Structural characterization of a trapped folding intermediate of pyrrolidone carboxyl peptidase from a hyperthermophile.

The refolding of cysteine-free pyrrolidone carboxyl peptidase (PCP-0SH) from a hyperthermophile is unusually slow. PCP-0SH is trapped in the denatured (D1) state at 4 °C and pH 2.3, which is different from the highly denatured state in the presence of concentrated denaturant. In order to elucidate the mechanism of the unusually slow folding, we investigated the structure of the D1 state using NMR techniques with amino acid selectively labeled PCP-0SH. The HSQC spectrum of the D1 state showed that most of the resonances arising from the 114-208 residues are broadened, indicating that conformations of the 114-208 residues are in intermediate exchange on the microsecond to millisecond time scale. Paramagnetic relaxation enhancement data indicated the lack of long-range interactions between the 1-113 and the 114-208 segments in the D1 state. Furthermore, proline scanning mutagenesis showed that the 114-208 segment in the D1 state forms a loosely packed hydrophobic core composed of α4- and α6-helices. From these findings, we conclude that the 114-208 segment of PCP-0SH folds into a stable compact structure with non-native helix-helix association in the D1 state. Therefore, in the folding process from the D1 state to the native state, the α4- and α6-helices become separated and the central β-sheet is folded between these helices. That is, the non-native interaction between the α4- and α6-helices may be responsible for the unusually slow folding of PCP-0SH.