Characterization of the denatured structure of pyrrolidone carboxyl peptidase from a hyperthermophile under nondenaturing conditions:: Role of the C-terminal α-helix of the protein in folding and stability

The cysteine-free pyrrolidone carboxyl peptidase (PCP-0SH) from a hyperthermophile, Pyrococcus furiosus, can be trapped in the denatured state under nondenaturing conditions, corresponding to the denatured structure that exists in equilibrium with the native state under physiological conditions. The denatured state is the initial state (D-1 state) in the refolding process but differs from the completely denatured state (D-2 state) in the concentrated denaturant. Also, it has been found that the D-1 state corresponds to the heat-denatured state. To elucidate the structural basis of the D-1 state, H/D exchange experiments with PCP-0SH were performed at pD 3.4 and 4 degrees C. The results indicated that amide protons in the C-terminal alpha 6-helix region hardly exchanged in the D-1 state with deuterium even after 7 days, suggesting that the alpha 6-helix (from Ser188 to Glu205) of PCP-0SH was stably formed in the D-1 state. In order to examine the role of the alpha 6-helix in folding and stability, H/D exchange experiments with a mutant, A199P, at position 199 in the alpha 6-helix region were performed. The alpha 6-helix region of A199P in the D-1 state was partially unprotected, while some hydrophobic residues were protected against the H/D exchange, although these hydrophobic residues were unprotected in the wild-type protein. These results suggest that the structure of A199P in the D-1 state formed a temporary stable denatured structure with a non-native hydrophobic cluster and the unstructured alpha 6-helix. Both the stability and the refolding rate decreased by the substitution of Pro for Ala199. We can conclude that the native-like helix (alpha 6-helix) of PCP-0SH is already constructed in the D-1 state and is necessary for efficient refolding into the native structure and stabilization of PCP-0SH.