Characterization of Chain Order of an Acylated-Lactoferricin Peptide by Solid-State NMR Spectroscopy and All-Atom Molecular Dynamics Simulations

Abstract

The hexapeptide, LfB6 (RRWQWR-NH2), derived from a cationic antimicrobial 25-residue fragment of bovine lactoferrin, retains broad spectrum activity. The two tryptophans and three arginines are thought to promote membrane interaction. Attachment of a short fatty acid to the N-terminus (C6-LfB6) further enhances membrane binding (Greathouse, et al. 2008. J. Pept. Sci. 14:1103). The mechanism by which antimicrobial peptides interact with bacterial cell membranes is proposed to depend on lipid composition. Mammalian membranes are comprised primarily of neutral lipids, whereas bacterial membranes contain a significant fraction of negatively charged lipids. We have shown using all-atom molecular dynamics simulations that association of C6-LfB6 with negatively charged membranes (3:1 POPE:POPG) begins with the arginines, followed by the tryptophans, with the C6 ‘tails’ inserting last into the membrane (Grossfield, et al. 2010. Biophys J. 98:92a). By contrast, the association with a zwitterionic membrane (POPC) is led by the C6 tail, followed by the tryptophans and arginines. Solid-state 2H NMR experimental results confirmed that the lipid order parameters are not significantly changed when C6-LfB6 is bound to negatively-charged membranes, while a slight decrease in order is observed with zwitterionic membranes. We now present the order parameters for a deuterated 6-carbon acyl chain (C6-d11) attached to either LfB6 or a single glycine control. Solid-state 2H NMR results shown an increase in acyl chain order for C6-LfB6 in POPE:POPG compared to POPC, whereas the chain order for C6-Gly is the same in POPE:POPG and POPC. Additionally, the quadrupolar splittings of individual methylene deuterons are resolved at acyl chain carbons 2-4 in C6-LfB but not in C6-Gly, suggesting restricted motion for the membrane-embedded acyl-peptide. Results from all-atom molecular dynamics simulations will be compared with experimental data from solid-state NMR.