Using thioamides to site-specifically interrogate the dynamics of hydrogen bond formation in β-sheet folding.

Thioamides are sterically almost identical to their oxoamide counterparts, but they are weaker hydrogen bond acceptors. Therefore, thioamide amino acids are excellent candidates for perturbing the energetics of backbone-backbone H-bonds in proteins and hence should be useful in elucidating protein folding mechanisms in a site-specific manner. Herein, we validate this approach by applying it to probe the dynamic role of interstrand H-bond formation in the folding kinetics of a well-studied β-hairpin, tryptophan zipper. Our results show that reducing the strength of the peptide's backbone-backbone H-bonds, except the one directly next to the β-turn, does not change the folding rate, suggesting that most native interstrand H-bonds in β-hairpins are formed only after the folding transition state.