Unusually Slow Denaturation and Refolding Processes of Pyrrolidone Carboxyl Peptidase from a Hyperthermophile Are Highly Cooperative: Real-Time NMR Studies

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• 作者：Satoshi Iimura ; Hiromasa Yagi ; Kyoko Ogasahara ; Hideo Akutsu ; Yasuo Noda ; Shin-ichi Segawa ; and Katsuhide Yutani
• 刊名：Biochemistry
• 出版年：2004
• 出版时间：September 21, 2004
• 年：2004
• 卷：43
• 期：37
• 页码：11906 - 11915
• 全文大小：218K
• 年卷期：v.43,no.37(September 21, 2004)
• ISSN：1520-4995

文摘

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 elucidatethe structural basis of the unusually slow kinetics of the protein, the denaturation and refolding processesof the PCP-0SH were investigated using a real-time 2D ¹H-¹⁵N HSQC and CD experiments. At 2 Murea denaturation of the PCP-0SH in the acidic region, all of the native peaks in the 2D HSQC spectrumcompletely disappeared. The conformation of the PCP-0SH just after removal of 6 M GuHCl could beobserved as a stable intermediate (D₁ state) in 2D HSQC and CD experiments, which is similar to amolten globule structure. The D₁ state of the PCP-0SH, which is the initial state of refolding, correspondedto the state at 2 M urea and seemed to be the denatured state in equilibrium with the native state underthe physiological conditions. The refolding of PCP-0SH from the D₁ state to the native state could beobserved to be highly cooperative without any intermediates between them, even if the refolding rate wasquite slow. In the higher concentration of denaturants, PCP-0SH showed HSQC and CD spectracharacteristic of completely unfolded proteins called the D₂ state. The unusually slow refolding rate wasdiscussed as originating in the conformations in the transition state and/or the retardation of reorganizationin an ensemble of nonrandom denatured structures in the D₁ state.