Trp residues near peptide termini enhance the membranolytic activity of cationic amphipathic α-helices.

KIA peptides were designed as a series of cationic antimicrobial agents of different lengths, based on the repetitive motif [KIAGKIA]. As amphiphilic helices, they tend to bind initially to the surface of lipid membranes. Depending on the conditions, they are proposed to flip, insert and form toroidal pores, such that the peptides are aligned in a transmembrane orientation. Tryptophan residues are often found near the ends of transmembrane helices, anchoring them to the amphiphilic bilayer interfaces. Hence, we introduced Trp residues near one or both termini of KIA peptides with lengths of 14-24 amino acids. Our hypothesis was that if Trp residues can stabilize the transmembrane orientation, then these KIA peptides will exhibit an increased propensity to form pores, with increased membranolytic activity. Using solid-state N NMR, we found that peptides with Trp near the ends are indeed more likely to be flipped into a transmembrane orientation, especially short peptides. Short KIA peptides also exhibited higher antimicrobial activity when modified with Trp, while longer peptides showed similar activities with and without Trp. The hemolytic activity of KIA peptides of all lengths was higher with Trp near the ends. Vesicle leakage was also increased (sometimes more than 10-fold) for the Trp-mutants, especially in thicker membranes. Higher functionality of amphiphilic helices may thus be achieved in general by exploiting the anchoring effect of Trp. These results demonstrate that the incorporation of Trp increases membranolytic activities (vesicle leakage, hemolysis and antimicrobial activity), in a way compatible with a transmembrane pore model of peptide activity.