Structure-function relationships of antimicrobial peptides and host-defense proteins

Pathogenic bacteria are increasingly becoming resistant to current antibiotic treatments. The vast number of antimicrobial peptides and host-defense proteins found in nature presents a novel class of potential therapeutics with a broad range of activity. In this work, a systematic evaluation was carried out to assess the antimicrobial activities and serum stabilities of several short Trp- and Arg-rich hexapeptides with simple differences in backbone modifications and circularized by either a peptide bond or by disulfide cross-linking. Both cyclization strategies were extended to slightly longer peptides based on the porcine antimicrobial peptide tritrpticin (VRRFPWWWPFLRR), which is also rich in Trp and Arg residues. These peptides showed small differences in their membrane-bound structures and, unexpectedly, had contrasting biological activities, with the backbone-cyclized peptide being antimicrobial and nonhemolytic and the disulfide-bonded peptide being non-antimicrobial and hemolytic. Further studies of tritrpticin focused on the roles of its Arg and Trp residues, which were substituted with various natural and unnatural amino acids. These analogues were studied with various biophysical techniques. In addition to electrostatic interactions with the anionic bacterial membranes, the guanidinium group of arginine seems to be optimal for hydrogen bonding with phospholipids, thereby contributing to membrane disruption. While the three Trp residues have a position-dependent contribution to the membrane permeabilizing activity of tritrpticin, most Ala- or Tyr-substituted peptides remained highly antimicrobial regardless. In a separate but complementary research project, a-helical peptides derived from different human chemokines, some with their own strong bactericidal activities, were studied as potential antimicrobials. Finally, the backbone dynamics of two related platelet chemokines, NAP-2 and TC-1, were studied by NMR spectroscopy to examine their very different antimicrobial activities despite a difference in sequence of only two amino acids. For the shorter chemokine with higher potency, the removal of these residues was shown to be sufficient to expose a more cationic surface and change its monomer-dimer equilibrium. Taken together, these studies showcase various structural and dynamic aspects that influence the functions of peptides and proteins with antimicrobial activity. Overall, these studies contribute towards the development of peptides as novel antibiotics while simultaneously gaining understanding about their roles in host-defense systems.