The dominant clusters involve π-π stacking of the substituents, suggesting such interactions promote pre-lasso stabilization. Contrary to the concern that larger substituents might introduce steric clashes and disfavor pre-ring assemblies, the π-π stacking of the MOD1 substituents seems to optimally match hydrophobic ring faces and solvated polar groups to reduce the likelihood of spontaneous pre-ring dissociation. This hypothesis is supported by the longer duration needed for the pre-lasso peptide to lose the β-hairpin and transition to unfolded structures, as indicated by the secondary structure pattern (Figure ). In the pre- lasso ensemble, additional clusters display sulfur interactions (Figure ). Thus, the pre-lasso folding propensity can be significantly promoted by π-π stacking of the MOD1 moieties that is further augmented by sulfur interactions in the pre-lasso ensemble.