Sphingomyelin Slows Interfacial Hydrogen-bonding Dynamics in Lipid Membranes

Hydrogen bonding at lipid interfaces partly determines membrane structure, heterogeneity, and dynamics. Given the chemical diversity of lipids, it is important to understand the relationship between lipid composition and interfacial H-bond dynamics. Here we investigate the role of palmitoyl sphingomyelin (PSM) in modulating interfacial H-bond networks in binary mixtures with dipalmitoyl phosphatidylcholine (DPPC) using a combination of ultrafast two-dimensional infrared (2D IR) spectroscopy and molecular dynamics (MD) simulations. We find that analysis of 2D IR spectra of the amide carbonyl is subtle, requiring a careful accounting of the heterogeneous H-bond environment of the amide. The amide groups in PSM, acting as H-bond donors, partially replace water-mediated interactions at lipid carbonyls, with the number of lipid-lipid H-bonds constituting up to 20% of the total. These interactions create comparatively stable hydrogen-bond networks that significantly slow interfacial dynamics. The comparison between experiments and simulations reveals composition-dependent H-bond ensembles for ester and amide carbonyls, with increased H-bond populations and slower dynamics with higher PSM concentrations. 2D IR spectra show that interfacial H-bond dynamics slow by up to 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. These 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.