Characterization of the Denatured Structure of Pyrrolidone Carboxyl Peptidase from a Hyperthermophile under Nondenaturing Conditions: Role of the C-Terminal <img src="http://pubs.acs.org/images/gifcha
详细信息 查看全文
- 作者：Satoshi Iimura ; Taro Umezaki ; Makoto Takeuchi ; Mineyuki Mizuguchi ; Hiromasa Yagi ; Kyoko Ogasahara ; Hideo Akutsu ; Yasuo Noda ; Shin-ichi Segawa ; Katsuhide Yutani
- 刊名：Biochemistry
- 出版年：2007
- 出版时间：March 27, 2007
- 年：2007
- 卷：46
- 期：12
- 页码：3664 - 3672
- 全文大小：258K
- 年卷期：v.46,no.12(March 27, 2007)
- ISSN：1520-4995
文摘
The cysteine-free pyrrolidone carboxyl peptidase (PCP-0SH) from a hyperthermophile,Pyrococcus furiosus, can be trapped in the denatured state under nondenaturing conditions, correspondingto 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 completelydenatured state (D2 state) in the concentrated denaturant. Also, it has been found that the D1 state correspondsto the heat-denatured state. To elucidate the structural basis of the D1 state, H/D exchange experimentswith PCP-0SH were performed at pD 3.4 and 4 <IMG SRC="/images/entities/deg.gif">C. The results indicated that amide protons in theC-terminal 
6-helix region hardly exchanged in the D1 state with deuterium even after 7 days, suggestingthat the 6-helix (from Ser188 to Glu205) of PCP-0SH was stably formed in the D1 state. In order toexamine the role of the 6-helix in folding and stability, H/D exchange experiments with a mutant, A199P,at position 199 in the 6-helix region were performed. The 6-helix region of A199P in the D1 state waspartially unprotected, while some hydrophobic residues were protected against the H/D exchange, althoughthese hydrophobic residues were unprotected in the wild-type protein. These results suggest that the structureof A199P in the D1 state formed a temporary stable denatured structure with a non-native hydrophobiccluster and the unstructured 6-helix. Both the stability and the refolding rate decreased by the substitutionof Pro for Ala199. We can conclude that the native-like helix (6-helix) of PCP-0SH is already constructedin the D1 state and is necessary for efficient refolding into the native structure and stabilization of PCP-0SH.
6-helix region hardly exchanged in the D1 state with deuterium even after 7 days, suggestingthat the 6-helix (from Ser188 to Glu205) of PCP-0SH was stably formed in the D1 state. In order toexamine the role of the 6-helix in folding and stability, H/D exchange experiments with a mutant, A199P,at position 199 in the 6-helix region were performed. The 6-helix region of A199P in the D1 state waspartially unprotected, while some hydrophobic residues were protected against the H/D exchange, althoughthese hydrophobic residues were unprotected in the wild-type protein. These results suggest that the structureof A199P in the D1 state formed a temporary stable denatured structure with a non-native hydrophobiccluster and the unstructured 6-helix. Both the stability and the refolding rate decreased by the substitutionof Pro for Ala199. We can conclude that the native-like helix (6-helix) of PCP-0SH is already constructedin the D1 state and is necessary for efficient refolding into the native structure and stabilization of PCP-0SH.