Measuring the Binding of an Antimicrobial Peptide with LPS by Fluoresence Correlation Spectroscopy

Abstract

Antimicrobial peptides are an important defense weapon of many organisms, which attack the membrane of bacteria, leading to inhibition of bacterial growth and finally, bacterial death. Despite their ancient origin, it is difficult for bacteria to develop resistance against these peptides. This makes antimicrobial peptides a promising candidate for new drugs against microbial diseases. The target of these peptides is lipopolysaccharides (LPS), which are the major component of the outer membrane of Gram–negative bacteria. Known peptide structures and computational models show an amphipathic cationic pattern BHPHB (B: basic; H: hydrophobic; P: polar residue, respectively), which is the possible binding site of antimicrobial peptides to LPS. A cyclic amphipathic cationic 19–residue peptide (V4) with one disulfide bond has been designed (Frecer et al., unpublished data), which has potential antimicrobial activity. Circular dichroism measurements showed that V4 has a β–sheet structure. The interaction of a fluorophore labeled–V4 with LPS has been investigated by fluorescence correlation spectroscopy (FCS). The study demonstrated that V4 can specifically bind LPS, in contrast to zwitterionic phosphatidylcholine (PC) of eukaryotic cells. FCS makes it possible to study the binding between peptide and LPS at low concentration in vitro. The dissociation constant of the peptide and LPS was obtained using this technique. Key words: antimicrobial peptides, LPS, peptide-lipid interaction, fluorescence correlation spectroscopy