BPS2026 – Investigating the effect of gramicidin A on membrane structure and lipid phase separation

Biomembranes define the boundaries of cells and organelles, serving as dynamic platforms for various biomolecular processes. The activity of many channel-forming peptides and proteins is influenced by the physical and chemical properties of the surrounding lipid bilayer, factors that in turn depend on lipid composition. This study focuses on gramicidin A (gA), a pore-forming peptide known to cause membrane deformation. Despite abundant evidence that lipid-protein interactions help shape membrane organization, the specific mechanisms by which pore-forming peptides influence lateral phase separation and bilayer properties remain unclear. We hypothesized that gA in ternary lipid mixtures would preferentially partition into the liquid-disordered (Ld) phase and induce bilayer thinning, potentially altering the phase fractions of liquid-ordered (Lo) and Ld phases. Using confocal microscopy, we found that at a peptide-to-lipid ratio of 1:150, gA induced macroscopic phase separation in DPPC/20:1-PC/cholesterol. However, in DPPC/DOPC/cholesterol, gA did not substantially impact the overall phase behavior. Fluorescence resonance energy transfer (FRET) revealed a subtle but statistically significant influence of gA, which increased the miscibility transition temperature (Tmix) of DPPC/20:1-PC/cholesterol in a concentration dependent manner. In contrast, gA in DPPC/DOPC/cholesterol had no significant effect. Cryo-EM imaging further showed that gA altered bilayer thickness and vesicle morphology in single-component 20:1-PC membranes, whereas no significant changes in the bilayer thickness were observed in DOPC vesicles.