Effect of multiple aliphatic amino acids substitutions on the structure, function, and mode of action of diastereomeric membrane active peptides

The initial stages leading to the binding and functioning of membrane-activ e polypeptides including hormones, signal sequences, and lytic peptides are mainly governed by electrostatic attraction and hydrophobic partitioning b etween water and lipid bilayers. Antimicrobial peptides serve as an importa nt model for studying the details of these initial steps. However, a system atic analysis of the contribution of multiple hydrophobic amino acids to th ese steps have been hindered by the propensity of many peptides to aggregat e and become inactivated in solution. To this end, we synthesized a series of model amphipathic all L-amino acid peptides and their diastereomers with the sequence KX3KWX2KX2K, where X = Gly, Ala, Val, Ile, or Leu. The effect of the aliphatic amino acids on the biological activity, binding, structur e, membrane localization, and mode of action of these peptides was investig ated. Most of the L-amino acid peptides oligomerized and adopted distinct s tructures in solution and in a membrane mimetic environment. Among this gro up only the Leu containing peptide was hemolytic and highly active on most bacteria tested. The Val- and Leu-containing peptides were hemolytic but in active toward most bacteria tested. In contrast, the diastercomeric peptide s were monomeric and unstructured in solution, but they adopted distinct st ructures upon membrane binding. While hemolytic activity was drastically re duced, the spectrum of antibacterial activity was preserved or increased. I mportantly, we found a direct correlation with the diastercomers between hy drophobicity and propensity to form a helical/distorted-helix and activity (induced membrane leakage and antibacterial activity), despite the fact tha t they contained 30% D-amino acids. Furthermore, efficient increase in memb rane permeability can proceed through different mechanisms. Specifically, t he Leu-containing diastereomeric peptide micellized vesicles and possibly b acterial membranes while the Ile-containing diastereomeric peptide fused mo del membranes and irregularly disrupted bacterial membranes.