Effects of Membrane Cholesterol on Stability of Transmembrane Helix Associations

Membrane cholesterol is an essential and abundant component of eukaryotic cell membranes. The unique chemical structure of cholesterol significantly influences the physicochemical properties of phospholipid bilayers, such as hydrophobic thickness and lateral pressure profile. However, the mechanisms by which these alterations regulate the balance of protein-lipid interactions in lipid bilayer environments remain unclear. To experimentally assess basic and common driving forces for helix associations in membranes, the self-associations of a de novo designed simple transmembrane helix (AALALAA)₃ and its derivative helices were examined. Single-pair fluorescence resonance energy transfer (sp-FRET) experiments were performed to monitor the thermodynamic and kinetic stabilities of helix associations in single liposomes. The addition of cholesterol exerted both stabilizing and destabilizing effects on these associations, up to a change in ΔG_a of approx. 10 kJ mol^-1, and these effects were dependent on the association topology, amino acid sequence, and number of helices. These results demonstrate that cholesterol in the membrane regulates the stability of transmembrane proteins in a protein context-dependent manner through physicochemical mechanisms.