Characterization of Antimicrobial Methylated Tryptophan Retro Lactoferricin Peptides by Solid State NMR and Fluorescence Spectroscopy

Abstract

Lactoferricin B (LfB) is a 25-residue peptide from the N-terminus of bovine lactoferrin with broad-spectrum antimicrobial activity. A hexapeptide has been found that retains antimicrobial activity (LfB6: RRWQWR-NH2) (Tomita, et al., Acta Paediatr Jpn, 1994, 36:585-91). Previous work in our lab has shown that reversing the sequence (Retro LfB6: RWQWRR-NH2), amino-acylation, or tryptophan-methylation (MeTrp) increase antimicrobial activity. We now compare the effects of Trp-methylation at position 2 (RMeWQWRR-NH2; Retro LfB6 MeTrp2) versus position 4 (RWQMeWRR-NH2; Retro LfB6 MeTrp4), and of N-acylation of each peptide with a C6-fatty acid tail. Selective deuteration of the MeTrp residues permits analysis of the peptides by solid-state 2H NMR spectroscopy in oriented bilayers. We find that the antimicrobial activity of peptides with MeTrp at position 2 is reduced compared to position 4, except in the case C6-Retro MeTrp2 which exhibits greater activity towards E. coli. 31P NMR spectra of oriented samples composed of DMPC and DMPC:DMPG (3:1), to mimic mammalian or bacterial cell membranes, respectively, reveal that the bilayer remains intact when exposed to the peptides. This suggests the mechanism for antimicrobial activity does not involve large scale membrane perturbation. 2H NMR spectra reveals that the MeTrp in the acyl and non-acyl MeTrp2 peptides is less well-aligned on the surface compared to the MeTrp4 peptides. Partitioning assays confirm weaker binding for Retro MeTrp2 peptides compared to MeTrp4. Fluorescence spectroscopy shows that, although the spectra of the Retro MeTrp4 and MeTrp2 peptides are similar, the emission max for the Trp and MeTrp residues in acyl and non-acyl Retro MeTrp2 peptides are blue-shifted (lower max) compared to the Retro MeTrp4 peptides, indicating a less polar environment.