title: Unusually slow denaturation and refolding processes of pyrrolidone carboxyl peptidase from a hyperthermophile are highly cooperative: real-time NMR studies.
abstract: The refolding rate of heat-denatured cysteine-free pyrrolidone carboxyl peptidase (PCP-0SH) from Pyrococcus furiosus has been reported to be unusually slow under some conditions. To elucidate the structural basis of the unusually slow kinetics of the protein, the denaturation and refolding processes of the PCP-0SH were investigated using a real-time 2D (1)H-(15)N HSQC and CD experiments. At 2 M urea denaturation of the PCP-0SH in the acidic region, all of the native peaks in the 2D HSQC spectrum completely disappeared. The conformation of the PCP-0SH just after removal of 6 M GuHCl could be observed as a stable intermediate (D(1) state) in 2D HSQC and CD experiments, which is similar to a molten globule structure. The D(1) state of the PCP-0SH, which is the initial state of refolding, corresponded to the state at 2 M urea and seemed to be the denatured state in equilibrium with the native state under the physiological conditions. The refolding of PCP-0SH from the D(1) state to the native state could be observed to be highly cooperative without any intermediates between them, even if the refolding rate was quite slow. In the higher concentration of denaturants, PCP-0SH showed HSQC and CD spectra characteristic of completely unfolded proteins called the D(2) state. The unusually slow refolding rate was discussed as originating in the conformations in the transition state and/or the retardation of reorganization in an ensemble of nonrandom denatured structures in the D(1) state.
authors.affiliation: School of Science and Technology, Kwansei Gakuin University, Sanda, Hyogo 669-1337, Japan.
authors.forename: Satoshi
authors.initials: S
authors.lastname: Iimura
authors.forename: Hiromasa
authors.initials: H
authors.lastname: Yagi
authors.forename: Kyoko
authors.initials: K
authors.lastname: Ogasahara
authors.forename: Hideo
authors.initials: H
authors.lastname: Akutsu
authors.forename: Yasuo
authors.initials: Y
authors.lastname: Noda
authors.forename: Shin-ichi
authors.initials: S
authors.lastname: Segawa
authors.forename: Katsuhide
authors.initials: K
authors.lastname: Yutani
mesh_terms: D019843:Archaeal Proteins / Q000737:chemistry / Q000378:metabolism*; D002942:Circular Dichroism; D019906:Nuclear Magnetic Resonance, Biomolecular; D011489:Protein Denaturation; D017510:Protein Folding; D020673:Protein Renaturation; D020027:Pyrococcus furiosus / Q000201:enzymology*; D011751:Pyroglutamyl-Peptidase I / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013816:Thermodynamics; D013997:Time Factors
chemical_list: D019843:Archaeal Proteins; D011751:Pyroglutamyl-Peptidase I