Membrane free-energy landscapes derived from atomistic dynamics explain nonuniversal cholesterol-induced stiffening.

All lipid membranes have inherent morphological preferences and resist deformation. Yet adaptations in membrane shape can and do occur at multiple length scales. While this plasticity is crucial for cellular physiology, the factors controlling the morphological energetics of lipid bilayers and the dominant mechanisms of membrane remodeling remain to be fully understood. An ongoing debate regarding the universality of the stiffening effect of cholesterol underscores the challenges facing this field, both experimentally and theoretically, even for simple lipid mixtures. On the computational side, we have argued that enhanced-sampling all-atom molecular dynamics simulations are uniquely suited for the quantification of membrane conformational energetics, as they minimize a priori assumptions and permit analysis of bilayers in deformed states. To showcase this approach, we examine reported inconsistencies between alternative experimental measurements of bending moduli for cholesterol-enriched membranes. Specifically, we analyze lipid bilayers with different chain saturation and compute free-energy landscapes for curvature deformations distributed over areas from ∼5 to ∼60 nm 2 . These enhanced simulations, totaling over 100 μs of sampling time, enable us to directly quantify both bending and tilt moduli and to dissect the contributing factors and molecular mechanisms of curvature generation at each length scale. Our results show that the effects of cholesterol on bending rigidity are lipid-specific and suggest that this specificity arises from differences in the torsional dynamics of the acyl chains. In summary, we demonstrate that quantitative relationships can now be established between lipid structure and bending energetics, paving the way for addressing open fundamental questions in cell membrane mechanics.