Extended Data Fig. 7: Transport of ncAAs in the ‘Z’ position of a Z-AisoK tripeptide.

a. X-ray crystal structure of OppA:G-SisoK reveals a cavity around the N-terminal glycine, suggesting that larger sidechains can be accommodated at the ‘Z’ position of a generalized Z-XisoK peptide. b. SDS-PAGE analysis of sfGFP-N150TAG expression in the presence of different Z-AisoK peptides with wt-MbPylRS/PylT. Full-length sfGFP expression indicates efficient Z-AisoK transport, intracellular processing into Z and AisoK and AisoK incorporation by the wt- MbPylRS/PylT. Top: sfGFP expression in wt-K12 cells. Z-AisoK tripeptides 1, 2, 3, 4, 6, 7, 8, 9, 10 and 11 lead to efficient full-length sfGFP expression comparable to wt-sfGFP expression. Bottom: sfGFP expression in ΔoppA cells. Full-length sfGFP expression in the presence of Z-AisoK peptides is completely abolished, indicating that Z-AisoK transport is dependent on OppA. c. SDS-PAGE analysis of sfGFP-N150TAG expression in evolved Z-strains (as in (b)). Top: Expression in K12-Z1. All Z-AisoK tripeptides, apart from 14 and 15, bearing negatively charged Z-residues lead to efficient AisoK incorporation, indicating that they are successfully imported and cleaved. Bottom: Expression in K12-Z2. All Z-AisoK tripeptides, including those bearing negatively charged Z-residues (14,15) lead to efficient full-length sfGFP expression and AisoK incorporation. b,c. Consistent results were obtained over three distinct replicate experiments.