Finally, we further evaluated the role of His5 on pre-lasso stability. Contact analysis of His5 with the ring residues 1-8 and tail residues 19-21, using a 4 Å cutoff to define a contact, shows consistent interactions with neighboring Val6 and Pro7 residues (Figure ). However, native pre-MccJ25 shows fewer interactions with the tail residues when His5 is protonated compared to neutral. This result suggests that conditions favoring protonation and added hydrophilicity could increase the hydration of His5 and decrease its interactions with neighboring and tail residues. This behavior changes, however, in simulations with MOD1, wherein protonated His5 interacts more with the tail residues. In particular, the last residue (Gly21) goes from 2-3 interactions in the neutral version to more than 20 interactions in the protonated peptide. The more hydrophilic character of His+ also influences this result as the side chain flipping out toward water molecules exposes the amide backbone such that it can hydrogen bond with the terminal carboxylate of Gly21. This interaction is facilitated by the enlargement in the ring circumference due to the size of the auxiliary and the thioester groups on residues 1 and 8, which decreases the steric hindrance caused by Tyr20 and promotes folding of the loop/tail. Thus, His5+ interactions stabilize the pre-lasso through backbone hydrogen bonding to the terminal carboxylate and side chain hydration (Figure ), suggesting that reaction conditions such as pH can facilitate stable pre-lasso folding. Collectively, alteration of the native residues in MccJ25 with unnatural modifications and ionizable groups significantly modulates the pre-lasso folding propensity. These findings suggest that the rational design of lasso peptides can target variations in sequence, unnatural modifications, and ionizable functionalities to increase the stability of the pre-lasso fold. CONCLUSIONS Lasso peptides are compact structures with unique properties that have the potential to become scaffolds for therapeutic development. MccJ25 is a model lasso peptide due to its antimicrobial properties and unique β-hairpin within its folded structure. In nature, MccJ25, like all characterized natural lasso peptides, access their threaded structures enzymatically.