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.