Characterization of the denatured structure of pyrrolidone carboxyl peptidase from a hyperthermophile under nondenaturing conditions: Role of the C-terminal α-helix of the protein in folding and stability

Satoshi Iimura, Taro Umezaki, Makoto Takeuchi, Mineyuki Mizuguchi*, Hiromasa Yagi, Kyoko Ogasahara, Hideo Akutsu, Yasuo Noda, Shin Ichi Segawa, Katsuhide Yutani*

*この論文の責任著者

研究成果: ジャーナルへの寄稿学術論文査読

5 被引用数 (Scopus)

抄録

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 °C. The results indicated that amide protons in the C-terminal α6-helix region hardly exchanged in the D1 state with deuterium even after 7 days, suggesting that the α6-helix (from Ser188 to Glu205) of PCP-0SH was stably formed in the D1 state. In order to examine the role of the α6-helix in folding and stability, H/D exchange experiments with a mutant, A199P, at position 199 in the α6-helix region were performed. The α6-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 α6-helix. Both the stability and the refolding rate decreased by the substitution of Pro for Ala199. We can conclude that the native-like helix (α6-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.

本文言語英語
ページ(範囲)3664-3672
ページ数9
ジャーナルBiochemistry
46
12
DOI
出版ステータス出版済み - 2007/03/27

ASJC Scopus 主題領域

  • 生化学

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