| Project/Area Number |
17350081
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Chemistry related to living body
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| Research Institution | University of Tsukuba |
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Principal Investigator |
YAMAMOTO Yasuhiko University of Tsukuba, Graduate School of Pure and Aplied Sciences, Professor (00191453)
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| Co-Investigator(Kenkyū-buntansha) |
NAGATOMO Shigenori University of Tsukuba, Graduate School of Pure and Applied Sciences, Assistant Professor (80373190)
KAWANO Shin University of Tsukuba, Graduate School of Pure and Applied Sciences, Research associate (90431676)
三田 肇 筑波大学, 大学院数理物質科学研究科, 講師 (00282301)
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| Project Period (FY) |
2005 – 2007
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| Project Status |
Completed (Fiscal Year 2007)
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| Budget Amount *help |
¥15,690,000 (Direct Cost: ¥15,000,000、Indirect Cost: ¥690,000)
Fiscal Year 2007: ¥2,990,000 (Direct Cost: ¥2,300,000、Indirect Cost: ¥690,000)
Fiscal Year 2006: ¥3,800,000 (Direct Cost: ¥3,800,000)
Fiscal Year 2005: ¥8,900,000 (Direct Cost: ¥8,900,000)
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| Keywords | Paramagnetic NMR / Heme comulex / Metalloprotein / Electrochemistry / Coordination bond / Redox potential / Thermostability / Thermodynamics / シトクロムc / ヘムタンパク質 / 酸化還元電位 / タンパク質 / ヘム錯体 / 耐熱性 / 電子移動 / NMR / 熱変性 / 非共有結合性相互作用 |
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| Research Abstract |
Pseudomonas aeruginosa cytochrome C551 and a series of its mutants exhibiting various thermostabilities have been studied by paramagnetic ^1H NMR and cyclic voltammetry in order to elucidate the molecular mechanisms responsible for control of the redox potentials (E^<o'>) of the proteins. The study revealed that the E^<o'> value of the protein is regulated by two molecular mechanisms operating independently of each other. One is based on the Fe-Met coordination bond strength in the protein, which is determined by the amino acid side-chain packing in the protein, and the other on the pKa value of the heme 17-propionic acid side-chain, which is affected by the electrostatic environment. The former mechanism alters the magnitude of the E^<o'> value throughout the entire pH range and the latter regulates the pK values reflected by the pH profile of the E^<o'> value. These findings provide novel insights into functional regulation of the protein, which could be utilized for tuning the E^<o'> value of the protein by means of protein engineering.
We also succeeded in designing cytochrome c. Thermophile Hydrogenobacter thermophilus cytochrome c552 (HT) is a stable protein with denaturation temperatures ? of 109.8 and 129.7 ℃ for the oxidized and reduced forms, respectively. The removal of a single hydroxyl group from the hydrophobic core of HT, through the replacement of a Tyr by Phe, resulted in further elevation of the Tm value of the oxidized form by 〜6℃, the T_m value of the reduced one remaining essentially unaltered. As a result, the redox potential of the mutant with higher stability in the oxidized form exhibited a negative shift of 〜20 mV relative to that of wild-type HT in an enthalpic manner. These findings indicated that the redox function of a protein can be enthalpically regulated through the stability of the oxidized form by altering the contextual stereochemical packing of hydrophobic residues in the protein interior using protein engineering.
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