This journal is © the Owner Societies 2019 Phys. Chem. Chem. Phys. Cite this: DOI: 10.1039/c8cp06280c Membrane disintegration by the antimicrobial peptide (P)GKY20: lipid segregation and domain formation† Rosario Oliva, a Pompea Del Vecchio, a Antonio Grimaldi, a Eugenio Notomista, b Valeria Cafaro, b Katia Pane, b Vitor Schuabb, c Roland Winter* c and Luigi Petraccone * a Antimicrobial peptides (AMPs) are membrane-active peptides with a broad spectrum of activity against different pathogenic organisms and they represent promising new drugs to overcome the emergence of resistance to antibiotics in bacteria. (P)GKY20 is an antimicrobial peptide with a low hemolytic effect on eukaryotic cells and a strong antimicrobial activity especially against Gram-negative bacteria. However, its mechanism of action is still unknown. Here, we use fluorescence spectroscopy and differential scanning calorimetry combined with atomic force microscopy to characterise the binding of (P)GKY20 with model biomembranes and its effect on the membrane’s microstructure and thermotropic properties. We found that (P)GKY20 selectively perturbs the bacterial-like membrane via a carpet-like mechanism employing peptide conformational changes, lipid segregation and domain formation as key steps in promoting membrane disruption. These results shed a first light on the action mechanism of (P)GKY20 and could represent an important contribution to the development of new peptides serving as antimicrobial agents. Introduction During the last few years, the emergence of resistance from bacteria to the conventional antibiotics has become a serious global problem. 1 The onset of resistance is mainly due to the massive and out-of-control use of these drugs in our community. In fact, microorganisms have developed a series of mechanisms that has rendered antibiotics ineffective. 2 Thus, there is a need for new anti-infective agents and among the proposed drugs, there are antimicrobial peptides (AMPs) that comprise a particular class of amino acid-based antibiotics. 3,4 They are a very heterogeneous group of antimicrobials with different lengths (usually from 12–13 to more than 70–80 residues) and structures that are involved in the defence mechanisms found in every form of life. 4,5 Since AMPs interact in a non-specific way with the lipid matrix of the bacterial membrane, they can overcome the problem of resistance to antibiotics in pathogenic microorganisms, 6,7 thereby representing an alternative to conventional antibiotics. Understanding the molecular basis of the interaction process with the membrane is critical in the development of new AMPs with an improved antimicrobial activity and low cytotoxicity. However, the mechanisms of action of antimicrobial peptides are incompletely understood and a number of competing but not necessarily mutually exclusive models have been proposed. As a matter of fact, AMPs interact selectively with prokaryotic cells and this behavior is believed to be a consequence of the difference in the chemical composition between prokaryotic and eukaryotic membranes. 8 Indeed, bacterial membranes contain a high percentage of negatively charged phospholipids, while eukaryotic membranes mainly contain zwitterionic phos- pholipids. The mechanism of the final killing step of AMPs depends strongly on a range of physico-chemical properties such as peptide concentration and type 9 as well as the secondary structure adopted in the presence of the membrane environments. 10–12 The antimicrobial peptide (P)GKY20 is a peptide modelled on the Gly 271 to Ile 290 sequence of the human thrombin. 13,14 It possesses a net positive charge of 5 at physiological pH, a low hemolytic effect on eukaryotic cells and a strong antimicrobial activity especially against Gram-negative bacteria. 13 Numerous studies have shown the biological activity of (P)GKY20 and of its five-residue-longer version GKY25; 15 however, the molecular basis of its action mechanism has not been explored, yet. a Department of Chemical Sciences, University of Naples ‘‘Federico II’’, Via Cintia 4, 80126 Napoli, Italy. E-mail: luigi.petraccone@unina.it b Department of Biology, University of Naples ‘‘Federico II’’, Via Cintia 4, 80126 Napoli, Italy c Physical Chemistry I, Technical University of Dortmund, Otto-Hahn-Str. 4a, 44227 Dortmund, Germany. E-mail: roland.winter@tu-dortmund.de † Electronic supplementary information (ESI) available. See DOI: 10.1039/c8cp06280c Received 9th October 2018, Accepted 23rd January 2019 DOI: 10.1039/c8cp06280c rsc.li/pccp PCCP PAPER Published on 24 January 2019. Downloaded by Universita Degli Studi di Napoli Federico II on 2/2/2019 9:08:01 AM. View Article Online View Journal