Extended Data Fig. 4: Design rationale and structures for mechanism-of-action control peptides.

In the absence of backbone N-methylation, as in peptide 11, the flat ring-shaped cyclic conformation favors peptide stacking and inter-subunit backbone hydrogen bonding, giving rise to tubular ensembles that can perturb transmembrane ion gradients to exert antimicrobial activities. Backbone N-methylation on each face of the macrocycle, as in peptide 30 and 33, creates a dual effect that prevents peptide self-assembly and membrane/antimicrobial activity. The modified ring structure not only lacks two amide hydrogen bonding sites but is also incapable of ring stacking and inter-subunit hydrogen bonding as a result of steric clashes by the N-methyl moieties. Therefore, peptides 30 and 33 are interesting control and mechanism of action probes because of their inability to self-assemble and exert membrane and antimicrobial activity, despite having identical amino acid sequence as peptide 11. For clarity, side chains are omitted from the molecular models shown. Likewise, switching the stereochemistry of one of the amino acid side chains yields a diastereomer of the parent peptide. Diastereomers, such as peptides 31, 32, and 35 lack the flat ring-shaped cyclic conformation that favors peptide stacking, thus diminishing the propensity for self-assembly. On the other hand, the alternating arrangement of D- and L-amino acids is present in enantiomers of the parent peptides, such as 34 and 36, producing the flat ring-shaped cyclic conformation that favors peptide stacking.