Structural determination of an antimicrobial peptide by circular dichroism and nuclear magnetic resonance spectroscopic techniques

Abstract

Antimicrobial peptides are ubiquitously produced throughout nature. Many of these relatively short peptides are lethal towards bacteria and fungi, yet they display minimal toxicity towards mammalian cells. It is widely believed that these peptides act through non-specific binding to biological membranes. With concerns over the increasing ineffectiveness of conventional antibiotics due to emergence of resistant bacterial strains and the overuse of chemical preservatives in foods, naturally occurring antimicrobial peptides such as lactoferricin may provide feasible alternatives. Our objective was to determine the three dimensional structure of an antimicrobial peptide, derived from human lactoferricin, by circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopies. The conformation of the peptide in membrane-like environments would also be monitored, thus providing a better understanding of their mode of actions. The structure of antimicrobial peptide was determined using CD spectroscopy and conventional two-dimensional NMR methods. CD measurements were taken on a Jasco J-720 CD spectropolarimeter using a cell with a 1-mm path length. The CD spectra were recorded at 0.2 nm intervals from 190 nm to 250 nm. DQF-COSY, TOCSY and NOESY spectra were acquired at 27ºC. The CD data revealed that human lactoferricin is partly structured with around 20% α-helical content. The NMR structure of human lactoferricin also revealed to adopt a helical conformation. This contrasts with the structure of bovine lactoferricin that adopts a sheetlike conformation. The helix stretching from Arg⁸ to Lys¹² is amphipathic, with polar residues line up along one side of the helix, while non-polar residues along the other side. The amphipathic structural arrangement was in agreement with other antimicrobial peptides that exert their antimicrobial activities through membrane disruption. Elucidation of the three-dimensional structure is expected to provide valuable insight into the antimicrobial mechanism of the peptide.