Understanding the Free Energy Landscape of Phase Separation in Lipid Bilayers using Molecular Dynamics.

Liquid-liquid phase separation (LLPS) inside the cell often results in biological condensates that can critically impact cell homeostasis. Such phase separation events occur all across a cell and often involve common biomolecules, including lipids in the cell membranes. Lipid phase separation at the cell membrane and subsequent ordered domain formation in a sea of disordered lipids led to the so-called "lipid raft hypothesis." 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 bulk phenomenon on a cell membrane with a diverse lipidome is exceptionally difficult. Hence simple model systems that can recapitulate similar behavior are widely used to study this phenomenon. Despite simplifying the problem, the relative timescale and system size required for simulating phase separation events pose a challenge for molecular dynamics (MD) simulations. Thus, most MD studies focus on spontaneous lipid phase separation as an adequate sampling of transition events between mixed and separated lipid states is hard to achieve in conventional MD simulations. Here, we propose a proof-of-concept pipeline that can realize such multiple-state transitions by combining coarse-grained model membranes with enhanced sampling protocols. Using this pipeline, we can ask not just whether a system phase is separated or not, but why it separates, with statistical rigor.