| This thesis has focused on the study of two classes of antimicrobial peptides found in vertebrates, the tryptophan-rich antimicrobial peptides (tritrpticin, indolicidin and lactoferricin B (LfcinB)) and the human defensins. The Trp-rich cationic antimicrobial peptides were observed to preferentially insert into anionic phospholipid bilayers, becoming localized to the interfacial region of the bilayer. Using solution NMR spectroscopy, the three-dimensional structures of tritrpticin and a hexa-peptide fragment of LfcinB, thought to be its antimicrobial-active center, were determined in membrane-mimetic SDS micelles. Additionally, a structure-function study was performed on seven analogues of tritrpticin, in which various amino acids were altered. The interactions between the peptides and membranes were characterized using various fluorescence assays, with comparisons being made to their antimicrobial activities, hemolytic activities and their three-dimensional structures in detergent micelles. The research on tryptophan-rich antimicrobial peptides expanded to include the Tip-rich membrane-proximal region of HIV gp41, postulated to disrupt lipid packing during the fusion of the viral and host-cell membranes. The NMR solution structure of a 19-residue peptide corresponding to this region was determined in dodecylphosphocholine micelles. The peptide adopts a helical structure, with the seven aromatic residues forming a collar around the axial length of the peptide that would likely position this peptide in the membrane-water interface of a phospholipid bilayer. Due to the capacity of the Trp-rich peptide to induce lysis of phospholipid vesicles, structure-function studies of this peptide were initiated with the goal of converting the peptide into an antimicrobial peptide. Defensins are 3--5 kDa, cationic antimicrobial peptides with three disulfide bonds. The NMR structure and oligomeric state of human beta-defensin-3 (HBD-3) in aqueous solution was determined and compared to the NMR structures of two other related beta-defensins. While a possible dimer interface was proposed for HBD-3, using homonuclear NMR techniques it was not possible to solve its dimeric structure. Therefore, synthetic genes were designed for HBD-3 and HNP-1 (an alpha-defensin that is also dimeric). proHNP-1, mature-HNP-1 and mature-HBD-3 were successfully expressed to high levels as thioredoxin fusion proteins. Their expression in the OrigamiB strain of E. coli resulted in the successful oxidation of three disulfide bonds. |
