Membrane Interactions of an Acylated and Non-Acylated Lactoferricin Peptide by Solid-State NMR and Fluorescence Spectroscopy and Molecular Dynamics Simulations

Abstract

LfB6 (RRWQWR-NH2) is a tryptophan- and arginine-rich cationic antimicrobial peptide, derived from bovine lactoferrin, with broad spectrum activity that can be enhanced by N-terminal acylation (CH3(CH2)4CO-RRWQWR-NH2; C6-LfB6). The arginines promote selective interaction with negative charges present in bacterial cell membranes, while the tryptophans prefer the membrane-water interface. We have combined solid-state 2H and 31P NMR and fluorescence experiments with all-atom and coarse-grained molecular dynamics simulations to investigate the interactions of LfB6 and C6-LfB6 peptides in bilayers composed of two distinct compositions: 3:1 POPE:POPG (anionic) and POPC (zwitterionic). Previously we reported that the arginines of C6-LfB6 are first to associate with POPE:POPG; whereas in POPC the C6 tail associates first. 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 for zwitterionic membranes (Romo, et al. 2011. Biochim. Biophys. Acta. 1808(8):2019-30). We now compare these results with those for non-acylated LfB6 peptide. Solid-state 2H NMR spectra of mechanically aligned samples reveal that the order of POPC and of POPG is reduced in the presence of 1 mol% LfB6. By contrast, POPE shows an increase in order and some line broadening. 31P NMR spectra indicate little effect on the lipid head group for either peptide. The Trp fluorescence emission maxima are blue-shifted for LfB6 (340 nm) and C6-LfB6 (336 nm) in POPE:POPG, compared to POPC (353 nm LfB6 and 348 nm C6-LfB6), suggesting that the Trp residues are less water-exposed in the anionic lipid membranes and when the C6 acyl chain is present.