Flexible membrane active antimicrobial tripeptides with stability towards chymotryptic degradation

Abstract

In recent years antimicrobial peptides have gained a lot of attention due to their potential as a new generation of antibiotics combating the growing problem of antibiotic resistance. It is believed that the amphipathic structure of cationic peptides is a key feature for antimicrobial activity, and that this enables them to interact with the bacterial cell membrane. The conformational space of a range of cationic tripeptides have in this project been studied in solvent using density functional theory and molecular dynamics simulations. The results indicate that the cationic tripeptides are able to change between different, but equally stable, conformations that are both amphipathic and non-amphipathic, a property referred to as face flipping. Based on this, face flipping is proposed to be a key feature for the membrane interaction mechanism. The tripeptides mode of interaction was therefore studied with cellular model systems in more detail using MD simulations. The results show that the peptides first interact with the negatively charged head groups of the membrane with their cationic charges and then flip the hydrophobic groups into the membrane bilayer. The results thus provide strong support to the face flipping hypothesis.

A problem with antimicrobial peptides is that oral administration is difficult due to the degradation by digestive enzymes. The stability towards chymotryptic degradation has therefore been investigated by probing the S1, S1' and S2' binding pockets with unnatural amino acid side chains. The effect of different side chain substitutions were examined by combining isothermal titration calorimetry, crystallization experiments and extensive molecular modelling. Through these studies it was possible to investigate the preferential binding of several relevant unnatural amino acid side chains to the subsites of chymotrypsin. Important structural and mechanistic features were revealed in a fashion not feasible through the use of native peptide substrates. It was also found that proteolytic stability can be controlled not only by probing the S1 pocket, but also the notably less studied S1' pocket.