Characterization of Membrane Interactions of Antimicrobial Lactoferricin Peptides with Central Residue Substitutions

Abstract

The rise in antibiotic-resistant bacteria has led to an active search for new and more effective antimicrobial drugs. A hexapeptide (LfB6: RRWQWR-NH2) derived from the iron-binding protein lactoferrin exhibits antimicrobial activity (Tomita, Acta Paediatr Jpn, 1994, 36:585). A related heptapeptide produced in our lab, with 4 positively charged arginines and 2 methylated tryptophans (RRMeWQMeWRR-NH2; MeTrp-LfB7), exhibits enhanced activity against gram negative and gram positive bacteria. Substitutions of the central glutamine (Gln4;Q) residue that may alter peptide conformational flexibility are now being investigated. When Gln4 was changed to glycine (Gly;G) or proline (Pro;P), significant changes in peptide-membrane interactions were observed, although the antimicrobial activity was not increased. We now examine the effects of replacing Gln4 with gamma amino butyric acid (GABA), to introduce more flexibility; or D-Pro-Gly, to constrain the backbone into a β-hairpin turn (Stanger and Gellman, J. Am. Chem. Soc. 1998, 120:4236). The increased positive ellipticity at ∼230 nm observed in the CD spectrum of the D-Pro-Gly peptide in anionic membranes suggests significant Trp-Trp interactions. Tryptophan fluorescence emission spectra indicate that peptides with either GABA or D-Pro-Gly substitutions are more deeply buried in anionic membranes. Although the GABA and D-Pro-Gly peptides were both more active against gram negative (E. coli), compared to gram positive (S. aureus) bacteria, they were not as active as the Gln4 peptide. Investigations of the peptide-lipid interactions are being continued by means of solid-state 2H and 31P NMR spectra.