Kinetics of membrane lysis by custom lytic peptides and peptide orientations in membrane

To aid the development of custom peptide antibiotics, a kinetic study of me mbrane lysis by cecropin B (CB) and its analogs, cecropin B1 (CB1) and cecr opin B3 (CB3) was carried out to determine the mechanism by which these pep tides disrupt the bilayer structure of liposomes of defined composition. Di sruption of the phospholipid bilayer was determined by a fluorescence assay involving the use of dithionite to quench the fluorescence of lipids label ed with N-7-nitro-2,1,3-benzoxadiazol-4-yl. Lytic peptides caused the disru ption of liposomes to occur in two kinetic steps. For liposomes composed of mixtures of phosphatidylcholine and phosphatidic acid, the time constants for each kinetic step were shorter for CB and CB1 than for CB3. Oriented ci rcular dichroism experiments showed that the peptides could exist in at lea st two different membrane-associated states that differed primarily in the orientation of the helical segments with respect to the bilayer surface. Th e results are discussed in terms of kinetic mechanisms of membrane lysis. T he mode of actions of these peptides used for the interpretation of their k inetic mechanisms were supported by surface plasmon resonance experiments i ncluding or excluding the pore-forming activities.