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時間分解共鳴ラマン分光法による光解離後の一酸化炭素結合型ミオグロビンとヘモグロビンのタンパク質動的構造の研究
https://ir.soken.ac.jp/records/261
https://ir.soken.ac.jp/records/26145cbdbab-c4f4-4cb2-bea3-b93450ccbb19
| 名前 / ファイル | ライセンス | アクション |
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要旨・審査要旨 / Abstract, Screening Result (370.0 kB)
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本文 / Thesis (2.7 MB)
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| Item type | 学位論文 / Thesis or Dissertation(1) | |||||
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| 公開日 | 2010-02-22 | |||||
| タイトル | ||||||
| タイトル | 時間分解共鳴ラマン分光法による光解離後の一酸化炭素結合型ミオグロビンとヘモグロビンのタンパク質動的構造の研究 | |||||
| タイトル | ||||||
| タイトル | Studies on the Protein Dynamics of Photodissociated Carbonmonoxy Myoglobin and Hemoglobin by Time-Resolved Resonance Raman 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_46ec | |||||
| 資源タイプ | thesis | |||||
| 著者名 |
目, 喜直
× 目, 喜直 |
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| フリガナ |
サカン, ヨシナオ
× サカン, ヨシナオ |
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| 著者 |
SAKAN, Yoshinao
× SAKAN, Yoshinao |
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| 学位授与機関 | ||||||
| 学位授与機関名 | 総合研究大学院大学 | |||||
| 学位名 | ||||||
| 学位名 | 博士（理学） | |||||
| 学位記番号 | ||||||
| 内容記述タイプ | Other | |||||
| 内容記述 | 総研大甲第36号 | |||||
| 研究科 | ||||||
| 値 | 数物科学研究科 | |||||
| 専攻 | ||||||
| 値 | 08 機能分子科学専攻 | |||||
| 学位授与年月日 | ||||||
| 学位授与年月日 | 1992-09-28 | |||||
| 学位授与年度 | ||||||
| 値 | 1992 | |||||
| 要旨 | ||||||
| 内容記述タイプ | Other | |||||
| 内容記述 | Myoglobin(Mb) that is monomer and Hemoglobin(Hb) that is tetramer are one of the most basic hemoproteins. Hb has a Physiological function as an oxygen carrier in blood, and Mb can store oxygen in mus- cle. Both of Mb and a subunit of Hb consist of one heme (iron protopor- phtrin IX) and a single polypeptide chain.Mb and Hb bind carbonmonox- ide(CO) at the oxygen binding site. Carbonmonoxy myoglobin (MbCO) and carbonmonoxy hemoglobin (HbCO) are photodissociable upon laser illumination,reversibly. According to the x-ray data of crystalline Mb,there is no pathway for the ligand entry from the solvent to the heme pocket. However,in some of derivative Mb another structure was also detected, in which distal histidine swings out toward the solvent and a pathway for the ligand entry is open. This structure is called "open"form, and the other structure that has no pathway is called "closed" form. When the ligand enters into the heme pocket , it is expected that the protein around the heme undergoes the conformational change. On the other hand, IR and Raman spectros- copy showed that in acidic MbCO, the C-O(v<small>c-o</small>) and Fe-CO(v<small>Fe-co</small>) stretchinig vibrational modes are located at different frequencies from these of neutral MbCO. In the present Raman experiment two v<small>Fe-co</small> bands were identified at around 490 cm<sup>-1</sup> and 510 cm<sup>-1</sup> for MbCO at pH 4.5, while only one peak was observed at 510 cm<sup>-1</sup> for MbCO at neutral pH. The value pH 4.5 happens to be equal to the pK<small>a</small> value of the distal histidine and thus, half of the distal histidine is protonated at pH 4.5. Therefore, it was inferred that the protonated distal histidine repelled lysine-45 in the vicinity of the heme distal side, and the protein conforma- tion changed to the "open" form. Thus, the higher and lower frequency of v<small>Fe-co</small> correspond to the "closed" and "open" form, respectively. The v<small>Fe-</small> <small>co</small>frequency is sensitive to the Fe-C-O angle. According to the XANES of MbCO solution at neutral pH, the Fe-C-O bent angle is 150. From a simple normal-coordinate calculation for isolated three-body oscillators, the v<small>Fe-co</small> frequencies for the bent angle of 160 and 180 were estimated at 500 cm<sup>-1</sup> and 490 cm<sup>-1</sup>, respectively. It is assumed that the Fe-C-O angle of the "open" form is linear and that of "closed" form is bent. If the photodissociated ligand recombines to the "open" form and relaxes to the "closed" form gradually at neutral pH, the v<small>Fe-co</small> should appear around 490 cm<sup>-1</sup> first and shift to 505 cm<sup>-1</sup>. In this study, the nanosecond time-resolved resonance Ranman(TR<sup>3</sup>) spectra were ob- served in order to catch the transient species of MbCO on the recombina- tion reaction. TR<sup>3</sup> spectroscopy is a powerful technique to analyze the dynamical conformation in short time scale, and information obtained by it is very basic and important in studies of the relationship between physio- logical functions and protein dynamics. MbCO at acidic pH(4.5), containing equal amounts of the "closed" and the "open" forms, were measured by the TR<sup>3</sup> system. If the assump- tion above is true, the recombination rate of the "open" form should be faster than that of the "closed" form. Indeed, the temporal behaviors of the v<small>Fe-co</small> bands of the "open" and "closed" forms were different. The v<small>Fe-co</small> band of the "open" form recovered faster than the "closed" form. Howev- er, at neutral pH, there were no transient bands around 490 cm<sup>-1</sup> in all time range observed (-20ns-1ms). It suggests either that the transient "open" form is absent or that the structural is change too fast to be detect ed(ChapterII). In the acide pH,the recombination kinetics may be affected by pH effect. It is desirable to measure both of "open" and "closed" forms under identical conditions including pH and temperatures. The human abnormal hemoglobins, "Boston" and "Saskatoon" are best models for this purpose, since abnormal hemoglobins contain two types subunits in one molecule, that is normal chains and abnormal chains. In the abnormal chain the distal histidine is replaced by tyrosine. In "Boston", the α-chain is abnor- mal, and in "Saskatoon" the β-chain is abnormal. Their v<small>Fe-co</small> bands arise around 490 and 505 cm<sup>-1</sup>, corresponding to the "open" and "closed" forms, respectively (ChapterIII). Some human MbCO mutants whose distal His was replaced by various amino acids residues through site-directed mutagenesis were also examined at neutral pH in order to make clarity the role of the distal histidine on the rebinding reaction. Some of them have the v<smal>Fe-co</small> band around 490 cm<sup>-1</sup> and the others have it around 510 cm<sup>-1</sup> (ChapterIV). The results from the experiments on these Hbs and Mbs also showed that MbCO with the lower v<small>Fe-co</small> band recovered faster than that with higher band. Transient bands observed the time range between 100-1000ns were significantly broad. The transient "open" form was not detected in all time range for the species with the "closed" equilibrium structure. These results suggested that the ligand enterd to the heme pocket through a pathway which was created by a conformational changes in the distal side, but the so-called "open" form was never produced during the recombina- tion reaction. The Fe-C-O angle in a transient form seems to be slightly perturbed around the equilibrium angle and it has appreciable distribu- tions. |
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| 値 | 有 | |||||
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| 内容記述タイプ | Other | |||||
| 内容記述 | application/pdf | |||||
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| 出版タイプ | AM | |||||
| 出版タイプResource | http://purl.org/coar/version/c_ab4af688f83e57aa | |||||
