| Project/Area Number |
16K21618
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| Research Category |
Grant-in-Aid for Young Scientists (B)
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| Allocation Type | Multi-year Fund |
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| Research Field |
Applied biochemistry
Biophysics
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| Research Institution | Institute of Physical and Chemical Research |
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Principal Investigator |
Yoshinori Matsuura 国立研究開発法人理化学研究所, 放射光科学研究センター, リサーチアソシエイト (50513462)
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| Project Period (FY) |
2016-04-01 – 2019-03-31
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| Project Status |
Completed (Fiscal Year 2018)
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| Budget Amount *help |
¥4,160,000 (Direct Cost: ¥3,200,000、Indirect Cost: ¥960,000)
Fiscal Year 2018: ¥1,040,000 (Direct Cost: ¥800,000、Indirect Cost: ¥240,000)
Fiscal Year 2017: ¥1,560,000 (Direct Cost: ¥1,200,000、Indirect Cost: ¥360,000)
Fiscal Year 2016: ¥1,560,000 (Direct Cost: ¥1,200,000、Indirect Cost: ¥360,000)
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| Keywords | 蛋白質工学 / 蛋白質の熱安定性 / 静電的相互作用 / バイオテクノロジー / 超好熱菌 / DSC / 蛋白質の安定化 / 静電相互作用 / 蛋白質 |
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| Outline of Final Research Achievements |
The goal was to see if one can achieve the same stability as for a CutA1 from hyperthermophile Pyrococcus horikoshii that has the denaturation temperature (Td) near 150 oC. The hydrophobic mutant of EcCutA1 (Ec0VV) with Td of 113.2 oC was used as a template for mutations. The highest stability of multiple mutants was a mutant substituted by 9 charged residues that had Td of 142.2 oC. To evaluate the energy of ion-ion interactions of mutant proteins, we used the structural ensemble obtained by molecular dynamics (MD) simulation at 300K. The Td of ionic mutants linearly increases with the increments of the computed energy of ion-ion interactions for ionic mutant proteins even up to the temperatures near 140 oC, suggesting that ion-ion interactions cumulatively contribute to the stabilization of a protein at high temperatures.
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| Academic Significance and Societal Importance of the Research Achievements |
蛋白質は生体を構成する最も重要な成分の一つであるため、熱安定性向上を意図した蛋白質改変は、基礎研究・応用研究両方にとって有用である。本研究では、大腸菌由来CutA1蛋白質に、荷電性残基を変異導入することによって変性温度150℃の超安定蛋白質を再現しようと試みた。その際、水溶液中における荷電性残基の挙動を検証するために、分子動力学(MD:Molecular Dynamics)シミュレーションを用いた。その結果、蛋白質の熱安定性における水溶液中での荷電性残基間の静電相互作用の重要性が示唆され、150℃に迫る変性温度を持つ蛋白質を再現することが出来た。
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