Lactoferricin Peptides: The Importance of Methyl-Tryptophan and Glutamine for Structure and Activity

Abstract

The rise in antibiotic-resistant strains of bacteria has led to an active search for more potent antimicrobial drugs. The hexapeptide (LfB6: RRWQWR-NH2) from the N-terminal of the protein lactoferricin was shown to be the antimicrobial ‘active core’ by Tomita (Acta Paediatr Jpn, 1994, 36:585). A heptapeptide synthesized in our lab, which contains 4 positively charged arginines and 2 methylated tryptophans (LfB7 MeTrp3,5: RRMeWQMeWRR-NH2), exhibits enhanced activity when compared to LfB6 against gram positive and gram negative bacteria. To determine if Trp-methylation is required for the increased efficacy, a control peptide without methylated Trp (LfB7: RRWQWRR-NH2) has been synthesized and characterized. In addition, glutamine was replaced with alanine in both peptides to examine the importance of H-bonding at the central position (LfB7 MeTrp3,5 Ala4: RRMeWAMeWRR-NH2and LfB7 Ala4: RRWAWRR-NH2). Replacement of the central Gln with Ala reduced activity 5-fold against E. coli. LfB7 MeTrp3,5 exhibits the highest activity, with a 4- and 6-fold increase compared to LfB7, against E. coli and S. aureus, respectively. The Ala, Trp and MeTrp residues were labeled with deuterium, and the peptide was studied by solid-state deuterium NMR in bacterial- and mammalian-like membranes. The 2H-quadrupolar splittings, whether from labeled Ala, MeTrp or Trp, are larger when MeTrp is present in the sequence, indicating a change in peptide conformation and/or dynamics upon Trp-methylation. Tryptophan emission fluorescence spectra reveal that the Trp and MeTrp residues are more deeply buried in anionic compared to neutral lipids, and circular dichroism spectra suggest that anionic lipids promote a conformational change that results in Trp-Trp (and MeTrp-MeTrp) interactions. These results emphasize the importance of Gln at the central position and of Trp-methylation for structure and activity of lactoferricin.