N- and O-methylation of sphingomyelin markedly affects its membrane properties and interactions with cholesterol

► Sphingomyelin analogs were prepared which had methylations on the 2NH- or the 3OH-function, or both. ► The bilayer properties of the SM analogs were determined experimentally and by molecular dynamics simulations. ► Methylations also markedly attenuated interactions with sterols. ► MD simulations further showed that cholesterol's depth and tilt angle in the bilayer were affected. ► We conclude that interfacial properties markedly influence molecular and domain-forming properties of SMs.