A Systematic Study of Phase Changes Induced by Trans-Membrane Peptide Gramicidin-A in Multi-Component Lipid Membranes

Abstract

What are the effects of proteins on lipid membrane domains? In order to answer this question, we systematically investigated the phase changes induced by trans-membrane peptide gramicidin-A in 16:0-18:2PC(PLPC)/di18:0PC(DSPC)/cholesterol and 16:0-18:2PC/di16:0PC(DPPC)/cholesterol lipid bilayers. Quaternary giant unilamellar vesicles (GUV) were prepared using our recently developed Damp-Film method. The phase boundaries of liquid-ordered and liquid-disordered (Lo+Ld) coexisting region as well as the critical points were determined using video fluorescence microscopy. Within the phase coexisting regions, thermodynamic tie-lines were determined using a fluorescence assay. Our results show that adding 1 mol% of gramicidin produces significant and complex phase changes to the lipid bilayers: at some lipid compositions, gramicidin can induce lipid domains; at others, gramicidin completely abolish the phase separation; even if the phase separation is preserved, gramicidin significantly alters the lipid compositions of membrane domains and tie-lines. In the biological relevant critical region, these changes could be quite dramatic. We also measured gramicidin-A partition coefficients between coexisting Lo+Ld lipid phases. Away from the critical point, the coefficient is close to 2, indicating that gramicidin slightly prefers the disordered Ld lipid domains with smaller bilayer thickness. However, the partition coefficient continuously changes with lipid composition. Near the critical point, the partition coefficient approaches to the theoretical value of 1.