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  1. 010 学術雑誌論文
  2. 桑島, 邦博 / KUWAJIMA, Kunihiro

Effects of proline mutations on the folding of staphylococcal nuclease

https://ir.soken.ac.jp/records/4288
https://ir.soken.ac.jp/records/4288
7d1151ff-9c76-403f-945c-865aeb3cdd0c
Item type 学術雑誌論文 / Journal Article(1)
公開日 2014-03-12
タイトル
タイトル Effects of proline mutations on the folding of staphylococcal nuclease
タイトル
タイトル Effects of proline mutations on the folding of staphylococcal nuclease
言語 en
言語
言語 eng
資源タイプ
資源タイプ識別子 http://purl.org/coar/resource_type/c_6501
資源タイプ journal article
アクセス権
アクセス権 metadata only access
アクセス権URI http://purl.org/coar/access_right/c_14cb
著者 MAKI, Kosuke

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MAKI, Kosuke

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IKURA, Teikichi

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IKURA, Teikichi

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HAYANO, Toshiya

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HAYANO, Toshiya

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TAKAHASHI, Nobuhiro

× TAKAHASHI, Nobuhiro

TAKAHASHI, Nobuhiro

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KUWAJIMA, Kunihiro

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KUWAJIMA, Kunihiro

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著者別名 桑島, 邦博

× 桑島, 邦博

桑島, 邦博

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抄録
内容記述タイプ Abstract
内容記述 Effects of proline isomerizations on the equilibrium unfolding and kinetic refolding of staphylococcal nuclease were studied by circular dichroism in the peptide region (225 nm) and fluorescence spectra of a tryptophan residue. For this purpose, four single mutants (P11A, P31A, P42A, and P56A) and four multiple mutants (P11A/P47T/P117G, P11A/P31A/P47T/P117G, P11A/P31A/P42A/P47T/P117G, and P11A/P31A/P42A/P47T/P56A/P117G) were constructed. These mutants, together with the single and double mutants for Pro47 and Pro117 constructed in our previous study, cover all six proline sites of the nuclease. The P11A, P31A, and P42A mutations did not change the stability of the protein remarkably, while the P56A mutation increased protein stability to a small extent by 0.5 kcal/mol. The refolding kinetics of the protein were, however, affected remarkably by three of the mutations, namely, P11A, P31A, and P56A. Most notably, the amplitude of the slow phase of the triphasic refolding kinetics of the nuclease observed by stopped-flow circular dichroism decreased by increasing the number of the proline mutations; the slow phase disappeared completely in the proline-free mutant (P11A/P31A/P42A/P47T/P56A/P117G). The kinetic refolding reactions of the wild-type protein assessed in the presence of Escherichia coli cyclophilin A showed that the slow phase was accelerated by cyclophilin, indicating that the slow phase was rate-limited by cis−trans isomerization of the proline residues. Although the fast and middle phases of the refolding kinetics were not affected by cyclophilin, the amplitude of the middle phase decreased when the number of the proline mutations increased; the percent amplitudes for the wild-type protein and the proline-free mutants were 43 and 13%, respectively. In addition to these three phases detected with stopped-flow circular dichroism, a very fast phase of refolding was observed with stopped-flow fluorescence, which had a shorter dead time (3.6 ms) than the stopped-flow circular dichroism. The following conclusions were drawn. (1) The effects of the P11A, P31A, and P56A mutations on the refolding kinetics indicate that the isomerizations of the three proline residues are rate-limiting, suggesting that the structures around these residues (Pro11, Pro31, and Pro56) may be organized at an early stage of refolding. (2) The fast phase corresponds to the refolding of the native proline isomer, and the middle phase whose amplitude has decreased when the number of proline mutations was increased may correspond to the slow refolding of non-native proline isomers. The occurrence of the fast- and slow-refolding reactions together with the slow phase rate-limited by the proline isomerization suggests that there are parallel folding pathways for the native and non-native proline isomers. (3) The middle phase did not completely disappear in the proline-free mutant. This suggests that the slow-folding isomer is produced not only by the proline isomerizations but also by another conformational event that is not related to the prolines. (4) The very fast phase detected with the fluorescent measurements suggests that there is an intermediate at a very early stage of kinetic refolding.
書誌情報 Biochemistry
en : Biochemistry

巻 38, 号 7, p. 2213-2223, 発行日 1999
出版者
出版者 American Chemical Society
ISSN
収録物識別子タイプ ISSN
収録物識別子 0006-2960
DOI
識別子タイプ DOI
関連識別子 http://doi.org/10.1021/bi981962+
関連名称 10.1021/bi981962+
権利
権利情報 © American Chemical Society
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