(A) Schematic structure of fully modified WT nisin. This form of the antimicrobial peptide lacks the N-terminal leader, which has been proteolytically cleaved by NisP, activating the lantibiotic as the last maturation step. The 34 amino acid peptide is displayed in the conventional numbering scheme from core peptide position I1 until K34. Black circles represent canonical amino acids in the one letter code and green filled circles represent dehydroalanine (Dha) or dehydrobutyrine (Dhb). Blue filled circles depict the (methyl)lanthionine rings comprising alanine (Ala) or aminobutyric acid (Abu) with thioether bridges depicted by yellow–orange circles. The analyzed locations for ncAA incorporation, nisinI4 and nisinK12, are highlighted in red. (B) Structure of Nε-Boc-L-lysine (BocK), the ncAA utilized in this study.