Computational protocol: Collapse of a lipid-coated nanobubble and subsequent liposome formation

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Protocol publication

[…] Lipid–water systems are composed of CG-saturated phosphatidylcholine (PC), which corresponds to PCs with tail lengths of C15-18, i.e., DPPC-like lipids. Water molecules are based on the MARTINI force field, where the molecules are represented by grouping four heavy atoms into a bead. The initial structure was a preformed nanobubble coated with a lipid monolayer in water. To this end, water beads within a given distance from the center of a cubic liquid water system, which was pre-equilibrated to 323 K and 1 bar, were hollowed out to generate a spherical void region. Then, the void interface was coated by placing lipid molecules where the hydrophilic head groups of the lipid molecules were stuck into the interface. The base system was composed of 600 lipid and 78,501 water CG models. Periodic boundary conditions were used in all directions.Subsequent to the energy minimization of the initial configuration, constant temperature and volume MD calculations were performed at 323 K to equilibrate the systems for at least 200 ns. The leapfrog algorithm was used for integrating the equations of motion and the time step was set to 20 fs. The bonded and nonbonded interaction settings were the same as in the original paper on the MARTINI force field. The temperatures of the DPPC and the water were kept constant separately using the velocity rescaling method with a 1.0 ps coupling constant. During the constant temperature and volume MD simulation, water beads were spontaneously evaporated to the void region and the lipid nanobubble contained the evaporated water beads (). Note that when the lipid monolayer collapsed or ruptured during the constant volume MD calculation, we changed the system size and restarted the calculation such that the water liquid–vapor interface was fully coated by the CG lipid monolayer. The number density of the CG beads for the 600-lipid system was 7.86 -/nm3.After the constant volume MD calculation, the pressure was relaxed to 1 bar using the Berendsen’s isotropic coupling method with a 2.0 ps coupling constant and a compressibility of 3 × 10−5 1/bar. Twenty different initial configurations taken from every 10 ns of the constant volume MD calculation were used to statistically investigate the liposome formation from the nanobubble. The time scale shown in the manuscript is the simulated time, although, the effective time could be greater. Note that the target pressure is arbitrary because the MARTINI water CG model does not properly reproduce the characteristics of the pure water liquid–vapor interface. We chose the MARTINI force field in an attempt to maintain the semi-quantitative features for phospholipids while reducing the computational costs to analyze the nanobubble collapse.To verify the effects of the number of lipids coating the nanobubble, the 1200-, 2400-, and 4800-lipid systems were constructed on the basis of the 600-lipid system, maintaining the water bead/lipid ratio. The preparation of a larger number of lipid systems is arbitrary. The schematic diagram for our approach is shown in . First, the base lipid system is stretched (Stage 1) using the position scaling method of the deform code in the GROMACS 4.6.7 software packages until the monolayer is ruptured and a part of the liquid water surface is directly exposed to the water vapor phase. Second, the stretched base system is duplicated so that the ruptured monolayers, i.e., the pore regions, face each other (Stage 2) and constant volume MD simulations are performed to merge the lipid monolayers (Stage 3). Then, the rectangular system is deformed into the cubic system (Stage 4). The number density of the CG beads is 7.86, 7.65, 7.38 and 7.13 -/nm3 for the 600-, 1200-, 2400-, and 4800-lipid systems, respectively.Before we set the density of the initial configuration, we performed a test calculation in which the initial configuration was a nanobubble partly coated with a lipid monolayer. Subjected to a positive pressure, the bubble shape became semi-stable when the liquid–vapor interface was fully coated with the lipid monolayer before the collapse (see ). Therefore, to analyze the lipid dynamics effectively, we set the fully lipid-coated bubble as the initial configuration in this study. The apparent area per lipid for the systems are in the range 0.6–0.7 nm2, where the coating monolayer is expected in the liquid-expanded phase.Unsteady and nonequilibrium MD simulations should be performed with great care for various simulation parameters and system sizes. We tested different coupling constants (10 ps and 20 ps for the thermostat and barostat, respectively), different compressibility parameters for the Berendsen’s coupling method (3 × 10−6 1/bar and 3 × 10−4 1/bar, see in the ), and larger water phase systems (at most 4800 lipids and 1,884,024 water beads). The essential formation dynamics were independent of the parameters and system sizes were tested here. Further, to investigate the impact of the saturation of phospholipid tails, unsaturated PC lipid systems, which were composed of the MARTINI dilinoleyl-PC model, were prepared. We performed CG MD simulations of 10 samples for the 600-lipid system and one sample for the 4800-lipid and a large number of water beads (1,884,024) system.The MD calculations and the trajectory processing were performed using the GROMACS 4.6.7 software packages. The in-house Python codes developed with the aid of the MDAnalysis library were used for the trajectory analysis and all snapshots were rendered using visual molecular dynamics. The in-house Python codes and the input files for the GROMACS are available from the authors upon request. […]

Pipeline specifications

Software tools MARTINI, GROMACS, MDAnalysis, VMD
Application Protein structure analysis