The plasma membrane (PM) of mammalian cells is made of two layers of structurally distinct lipid molecules. The existence of the bilayer’s compositional asymmetry has been known since the 1970s but we recently discovered that it can be accompanied by a large imbalance in the total lipid abundances in the two monolayers [1]. Transient and localized exposure of cytoplasmic leaflet lipids on the cell surface has been observed under a wide range of physiological conditions, indicating that loss of PM asymmetry plays important roles in biological processes [2]. In cells the release of asymmetry is achieved via the regulated activity of specialized proteins in the PM that catalyze the non-specific bi-directional movement of phospholipids between the two leaflets. While these proteins allow lipids to redistribute according to their chemical potential, glycolipids which are present only in the exoplasmic leaflet and have large sugar moieties attached to the lipid headgroups, are unlikely to easily translocate to the cytoplasmic leaflet. One hypothesis is that the asymmetric distribution of glycolipids following complete scrambling of all other PM lipids drives the generation of curvature and ultimate budding of the vesicles. We propose to test this hypothesis with molecular dynamics (MD) simulations. We want to run simulations of glycolipid-containing bilayers (and controls), and use the simulation trajectories to analyze the propensity of the lipid membrane to curve via calculation of the lateral pressure distribution in the systems. We therefore plan to construct simplified membrane models with one type of bulk lipid present in both leaflets, e.g. 16,0-18,1 PC (POPC), and another lipid constrained only to one leaflet: either GM1, a prototypical glycolipid, or sphingomyelin (SM) which has the same structure as GM1 except that the bulky sugar headgroup is replaced with a choline headgroup (like that of POPC). Three different systems will be constructed for each composition by varying the initial relative number of POPC lipids in the two leaflets while keeping the total number of POPC+GM1/SM lipids constant. Then the same 6 systems will be constructed but with the addition of 30 mol% Chol which is known to redistribute very quickly between the leaflets and is likely to affect the curvature stress in the membrane. The goal would be to simulate the 12 bilayers for multiple microseconds to equilibrate the lipid packing (and cholesterol distribution) and calculate the first moment of the pressure profile in each system (i.e. the bilayer torque density) which will show if the bilayer wants to curve in either direction or prefers to be flat. References [1] Doktorova M, Symons JL, …, Levental KR, Levental I. 2023. Cell membranes sustain phospholipid imbalance via cholesterol asymmetry. bioRxiv. https://doi.org/10.1101/2023.07.30.551157 [2] Doktorova M, Symons JL, Levental I. 2020. Structural and functional consequences of reversible lipid asymmetry in living membranes. Nat. Chem. Biol. 16:1321-1330