Structure and organization of hemolytic and nonhemolytic diastereomers of antimicrobial peptides in membranes

Recently, we reported on a new group of diastereomers of short-model peptid es (12 amino acids long) composed of leucine and lysine with varying ratios , possessing several properties that make them potentially better than nati ve or de novo-designed all L-amino acid antimicrobial peptides. Preliminary studies have revealed that modulating the hydrophobicity and positive char ges of these diastereomers is sufficient to confer antibacterial activity a nd cell selectivity. However, the relationship between their biological fun ction, structure, and mode of action was not investigated. Here we synthesi zed and investigated three types of linear model diastereomers (12 amino ac ids long) with varying lysine:leucine (or tryptophan) ratios (i.e., K3L8W, K5L6W, and K7L4W), which confer different levels of lytic activities. For e ach K:L ratio, tryptophan was introduced in the middle or the N- or C-termi nus of the peptides, as an intrinsic fluorescent probe. Only the hemolytic peptide K3L8W binds to both negatively charged and zwitterionic phospholipi d membranes. K5L6W and K7L4W bind similarly, but only to negatively charged membranes, despite the fact that K5L6W is substantially more lytic to bact eria than K7L4W. Interestingly, although K3L8W contains 33% D-amino acids, ATR-FTIR spectroscopy revealed a structure of similar to 90% alpha-helix in both types of membranes. In addition, K5L6W contains similar to 40% 3(10)- helix and K7L4W is predominantly a random coil in membranes. Polarized ATR- FTIR and tryptophan-quenching experiments, using brominated phospholipids, revealed a similar depth of penetration and an orientation that was paralle l to the membrane surface for all the peptides, but with K3L8W affecting th e lipid order more than the others. The results provide insight into the mo de of action of this group of diastereomeric peptides, and the effect of hy drophobicity and positive charges on their membrane structure, function, an d cell selectivity. Moreover, this research should assist in the developmen t of suitable diastereomeric peptide antibiotics for therapeutic use that w ould overcame the problem the increasing resistance of bacteria to conventi onal antibiotics.