Application of biophysical techniques in field of petide interactions with lipid membranes antimicrobial peptides

Abstract

Protein interactions with biological membranes are one of the major regulatory mechanisms in live species. Among the membrane-active proteins there is a group of peptides destabilizing the lipid membrane called antimicrobial peptides (AMP’s). Versality and the unique mode of action of AMP‘s makes them one of the most promising candidates for the new class of modern antibiotics.

To characterize the changes in model biological membrane upon antimicrobial peptide addition various biophysical techniques were applied including spectroscopic and calorimetric measurements. Peptide amino acid sequence analysis indicates the possibility of helical secondary structuture formation. Circular dichroism (CD) spectroscopy measurements in far UV enabled observations of the changes in the peptide secondary structure upon its addition to the lipid bilayer. Initially unordered peptide due to the interactions with lipid molecules adopts highly organised structure. Lipid-peptide interactions are accompanied by the changes in lipid bilayer phase transition profiles as shown by the differential scanning calorimetry (DSC). Finally, modern biological 14N and 31P NMR approach enabled the observations of peptide induced membrane changes on the molecular level.
Autorzy: Tomasz Borowik, Per Eugen Kristiansen, Gerhard Gröbner

1. ANTIMICROBIAL PEPTIDES

1.1. GENERAL DESCRIPTION

Antimicrobial peptides belong to the innate immune system and host defence mechanism of a multitude of animals and plants. These are small molecules that are fast and lethal towards a broad spectrum of pathogens but quite inactive on eukaryotic cells. AMP’s have recently emerged as a potential class of novel agents that could either complement existing antibiotics or possibly even be used as alternatives [1]. Various experimental data suggest that, regardless of peptide origin, primary and secondary structures, antimicrobial activity is a result of specific interactions with pathogenic membranes [2] and the membrane destabilization is generally accepted as the primary lytic factor [3]. A great effort has been made to understand the basis of AMP’s membrane activity and selectivity with the goal of optimizing their cell-lytic properties. The available evidence suggests that the peptides exert their cell-lytic effect by a two-step mechanism consisting of electrostatic binding to the cell surface and membrane permeabilization. Membrane permeabilization leads to a breakdown of the transmembrane potential and causes leakage of cell contents finally resulting in cell death. The net positive charge of antimicrobial peptides causes their preferential binding to negatively charged targets on bacteria, which may account for the selectivity of antimicrobial peptides [4]. It has been reported that not only the nature of the peptide but also the characteristics of cell membrane, as well as the metabolic state of the target cell determine the mechanisms of action of antimicrobial peptides.

1.2. AMP’s MODES OF ACTION

Two general mechanisms were originally proposed to describe the process of phospholipid membrane permeation by membrane-active peptides, ‘barrel-stave’ and ‘carpet’ mechanisms (Fig.1).


The ‘barrel stave’ mechanism describes the formation of transmembrane pores by peptide clusters. Recruitment of additional peptide monomers to the membrane bound clusters leads to a progressive pore size increase. A crucial step in the barrel stave mechanism requires peptides to recognize one another in the membrane bound state. Peptide assembly can occur on the surface or within the hydrophobic core of the membrane, since hydrophobic peptides can span membranes as monomers. In contrary for a single amphiphilic α-helix it is energetically unfavourable to transverse the membrane as a monomer. Therefore such monomers must associate on the surface of the membrane before the insertion.

According to the ‘carpet model’, the peptides first bind onto the surface of the target microbial cell membrane with their hydrophobic surfaces facing the membrane and their hydrophilic surfaces facing the solvent. This process causes that membrane is subsequently covered by a ‘carpet-like’ cluster of peptides. The presence of negatively charged lipids is important for a peptide carpet to form, as they help to reduce the repulsive electrostatic forces between positively charged peptides. In the second step, after a threshold concentration has been reached, the peptides cause membrane permeation and finally even disintegration. High local concentration on the surface of the membrane depends upon the type of the target membrane and can occur either after all the surface of the membrane is covered with peptide monomers, or alternatively, antimicrobial peptides that associate on the surface of the membrane can form a local carpet.