2.1. Dendrimer Setup

We used PyMOL²⁴ to build the initial conformations of the dendrimers. The free lysine side chains, as well as the free N-termini, are titrated, while the C-terminus is capped with NH₂ groups. For both the ramified and palmitoylated lysines, we built additional blocks on our force field. In the ramified lysine, the amine group is changed for an amide bond, with identical parameters to the ones used in the main chain. For the palmitoylated lysine, used as a hydrophobic core of some peptide dendrimer sequences, we adapted the palmitoyl parameters from 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC) lipids and coupled them to a ramified lysine, previously described.

For the membrane simulations, a lipid bilayer of 304 POPC molecules was prepared. To build this membrane patch, we started with a pre-equilibrated bilayer with 128 POPC lipids²⁵ and multiplied it by 4 in the x/y plane using the GROMACS tool genconf, obtaining a membrane patch of 512 lipids. From this, we applied a shrinking procedure where 8 lipids, 4 from each monolayer, are randomly removed in a stepwise manner until we reached our target number of 304 lipids.²⁵ In each step, we relaxed our system using a short MD segment (∼1 ns) and using a stronger pressure (50 bar) in the x/y plane to induce rapid convergence of the total area. In all of these steps, water molecules can also be randomly removed to satisfy the water/lipid ratio requested. We prepared our systems with a very high ratio of 78:1 to preserve all the initial water molecules since we wanted to create a box large enough in height to allow the dendrimer to be placed far away from the membrane in the starting configurations.²⁶ After the final membrane patch of 304 lipids was obtained, the membrane was then equilibrated at normal pressure.

The dendrimers were prepared in a fully protonated form, placed ∼3 nm away from the membrane, and simulated using unbiased MD to sample their unbiased membrane approach pathways. The simulations were stopped when the dendrimer reached the nonbonded interactions cutoff distance (∼1.0 nm) from the membrane, and we removed the extra water molecules from the system in the last configurations, leaving a distance of ∼1.6 nm to the periodic images in the Z axis. This protocol leads to the removal of half the number of water molecules to ∼20k. Since the CpHMD simulations are more computationally demanding, this system reduction step was essential to increase their computational performance. The final system configurations were then subject to normal minimization and initialization protocols (see below).