Despite the presence of π-π stacking interactions in the MOD2 pre-lasso clusters, the larger size of the MOD2 thioester side chain causes its carboxylate group to be closer to the backbone oxygen of the Gly1 auxiliary (Figure ) than its R2NH2 + group. This spatial arrangement creates a small but sufficient repulsion that decreases the stability of the pre-lasso ring relative to MOD1. With greater instability, the pre-ring opens, and the β-hairpin dissociates more readily. Once opened, the interactions of Gly1 and Glu8 residues via π-π stacking (Figure ) stand out. The peptide then assumes several pre-tadpole forms, which are more numerous than those observed with the native sequence. Thus, modification of MccJ25 with auxiliary and thioester to Gly1 and Glu8 decreases the pre-lasso stability for MOD2, while MOD1 shows increased stabilization of the pre-lasso fold.