Out of Sight, Out of Mind: The Effect of the Equilibration Protocol on the Structural Ensembles of Charged Glycolipid Bilayers.

Molecular dynamics (MD) simulations represent an essential tool in the toolbox of modern chemistry, enabling the prediction of experimental observables for a variety of chemical systems and processes and majorly impacting the study of biological membranes. However, the chemical diversity of complex lipids beyond phospholipids brings new challenges to well-established protocols used in MD simulations of soft matter and requires continuous assessment to ensure simulation reproducibility and minimize unphysical behavior. Lipopolysaccharides (LPS) are highly charged glycolipids whose aggregation in a lamellar arrangement requires the binding of numerous cations to oppositely charged groups deep inside the membrane. The delicate balance between the fully hydrated carbohydrate region and the smaller hydrophobic core makes LPS membranes very sensitive to the choice of equilibration protocol. In this work, we show that the protocol successfully used to equilibrate phospholipid bilayers when applied to complex lipopolysaccharide membranes occasionally leads to a small expansion of the simulation box very early in the equilibration phase. Although the use of a barostat algorithm controls the system dimension and particle distances according to the target pressure, fluctuation in the fleeting pressure occasionally enables a few water molecules to trickle into the hydrophobic region of the membrane, with spurious solvent buildup. We show that this effect stems from the initial steps of NPT equilibration, where initial pressure can be fairly high. This can be solved with the use of a stepwise-thermalization NVT/NPT protocol, as demonstrated for atomistic MD simulations of LPS/DPPE and lipid-A membranes in the presence of different salts using an extension of the GROMOS forcefield within the GROMACS software. This equilibration protocol should be standard procedure for the generation of consistent structural ensembles of charged glycolipids starting from atomic coordinates not previously pre-equilibrated. Although different ways to deal with this issue can be envisioned, we investigated one alternative that could be readily available in major MD engines with general users in mind.