BPS2026 – Simulating pH-dependent membrane behavior through co-titration of proteins and lipids in CpHMD

Biological membranes are dynamic environments where local pH strongly influences ion transport, signaling, and protein-lipid interactions. pH effects on protein function are being studied but still the protonation behavior of membrane lipids remains poorly understood and is rarely considered in molecular simulations. Many lipids, including phosphatidylserine (PS) and phosphatidylethanolamine (PE), possess titratable headgroups that can undergo protonation or deprotonation depending on local conditions. Constant pH molecular dynamics (CpHMD) has emerged as a powerful tool to study proton-coupled conformational dynamics in proteins by enabling the dynamic sampling of protonation states. However, its application has been largely restricted to amino acid side chains, leaving lipid headgroups underexplored. Recent advances have shown the feasibility of parameterizing titratable cationic ionizable lipids—a critical component of lipid nanoparticles, but a systematic framework for biologically relevant anionic and zwitterionic phospholipids is lacking. Here, we extend a recent CpHMD protocol by developing parameters for ionizable phospholipids, enabling protonation sampling of anionic and zwitterionic membrane components and its applications to coupled protein-lipid systems. Specifically, we (1) selected representative lipids with titratable headgroups, (2) we derive CHARMM36-compatible force field parameters for the protonated state of lipid headgroups, (3) implement these parameters in the GROMACS CpHMD λ-dynamics framework, (4) validate the approach through bilayer simulations across pH different conditions, and (5) apply the framework to study how lipid protonation modulates the dynamics and residue pKa values of the OmpF porin. By enabling co-titration of proteins and lipids, this work will provide a robust computational framework for simulating pH-dependent membrane processes, offering new insights into how protonation dynamics shape membrane electrostatics and protein function.