Covalent reinforcement of a fragile region in the dimeric enzyme thymidylate synthase stabilizes the protein against chaotrope-induced unfolding

The mechanisms of urea- and guanidinium chloride-induced protein unfolding of the dimeric enzyme thymidylate synthase were investigated by far- and near-UV circular dichroism and intrinsic tryptophan fluorescence spectroscopy. The results revealed remarkable differences in the unfolding profiles of the proteins, which were attributed to the stability of the small structural domain in the active site. The rate of protein unfolding was observed to proceed rapidly under denaturant conditions which promote dissociation and loss of structure.