Membrane Interactions of Antimicrobial Retro-Lactoferricin Peptides by Solid-State NMR and Partitioning Assays

Abstract

Bovine lactoferricin (LfB25) is a 25-residue peptide released from the N-terminal of bovine lactoferrin which exhibits broad spectrum antimicrobial properties. A hexapeptide, LfB6 (RRWQWR-NH2), is a small fragment that retains significant antimicrobial activity (Tomita et al. 1994. Acta Paediatr Jpn. 36:585). We seek to modify and understand the antimicrobial properties of LfB6 through various modifications. Previous studies from our lab have shown that antimicrobial activity can be increased by N-acylation and Trp-methylation (Greathouse et al. 2008. J. Pept. Sci. 14:1103), as well as by reversal of the peptide sequence (Retro-LfB6). Acylation and Trp-methylation enhance the binding of the peptides to bacterial-like membranes. In addition, Trp-methylation allows for selective ring labeling with deuterium for solid-state NMR studies. To determine whether combining the three modifications (sequence reversal, Trp methylation, and acylation) will further enhance activity, two Retro-LfB6 peptides have been synthesized with a methylated Trp at sequence position 4. One of the peptides has been further modified by N-acylation with a 6-carbon fatty acid. Retro-LfB6 (RWQMeWRR-NH2) and C6 Retro-LfB6 (C6-RWQMeWRR-NH2) were examined by solid-state 31P and 2H NMR in mechanically aligned bilayers composed of a neutral membrane with a zwitterionic head group (DMPC) and a bacterial-like membrane composed of a 3:1 mixture of neutral (DMPC) and anionic lipids (DMPG). 2H NMR spectra suggest weaker binding or less well-defined orientations of the Retro-LfB6 peptides compared to native LfB6 peptides. 31P NMR spectra reveal that the lipids remain primarily in a bilayer arrangement, with the peptides causing little change to the phosphate head groups. Antimicrobial assays demonstrate that the C6-acylated, Trp-methylated, Retro-LfB6 peptide has enhanced activity relative to the native peptide against S. aureus. Results from partitioning assays will be compared with the NMR and antimicrobial data.