Abstract
In view of the emerging role of metal ions in improper protein folding (a phenomenon associated with a variety of diseases), new tools to characterize structural changes that accompany folding transitions are highly sought. Using a combination of fluorescence spectroscopy and studies involving the chromophoric chelator 4-(2-pyridylazo)resorcinol (PAR), we here show that the prototypical zinc protease carboxypeptidase A (CPA) unfolds in the presence of guanidine hydrochloride via a previously unidentified folding intermediate that resembles a molten globular state and retains the zinc ion. The spontaneous dissociation of the metal ion from CPA was observed only upon transition of the intermediate to the fully unfolded state of the protein. Furthermore, an analysis of zinc ion binding during CPA unfolding using PAR revealed the intermediate state to directly correlate with the ability of the chelator to gain access to the active site and to associate with the protein-bound metal ion. This observation is indicative of CPA's active site being PAR-inaccessible in the native state but becoming PAR-accessible in the folding intermediate. Taken together, the current study demonstrates the usefulness of PAR as a simple spectrophotometric tool to assess structural changes during the unfolding of CPA and potentially other zinc proteins. Hence, zinc accessibility probes (ZAPs) such as PAR may find utility in gaining further insight into the mechanism(s) of metalloprotein folding.