Gramicidin Peptides Alter Global Lipid Compositions and Bilayer Thicknesses of Coexisting Liquid-Ordered and Liquid-Disordered Membrane Domains.

Effects of adding 1 mol % of gramicidin-A on the biochemical properties of coexisting liquid-ordered and liquid-disordered (L + L) membrane domains were investigated. Quaternary giant unilamellar vesicles (GUV) of di18:1PC(DOPC)/di18:0PC(DSPC)/cholesterol/gramicidin-A were prepared using our recently developed damp-film method. The phase boundary of L + L coexisting region was determined using video fluorescence microscopy. Through fitting Nile Red fluorescence emission spectra, the thermodynamic tie-lines in the L + L two-phase region were determined. We found that at 1 mol % (i.e., ∼7% of membrane area), gramicidin peptides alter the phase boundary and thermodynamic tie-lines. Gramicidin abolishes the coexisting phases at some lipid compositions but induces phase separation at others. Previous studies of gramicidin emphasize the local perturbation of bilayer thickness adjacent to the protein through the interaction of "hydrophobic mismatch". For the first time, it becomes clear that adding gramicidin produces significant long-range and global effects on the structure of membrane domains: it alters the overall lipid compositions and bilayer thicknesses of coexisting L and L domains. We also found that gramicidin partitions favorably into the L phase. Adding gramicidin decreases cholesterol in the L phase and increases cholesterol in the L phase. Those compositional changes broaden the bilayer thickness difference between L and L domains and facilitate preferential partition of gramicidin into thinner L domains. Our results demonstrate that membrane proteins play significant roles in determining lipid compositions and bilayer thicknesses of biomembrane domains.