Disulfide bridges are commonly found covalent bonds that are usually believed to maintain structural stability of proteins. Here, we investigate the influence of disulfide bridges on protein dynamics through molecular dynamics simulations on the cysteine-rich trypsin inhibitor MCoTI-II with three disulfide bridges. Correlation analysis of the reduced cyclic peptide shows that two of the three disulfide distances (Cys11-Cys23 and Cys17-Cys29) are anticorrelated within ∼1 μs of bridge formation or dissolution: when the peptide is in nativelike structures and one of the distances shortens to allow bond formation, the other tends to lengthen. Simulations over longer timescales, when the denatured state is less structured, do not show the anticorrelation. We propose that the native state contains structural elements that frustrate one another's folding, and that the two bridges are critical for snapping the frustrated native structure into place. In contrast, the Cys4-Cys21 bridge is predicted to form together with either of the other two bridges. Indeed, experimental chromatography and nuclear magnetic resonance data show that an engineered peptide with the Cys4-Cys21 bridge deleted can still fold into its near-native structure even in its noncyclic form, confirming the lesser role of the Cys4-Cys21 bridge. The results highlight the importance of disulfide bridges in a small bioactive peptide to bring together frustrated structure in addition to maintaining protein structural stability.
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