The Two Faces of the Liquid Ordered Phase

[Image: see text] Coexisting liquid ordered (L(o)) and liquid disordered (L(d)) lipid phases in synthetic and plasma membrane-derived vesicles are commonly used to model the heterogeneity of biological membranes, including their putative ordered rafts. However, raft-associated proteins exclusively partition to the L(d) and not the L(o) phase in these model systems. We believe that the difference stems from the different microscopic structures of the lipid rafts at physiological temperature and the L(o) phase studied at room temperature. To probe this structural diversity across temperatures, we performed atomistic molecular dynamics simulations, differential scanning calorimetry, and fluorescence spectroscopy on L(o) phase membranes. Our results suggest that raft-associated proteins are excluded from the L(o) phase at room temperature due to the presence of a stiff, hexagonally packed lipid structure. This structure melts upon heating, which could lead to the preferential solvation of proteins by order-preferring lipids. This structural transition is manifested as a subtle crossover in membrane properties; yet, both temperature regimes still fulfill the definition of the L(o) phase. We postulate that in the compositionally complex plasma membrane and in vesicles derived therefrom, both molecular structures can be present depending on the local lipid composition. These structural differences must be taken into account when using synthetic or plasma membrane-derived vesicles as a model for cellular membrane heterogeneity below the physiological temperature.