Determination Of The Mechanism Of Action Of Peptides With Antimicrobial Potential By 31P And 2H Solid-state NMR

Abstract

A wide variety of organisms produce antimicrobial peptides as part of their first line of defense. These short cationic peptides are being considered as a new generation of antibiotics and represent great hopes against multiresistant-resistant bacteria which are an important clinical problem. Despite their diversity, the main target of antimicrobial peptides is the membrane(s) of pathogens. Previous studies have shown that a non-natural peptide composed of 14 residues (10 leucines and 4 phenylalanines modified with a crown ether) is able to disrupt negatively charged lipid bilayers. This peptide, called 14-mer, is of particular interest to lyse bacterial membranes. Biophysical studies suggested that the peptide binds to the membrane surface and induces pores stabilized by the peptide inverse-cone shape. However, the 14-mer is also able to disrupt neutral bilayers, limiting its application as antibiotic. To gain specificity against negatively charged membranes, several leucines have been substituted by positively charged residues (lysine, arginine, histidine).Solid-state NMR experiments performed in model membranes were used to better characterize the mode of action of the charged peptides. More specifically, 31P NMR provided information about the phospholipid polar head group, while 2H NMR was used to measure the effect on the lipid acyl chains. Results obtained by a combination of 2H, 31P and 15N NMR spectroscopy suggest that the peptides arrange themselves preferentially near the bilayer interface perturbing the membrane by the formation of pores. Lipid bilayers oriented between glass-plates were used to verify this hypothesis, while REDOR NMR experiments will be used to determine specifically which type of helical conformation is favored by these peptides.