Amphipathic Turn Conformations in Linear Tryptophan-Rich Cationic Antimicrobial Peptides: Implications for Rational Design of Novel Antimicrobials

Abstract

Drug resistant bacteria have become a rising global issue responsible for a large number of unexpected and lethal infections. Cationic antimicrobial peptides (CAP) represent a promising source for new antibiotic treatment of these “superbugs”. Initial testing by Cherkasov et al. (ACS Chemical Biology 4: 65-74, 2008), using peptide libraries and artificial neural network analysis, identified two artificially designed nonapeptides, HHC-10 (KRWWKWIRW) and HHC-36 (KRWWKWWRR), as effective antimicrobial peptides equalling or outperforming conventional antibiotics. These small peptides are attractive as potential therapeutics to combat the multiple drug resistant bacterial strains. In this study, conformational properties and the modes of interaction of these peptide constructs with lipid membranes are investigated in detail. Circular dichroism (CD) experiments and molecular modelling in different environments have revealed a flexible turn structures (β or γ) and a strong influence of close tryptophan interactions, likely stabilizing the turns. CD spectra have also revealed strong evidence for the interaction of these peptides with lipid membranes and their self-association in aqueous milieu. Isothermal titration calorimetry experiments have shown a strong preference of these peptides for negatively charged vesicles, representing bacterial membranes, over neutrally charged vesicles. Temperature-dependent CD spectra in the presence and absence of membranes have also revealed the conformational flexibility of the turn structures due to changes in Trp-Trp interactions. We therefore propose that these peptides adopt amphipathic turn structures (with the Trp residues and the positive charges on opposite sides of the turn's plane) that are crucial for their membrane interactions and antimicrobial activity. The results of this biophysical study leads to a rational design approach for novel broad-spectrum small CAPs, in which modifications in the aromatic amino acid composition is linked to their biological activity and interaction with biological membranes.