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Transient intermediates in the folding of dihydrofolate reductase as detected by far-ultraviolet circular dichroism spectroscopy
https://ir.soken.ac.jp/records/4148
https://ir.soken.ac.jp/records/4148108933b4-f891-4779-9969-28c98a80397c
| Item type | 学術雑誌論文 / Journal Article(1) | |||||
|---|---|---|---|---|---|---|
| 公開日 | 2014-01-17 | |||||
| タイトル | ||||||
| タイトル | Transient intermediates in the folding of dihydrofolate reductase as detected by far-ultraviolet circular dichroism spectroscopy | |||||
| タイトル | ||||||
| タイトル | Transient intermediates in the folding of dihydrofolate reductase as detected by far-ultraviolet circular dichroism spectroscopy | |||||
| 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 |
KUWAJIMA, Kunihiro
× KUWAJIMA, Kunihiro× GARVEY, Edward P× FINN, Bryan E× MATTHEWS, C. Robert× SUGAI, Shintaro |
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| 著者別名 |
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 | The kinetics of the reversible folding and unfolding of Escherichia coli dihydrofolate reductase have been studied by stopped-flow circular dichroism in the peptide region at pH 7.8 and 15 °C. The reactions were induced by concentration jumps of a denaturant, urea. The method can detect various intermediates transiently populated in the reactions although the equilibrium unfolding of the protein is apparently approximated by a two-state reaction. The results can be summarized as follows. (1) From transient circular dichroism spectra measured as soon as the refolding is started, a substantial amount of secondary structure is formed in the burst phase, i.e., within the dead time of stopped-flow mixing (18 ms). (2) The kinetics from this burst-phase intermediate to the native state are multiphasic, consisting of five phases designated as τ1, τ2, τ3, τ4, and τ5 in increasing order of the reaction rate. Measurements of the kinetics at various wavelengths have provided kinetic difference circular dichroism spectra for the individual phases. (3) The τ5 phase shows a kinetic difference spectrum consistent with an exciton contribution of two aromatic residues in the peptide CD region. The absence of the τ5 phase in a mutant protein, in which Trp 74 is replaced by leucine, suggests that Trp 74 is involved in the exciton pair and that the τ5 phase reflects the formation of a hydrophobic cluster around Trp 74. From the similarity of the kinetic difference spectrum to the difference between the native spectra of the mutant and wild-type proteins, it appears that Trp 47 is the partner in the exciton pair and that the structure formed in the τ5 phase persists during the later stages of folding. (4) The later stages of folding show kinetic difference spectra that can be interpreted by rearrangement of secondary structure, particularly the central β sheet of the protein. The pairwise similarities in the spectrum between the τ3 and τ4 phases, and between the τ1 and τ2 phases, also suggest the presence of two parallel folding channels for refolding. (5) The unfolding kinetics show three to four phases and are interpreted in terms of the presence of multiple native species. The total ellipticity change in kinetic unfolding reaction, however, agrees with the ellipticity difference between the native and unfolding states, indicating the absence of the burst phase in unfolding. These results on the formation of secondary structure are compared with those on the tertiary structure obtained in previous studies by absorption and fluorescence spectroscopies and provide a more detailed picture of the folding of dihydrofolate reductase. | |||||
| 書誌情報 |
Biochemistry en : Biochemistry 巻 30, 号 31, p. 7693-7703, 発行日 1991 |
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| 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 | 00062960 | |||||
| PubMed番号 | ||||||
| 識別子タイプ | PMID | |||||
| 関連識別子 | 1868049 | |||||
| DOI | ||||||
| 識別子タイプ | DOI | |||||
| 関連識別子 | http://doi.org/10.1021/bi00245a005 | |||||
| 関連名称 | 10.1021/bi00245a005 | |||||
| 権利 | ||||||
| 権利情報 | © 1991 American Chemical Society | |||||
