Protein–Lipid Interactions in a Three-Component POPC–Cholesterol–Sphingomyelin Modulated Membrane

Biomolecules and excipients can modulate membrane properties, initiate a string of events, and regulate their functionality. To elucidate these phenomena ex vivo, we prepared a three-component lipid model system that consisted of varying proportions of cholesterol, sphingomyelin, and 1-palmitoyl-2-oleoyl-<i>sn</i>-glycero-3-phosphocholine (POPC). To decipher the membrane dynamics, we included a nonmembranous protein, hemoglobin, in our study. Our initial investigation revealed the formation of self-assembled structures and phase separation in a pure POPC-based synthetic membrane when hemoglobin was reconstituted via a detergent-mediated method. A slight unfolding helped the protein adapt to the huge hydrophobic stress of the lipid environment and led to the formation of these self-assembled structures. To identify an optimal lipid composition that mimics the biological membrane, we employed three varying proportions of lipid mixtures: POPC, sphingomyelin, and cholesterol. We examine events like the formation of lipid bilayers, supramolecular structures, and phase separation using techniques like FRAP, FCS, AFM, Z-stacking, and rheology. We observed the variation in condensate formation and its distribution within the membrane, which differs upon an increase in concentration of sphingomyelin and cholesterol. Such membrane behavior is important for raft formation and signaling.