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Characterization of the denatured structure of pyrrolidone carboxyl peptidase from a hyperthermophile under nondenaturing conditions: role of the C-terminal alpha-helix of the protein in folding and stability.
Biochemistry. 2007 Mar 27; 46(12):3664-72.B

Abstract

The cysteine-free pyrrolidone carboxyl peptidase (PCP-0SH) from a hyperthermophile, Pyrococcus furiosus, can be trapped in the denatured state under nondenaturing conditions, corresponding to the denatured structure that exists in equilibrium with the native state under physiological conditions. The denatured state is the initial state (D1 state) in the refolding process but differs from the completely denatured state (D2 state) in the concentrated denaturant. Also, it has been found that the D1 state corresponds to the heat-denatured state. To elucidate the structural basis of the D1 state, H/D exchange experiments with PCP-0SH were performed at pD 3.4 and 4 degrees C. The results indicated that amide protons in the C-terminal alpha6-helix region hardly exchanged in the D1 state with deuterium even after 7 days, suggesting that the alpha6-helix (from Ser188 to Glu205) of PCP-0SH was stably formed in the D1 state. In order to examine the role of the alpha6-helix in folding and stability, H/D exchange experiments with a mutant, A199P, at position 199 in the alpha6-helix region were performed. The alpha6-helix region of A199P in the D1 state was partially unprotected, while some hydrophobic residues were protected against the H/D exchange, although these hydrophobic residues were unprotected in the wild-type protein. These results suggest that the structure of A199P in the D1 state formed a temporary stable denatured structure with a non-native hydrophobic cluster and the unstructured alpha6-helix. Both the stability and the refolding rate decreased by the substitution of Pro for Ala199. We can conclude that the native-like helix (alpha6-helix) of PCP-0SH is already constructed in the D1 state and is necessary for efficient refolding into the native structure and stabilization of PCP-0SH.

Authors+Show Affiliations

School of Science and Technology, Kwansei Gakuin University, 2-1 Gakuen, Sanda, Hyogo 669-1337, Japan.No affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

17309236

Citation

Iimura, Satoshi, et al. "Characterization of the Denatured Structure of Pyrrolidone Carboxyl Peptidase From a Hyperthermophile Under Nondenaturing Conditions: Role of the C-terminal Alpha-helix of the Protein in Folding and Stability." Biochemistry, vol. 46, no. 12, 2007, pp. 3664-72.
Iimura S, Umezaki T, Takeuchi M, et al. Characterization of the denatured structure of pyrrolidone carboxyl peptidase from a hyperthermophile under nondenaturing conditions: role of the C-terminal alpha-helix of the protein in folding and stability. Biochemistry. 2007;46(12):3664-72.
Iimura, S., Umezaki, T., Takeuchi, M., Mizuguchi, M., Yagi, H., Ogasahara, K., Akutsu, H., Noda, Y., Segawa, S., & Yutani, K. (2007). Characterization of the denatured structure of pyrrolidone carboxyl peptidase from a hyperthermophile under nondenaturing conditions: role of the C-terminal alpha-helix of the protein in folding and stability. Biochemistry, 46(12), 3664-72.
Iimura S, et al. Characterization of the Denatured Structure of Pyrrolidone Carboxyl Peptidase From a Hyperthermophile Under Nondenaturing Conditions: Role of the C-terminal Alpha-helix of the Protein in Folding and Stability. Biochemistry. 2007 Mar 27;46(12):3664-72. PubMed PMID: 17309236.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Characterization of the denatured structure of pyrrolidone carboxyl peptidase from a hyperthermophile under nondenaturing conditions: role of the C-terminal alpha-helix of the protein in folding and stability. AU - Iimura,Satoshi, AU - Umezaki,Taro, AU - Takeuchi,Makoto, AU - Mizuguchi,Mineyuki, AU - Yagi,Hiromasa, AU - Ogasahara,Kyoko, AU - Akutsu,Hideo, AU - Noda,Yasuo, AU - Segawa,Shin-ichi, AU - Yutani,Katsuhide, Y1 - 2007/02/20/ PY - 2007/2/21/pubmed PY - 2007/5/10/medline PY - 2007/2/21/entrez SP - 3664 EP - 72 JF - Biochemistry JO - Biochemistry VL - 46 IS - 12 N2 - The cysteine-free pyrrolidone carboxyl peptidase (PCP-0SH) from a hyperthermophile, Pyrococcus furiosus, can be trapped in the denatured state under nondenaturing conditions, corresponding to the denatured structure that exists in equilibrium with the native state under physiological conditions. The denatured state is the initial state (D1 state) in the refolding process but differs from the completely denatured state (D2 state) in the concentrated denaturant. Also, it has been found that the D1 state corresponds to the heat-denatured state. To elucidate the structural basis of the D1 state, H/D exchange experiments with PCP-0SH were performed at pD 3.4 and 4 degrees C. The results indicated that amide protons in the C-terminal alpha6-helix region hardly exchanged in the D1 state with deuterium even after 7 days, suggesting that the alpha6-helix (from Ser188 to Glu205) of PCP-0SH was stably formed in the D1 state. In order to examine the role of the alpha6-helix in folding and stability, H/D exchange experiments with a mutant, A199P, at position 199 in the alpha6-helix region were performed. The alpha6-helix region of A199P in the D1 state was partially unprotected, while some hydrophobic residues were protected against the H/D exchange, although these hydrophobic residues were unprotected in the wild-type protein. These results suggest that the structure of A199P in the D1 state formed a temporary stable denatured structure with a non-native hydrophobic cluster and the unstructured alpha6-helix. Both the stability and the refolding rate decreased by the substitution of Pro for Ala199. We can conclude that the native-like helix (alpha6-helix) of PCP-0SH is already constructed in the D1 state and is necessary for efficient refolding into the native structure and stabilization of PCP-0SH. SN - 0006-2960 UR - https://www.unboundmedicine.com/medline/citation/17309236/Characterization_of_the_denatured_structure_of_pyrrolidone_carboxyl_peptidase_from_a_hyperthermophile_under_nondenaturing_conditions:_role_of_the_C_terminal_alpha_helix_of_the_protein_in_folding_and_stability_ L2 - https://doi.org/10.1021/bi602456y DB - PRIME DP - Unbound Medicine ER -