Understanding the free-energy landscape of phase separation in lipid bilayers using molecular dynamics

Liquid-liquid phase separation inside the cell often results in biological condensates that can critically affect cell ho-meostasis. Such phase separation events occur in multiple parts of cells, including the cell membranes, where the "lipid raft"hy-pothesis posits the formation of ordered domains floating in a sea of disordered lipids. The resulting lipid domains often have functional roles. However, the thermodynamics of lipid phase separation and their resulting mechanistic effects on cell function and dysfunction are poorly understood. Understanding such complex phenomena in cell membranes, with their diverse lipid com-positions, is exceptionally difficult. For these reasons, simple model systems that can recapitulate similar behavior are widely used to study this phenomenon. Despite these simplifications, the timescale and length scales of domain formation pose a challenge for molecular dynamics (MD) simulations. Thus, most MD studies focus on spontaneous lipid phase separation-essentially measuring the sign (but not the amplitude) of the free-energy change upon separation-rather than directly interrogating the ther-modynamics. Here, we propose a proof-of-concept pipeline that can directly measure this free energy by combining coarse-grained MD with enhanced sampling protocols using a novel collective variable. This approach will be a useful tool to help connect the ther-modynamics of phase separation with the mechanistic insights already available from MD simulations.SIGNIFICANCE Standard molecular dynamics simulations can determine the sign of the free-energy change upon phase separation but not the amplitude. We present a new method to determine the phase separation free energy for lipid membranes, based on an enhanced sampling using the weighted ensemble method combined with a novel collective variable, validated using coarse-grained simulations applied to several simple systems. The new method will be valuable as a way to develop models that connect molecular-level structural features to the thermodynamics of phase separation.