The cysteine-free pyrrolidone carboxyl peptidase (PCP-0SH) from a hyperthermophile,
Pyrococcus furiosus, can be trapped in the denatured state under nondenaturing conditions, correspondingto 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 completelydenatured state (D
2 state) in the concentrated denaturant. Also, it has been found that the D
1 state correspondsto the heat-denatured state. To elucidate the structural basis of the D
1 state, H/D exchange experimentswith PCP-0SH were performed at pD 3.4 and 4
C. The results indicated that amide protons in theC-terminal
6-helix region hardly exchanged in the D
1 state with deuterium even after 7 days, suggestingthat the
6-helix (from Ser188 to Glu205) of PCP-0SH was stably formed in the D
1 state. In order toexamine the role of the
6-helix in folding and stability, H/D exchange experiments with a mutant, A199P,at position 199 in the
6-helix region were performed. The
6-helix region of A199P in the D
1 state waspartially unprotected, while some hydrophobic residues were protected against the H/D exchange, althoughthese hydrophobic residues were unprotected in the wild-type protein. These results suggest that the structureof A199P in the D
1 state formed a temporary stable denatured structure with a non-native hydrophobiccluster and the unstructured
6-helix. Both the stability and the refolding rate decreased by the substitutionof Pro for Ala199. We can conclude that the native-like helix (
6-helix) of PCP-0SH is already constructedin the D
1 state and is necessary for efficient refolding into the native structure and stabilization of PCP-0SH.