Solid-State NMR and Fluorescence Spectroscopy of Antimicrobial Methylated-Tryptophan Lactoferricin Peptides with Gln, Gly or Pro as the Central Residue

Abstract

In recent years pathogenic bacteria have become increasingly resistant to common antibiotics, leading to a demand for new, more potent antibacterial drugs. The 25 amino acid peptide lactoferricin (LfB25) exhibits broad spectrum antimicrobial properties and is of interest for drug development. A cationic amidated hexapeptide, (LfB6: RRWQWR-NH2), containing three arginines and two tryptophans has been identified as the “antimicrobial active site.” Our lab has shown that the antimicrobial activity of LfB6 can be enhanced by methylation of the tryptophan indole nitrogen (MeTrp). After selective deuteration of the MeTrp residues, membrane interactions can be examined by solid-state 2H NMR spectroscopy in oriented bilayers composed of mammalian- and bacterial-like membranes. A symmetric heptapeptide with four arginines and two methyl-tryptophans (RRMeWQMeWRR-NH2; LfB7 MeTrp 3,5) was designed and found to increase antimicrobial activity relative to LfB6. 2H NMR spectra reveal that the MeTrp residues are aligned at the membrane surface, while 31P NMR spectra indicate very little perturbation to the lipid head groups. Partitioning assays in multilamellar vesicles demonstrate an increase in membrane binding correlated with percent anionic lipid, and Trp emission fluorescence spectroscopy confirms that the MeTrps are more water exposed in neutral compared to anionic lipid membranes. To investigate the importance of backbone flexibility for activity and membrane interaction, the central glutamine of the heptapeptide has been changed to either Gly or Pro. Initial results indicate that the Pro-constrained peptide, RRMeWPMeWRR, is less antimicrobial and has weaker membrane alignment and binding than when Gln is the central residue. We will present results for the more flexible peptide with Gly as the central residue, RRMeWGMeWRR.