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Unusually slow denaturation and refolding processes of pyrrolidone carboxyl peptidase from a hyperthermophile are highly cooperative: real-time NMR studies.
Biochemistry. 2004 Sep 21; 43(37):11906-15.B

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+Show Affiliations

School of Science and Technology, Kwansei Gakuin University, Sanda, Hyogo 669-1337, Japan.No affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article

Language

eng

PubMed ID

15362877

Citation

Iimura, Satoshi, et al. "Unusually Slow Denaturation and Refolding Processes of Pyrrolidone Carboxyl Peptidase From a Hyperthermophile Are Highly Cooperative: Real-time NMR Studies." Biochemistry, vol. 43, no. 37, 2004, pp. 11906-15.
Iimura S, Yagi H, Ogasahara K, et al. Unusually slow denaturation and refolding processes of pyrrolidone carboxyl peptidase from a hyperthermophile are highly cooperative: real-time NMR studies. Biochemistry. 2004;43(37):11906-15.
Iimura, S., Yagi, H., Ogasahara, K., Akutsu, H., Noda, Y., Segawa, S., & Yutani, K. (2004). Unusually slow denaturation and refolding processes of pyrrolidone carboxyl peptidase from a hyperthermophile are highly cooperative: real-time NMR studies. Biochemistry, 43(37), 11906-15.
Iimura S, et al. Unusually Slow Denaturation and Refolding Processes of Pyrrolidone Carboxyl Peptidase From a Hyperthermophile Are Highly Cooperative: Real-time NMR Studies. Biochemistry. 2004 Sep 21;43(37):11906-15. PubMed PMID: 15362877.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Unusually slow denaturation and refolding processes of pyrrolidone carboxyl peptidase from a hyperthermophile are highly cooperative: real-time NMR studies. AU - Iimura,Satoshi, AU - Yagi,Hiromasa, AU - Ogasahara,Kyoko, AU - Akutsu,Hideo, AU - Noda,Yasuo, AU - Segawa,Shin-ichi, AU - Yutani,Katsuhide, PY - 2004/9/15/pubmed PY - 2004/11/2/medline PY - 2004/9/15/entrez SP - 11906 EP - 15 JF - Biochemistry JO - Biochemistry VL - 43 IS - 37 N2 - 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. SN - 0006-2960 UR - https://www.unboundmedicine.com/medline/citation/15362877/Unusually_slow_denaturation_and_refolding_processes_of_pyrrolidone_carboxyl_peptidase_from_a_hyperthermophile_are_highly_cooperative:_real_time_NMR_studies_ L2 - https://doi.org/10.1021/bi048762k DB - PRIME DP - Unbound Medicine ER -