Abstract
Interfacial hydrogen bonding (H-bonding) partly determines membrane structure, heterogeneity, and dynamics. Given the chemical diversity of lipids, it is important to understand how composition determines lipid-lipid interactions and how those are translated to H-bond populations and dynamics. Here, we investigate the role of palmitoyl sphingosylphosphorylcholine (PSM) in modulating lipid H-bond networks in combination with dipalmitoyl phosphatidylcholine (DPPC) using ultrafast two-dimensional infrared (2D IR) spectroscopy and molecular dynamics simulations. We report composition-dependent H-bond ensembles for ester and amide carbonyls, with increased H-bond populations and slower dynamics with higher PSM concentrations. Specifically, amide carbonyl 2D IR spectra indicate that PSM, acting as an H-bond donor, partially replaces water-mediated interactions, with the number of direct lipid-lipid H-bonds constituting up to 20% of the total. These interactions create comparatively stable H-bond networks that significantly slow interfacial dynamics. 2D IR spectra show an H-bond lifetime slowdown of 45% in an equimolar mixture of the two lipids compared to DPPC alone. This study highlights PSM's dual role in H-bonding, which increases membrane viscosity and stabilizes lipid interfaces, providing molecular insights into the role of sphingolipids in cell membranes. The findings further emphasize the synergy of experimental and computational approaches for extracting molecular-level insights into interfacial lipid-lipid and lipid-water interactions in heterogeneous membranes.