Source data and molecular dynamic trajectories for the article "In Situ Captured Antibacterial Action of Membrane-Incising Peptide Lamellae"New compounds with unique mechanisms of action are needed to combat the growing issue of antimicrobial resistance. Supramolecular assemblies, which combine the complex membrane attacking mechanisms of natural host defense peptides with the improved biostability of non-natural compounds offer a promising alternative to current small molecule antibiotics. However, for such membrane-targeting compounds the direct visual insight on the toxic agents in bacteria is still lacking. To this end, we employed a design strategy focusing on an inducible assembly mechanism and utilized electron microscopy (EM) to follow the formation of supramolecular peptide structures triggered by bacterial cell surface lipopolysaccharides (LPS). Inspired by the alternating chirality backbone pattern of some effective peptide antimicrobials, we designed lysine-rich heterochiral β3-peptides, termed lamellin-2K and lamellin-3K, with optimal residual spacing for enhanced coordination on the phosphate groups of LPS. Combined molecular dynamics simulations (MD), EM and bacterial assays confirmed that the phosphate-induced conformational change of these lamellins led to the formation of thin, striped lamellar layers, where each stripe represents double arrays of H-bonded peptide molecules that are interconnected by phosphate ions. EM micrographs of Gram-negative bacteria show that the lamellae incised the cell envelope, while leakage and antibacterial activity assays prove that growth inhibition starts already at submicromolar concentrations. Detailed image analysis demonstrated that the lamellae penetrating deep into the bacterial cell have a rather uniform size distribution and, surprisingly, only a few of these supramolecules are sufficient to cause major cell wall damage making them efficient in destroying target cells. Our findings also provide a missing mechanistic link for membrane-targeting agents, connecting how the antibiotic mechanism is built up from individual molecules through on-site formation of the active supramolecules that lead to bactericidal activity.Molecular dynamics trajectories from the production runs of single beta-peptide strands in water + 150 mM NaCl + 50:1 MePO4^(2-):peptide. Altogether 8 runs were performed, each one starting from a different helical conformation (H10, H12 and H14, both positive and negative winding) of the peptide. In the first half (500 ns) of the simulation, the intra-chain hydrogen bonds responsible for the actual helix were kept together by distance restraints. In the second half (again 500 ns), these restraints were instantaneously lifted and the system was left to evolve from the same state. In all cases the helical structure unwound in a very short time (under 100 ns), and did not refold in any helix.

Source data and molecular dynamic trajectories for the article "In Situ Captured Antibacterial Action of Membrane-Incising Peptide Lamellae"New compounds with unique mechanisms of action are needed to combat the growing issue of antimicrobial resistance. Supramolecular assemblies, which combine the complex membrane attacking mechanisms of natural host defense peptides with the improved biostability of non-natural compounds offer a promising alternative to current small molecule antibiotics. However, for such membrane-targeting compounds the direct visual insight on the toxic agents in bacteria is still lacking. To this end, we employed a design strategy focusing on an inducible assembly mechanism and utilized electron microscopy (EM) to follow the formation of supramolecular peptide structures triggered by bacterial cell surface lipopolysaccharides (LPS). Inspired by the alternating chirality backbone pattern of some effective peptide antimicrobials, we designed lysine-rich heterochiral β3-peptides, termed lamellin-2K and lamellin-3K, with optimal residual spacing for enhanced coordination on the phosphate groups of LPS. Combined molecular dynamics simulations (MD), EM and bacterial assays confirmed that the phosphate-induced conformational change of these lamellins led to the formation of thin, striped lamellar layers, where each stripe represents double arrays of H-bonded peptide molecules that are interconnected by phosphate ions. EM micrographs of Gram-negative bacteria show that the lamellae incised the cell envelope, while leakage and antibacterial activity assays prove that growth inhibition starts already at submicromolar concentrations. Detailed image analysis demonstrated that the lamellae penetrating deep into the bacterial cell have a rather uniform size distribution and, surprisingly, only a few of these supramolecules are sufficient to cause major cell wall damage making them efficient in destroying target cells. Our findings also provide a missing mechanistic link for membrane-targeting agents, connecting how the antibiotic mechanism is built up from individual molecules through on-site formation of the active supramolecules that lead to bactericidal activity.Molecular dynamics trajectories from the production runs of single beta-peptide strands in water + 150 mM NaCl + 50:1 MePO4^(2-):peptide. Altogether 8 runs were performed, each one starting from a different helical conformation (H10, H12 and H14, both positive and negative winding) of the peptide. In the first half (500 ns) of the simulation, the intra-chain hydrogen bonds responsible for the actual helix were kept together by distance restraints. In the second half (again 500 ns), these restraints were instantaneously lifted and the system was left to evolve from the same state. In all cases the helical structure unwound in a very short time (under 100 ns), and did not refold in any helix.

Molecular dynamics trajectories from the production runs of single beta-peptide strands in water + 150 mM NaCl + 50:1 MePO4^(2-):peptide. Altogether 8 runs were performed, each one starting from a different helical conformation (H10, H12 and H14, both positive and negative winding) of the peptide. In the first half (500 ns) of the simulation, the intra-chain hydrogen bonds responsible for the actual helix were kept together by distance restraints. In the second half (again 500 ns), these restraints were instantaneously lifted and the system was left to evolve from the same state. In all cases the helical structure unwound in a very short time (under 100 ns), and did not refold in any helix.

Molecular dynamics trajectories from the production runs of single beta-peptide strands in water + 150 mM NaCl + 50:1 MePO4^(2-):peptide. Altogether 8 runs were performed, each one starting from a different helical conformation (H10, H12 and H14, both positive and negative winding) of the peptide. In the first half (500 ns) of the simulation, the intra-chain hydrogen bonds responsible for the actual helix were kept together by distance restraints. In the second half (again 500 ns), these restraints were instantaneously lifted and the system was left to evolve from the same state. In all cases the helical structure unwound in a very short time (under 100 ns), and did not refold in any helix.