Quantitative studies of antimicrobial peptide-lipid membrane interactions

The increasing occurrence of multi-drug-resistant bacteria poses a serious threat to modern society. Therefore, novel types of anti-infective therapeutics are highly warranted. Antimicrobial peptides are a class of naturally occurring host-defense molecules that potentially might be developed into such novel therapeutics. However, limited understanding of the mechanisms underlying microbicidal activity of antimicrobial peptides has slowed down this development. A central step toward understanding the microbicidal mechanisms of action of antimicrobial peptides is to understand the mechanisms by which antimicrobial peptides interact with phospholipid membranes. Motivated by that fact, the scope of this thesis is to study these antimicrobial peptide-lipid membrane interactions. In particular, we attempt to study these interactions with a quantitative approach. For that purpose, we consider the three archetypal α-helical antimicrobial peptides mastoparan X, melittin, and magainin 2 as model peptides. These three peptides are investigated by three different experimental techniques. The first of these experimental techniques is analytical HPLC. We use this technique to document an effect that might pose a significant problem for quantitative studies of antimicrobial peptide-lipid membrane interactions; namely that antimicrobial peptides adsorb to surfaces of glass and plastic. Specifically, we demonstrate that under standard experimental conditions, this effect is significant for mastoparan X, melittin and magainin 2. Consequently, we conclude that investigators should always take this adsorptive effect into account when designing and interpreting their experiments on antimicrobial peptides. The second experimental technique is fluorescence correlation spectroscopy (FCS). We optimize this technique so that it can be used to quantify antimicrobial peptide-induced leakage of fluorescent markers from large unilamellar lipid vesicles in solution. For that purpose, we derive the mathematical framework required to calculate leakage from the FCS data, and we identify a number of experimental pitfalls that might lead to inaccurate conclusions, or even completely wrong conclusions, when interpreting the FCS data. We show that, if all of the pitfalls are avoided, then FCS is a technique with a large potential for quantitative studies of antimicrobial peptide-induced leakage of fluorescent markers from large unilamellar lipid vesicles in solution. Particularly interesting is our finding that FCS might be used for studying peptide-induced leakage of markers of different sizes, thereby providing a novel approach for rapid sizing of transmembrane pores formed by antimicrobial peptides. We demonstrate