Co-Titration of Phosphatidylserine Lipids and a Channel Protein Using Constant pH Molecular Dynamics

The protonation state of anionic lipids plays a central role in regulating membrane electrostatics, structure, and interactions with membrane proteins. Lipid titration in membranes is inherently collective and strongly influenced by local composition and lateral organization. Experimentally, determining lipid p K ₐ values in heterogeneous and dynamically organized membranes is challenging, making it difficult to disentangle intrinsic lipid properties from collective membrane effects. Moreover, lipid and protein protonation states can be electrostatically coupled, such that changes in the ionization state of one component can influence the titration behavior of the other. Here, we use constant pH molecular dynamics (CpHMD) simulations to investigate the titration behavior of phosphatidylserine (PS) lipids in bilayers with varying PS content, ranging from dilute mixtures with phosphatidylcholine (PC) to fully anionic membranes, as well as the cotitration of proteins and lipids using the OmpF porin embedded in a titratable PS membrane. We show that the apparent pKa of the PS headgroup depends strongly on membrane composition, increasing systematically with PS surface density. We compare the CpHMD-derived pKa values of OmpF acidic residues in various lipid compositions, ranging from fixed-charge PS to mixed PS/PC and fully titratable PS membranes. The resulting pKa shifts demonstrate that the protonation state of the surrounding lipid environment can substantially modulate protein titration, demonstrating coupled lipid–protein ionization. Together, these results highlight the electrostatic coupling between membrane lipids and proteins and establish CpHMD as a framework for capturing composition-dependent protonation behavior in complex membrane environments.