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 (D₁ state) in the refolding process but differs from the completely denatured state (D₂ state) in the concentrated denaturant. Also, it has been found that the D₁ state corresponds to the heat-denatured state. To elucidate the structural basis of the D₁ state, H/D exchange experiments with PCP-0SH were performed at pD 3.4 and 4 °C. The results indicated that amide protons in the C-terminal α6-helix region hardly exchanged in the D₁ state with deuterium even after 7 days, suggesting that the α6-helix (from Ser188 to Glu205) of PCP-0SH was stably formed in the D₁ state. In order to examine the role of the α6-helix in folding and stability, H/D exchange experiments with a mutant, A199P, at position 199 in the α6-helix region were performed. The α6-helix region of A199P in the D₁ 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 D₁ state formed a temporary stable denatured structure with a non-native hydrophobic cluster and the unstructured α6-helix. Both the stability and the refolding rate decreased by the substitution of Pro for Ala199. We can conclude that the native-like helix (α6-helix) of PCP-0SH is already constructed in the D₁ state and is necessary for efficient refolding into the native structure and stabilization of PCP-0SH.