Selective Effects of Backbone Cyclization and Disulfide Bonding as Global Covalent Constraints on the Conformational Ensemble of Sunflower Trypsin Inhibitor-1.

Backbone cyclization and disulfide bonding are key global covalent constraints in many bioactive peptides, yet their individual and combined effects on peptide conformational ensembles remain incompletely understood. Here, we combine all-atom molecular dynamics (MD) simulations, geometric analysis, and interpretable machine learning to examine these effects in sunflower trypsin inhibitor-1 (SFTI-1) as a minimal model system. Comparison of native SFTI-1 with a cyclic analogue lacking the disulfide bridge, an acyclic analogue lacking backbone cyclization, and a linear analogue lacking both shows that these constraints do not merely rigidify the peptide uniformly, but instead selectively reorganize its conformational ensemble across multiple structural levels. Backbone cyclization favors overall shape isotropy and determines the dominant global twist preference and the multimodal character of the twist distribution. The disulfide bridge plays a central role in maintaining the canonical Arg2-Phe12 and Thr4-Ile10 backbone hydrogen bonds. Coupling between local and global constraints is site-specific: the cis preference of the Ile7-Pro8 peptide bond is observed when either global constraint is present. Finally, by integrating unbiased MD with the Covalent protocol for interpretable collective variable discovery for enhanced sampling, we suggest that prefolding of the linear analogue facilitates compaction but is insufficient to generate the thiol-thiol proximity and orientation required for disulfide formation.