostatic interactions were cutoff at 1.2 nm and long-range electrostatic handled with the particle mesh Ewald (PME) method.⁵⁵

For analysis, averages over the last 100 ns of each simulation were used and SE estimated between the last four, 25 ns, segments. The analysis was carried out using GROMACS tools and custom tools written in Python and using the MDAnalysis package.^56,57 The area per lipid was estimated as the box area (along the bilayer plain) divided by the number of lipids per monolayer. The bilayer thickness was defined as the average phosphate distance between the bilayer leaflets. The bilayer area compressibility (K_A) was calculated from the amplitude of the box area fluctuations KA=kBTA/NA-A02, where k_B is the Boltzmann constant, T is the absolute temperature in Kelvin, N is the number of lipids in each monolayer, and A₀ is the equilibrium area. The bilayer water access was quantified as average number of water oxygen atoms within 0.4 nm of the bilayer hydrophobic core (defined as the fatty acid carbon atoms). Simulation snapshots were rendered in Visual Molecular Dynamics (VMD).⁵⁸