Development of novel time-resolved diffusion method and application to detection of protein conformation and intermolecular interaction
| Project/Area Number |
17H03008
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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 |
Physical chemistry
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| Research Institution | Kyoto University |
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Principal Investigator |
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| Project Period (FY) |
2017-04-01 – 2020-03-31
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| Project Status |
Completed (Fiscal Year 2019)
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| Budget Amount *help |
¥18,070,000 (Direct Cost: ¥13,900,000、Indirect Cost: ¥4,170,000)
Fiscal Year 2019: ¥5,330,000 (Direct Cost: ¥4,100,000、Indirect Cost: ¥1,230,000)
Fiscal Year 2018: ¥8,060,000 (Direct Cost: ¥6,200,000、Indirect Cost: ¥1,860,000)
Fiscal Year 2017: ¥4,680,000 (Direct Cost: ¥3,600,000、Indirect Cost: ¥1,080,000)
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| Keywords | タンパク質反応 / 分子間相互作用 / 光反応 / 拡散 / 熱力学 / 拡散係数 / タンパク質 / 反応 / 時間分解 |
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| Outline of Final Research Achievements |
A method of time-resolved rotational diffusion coefficient measurement was developed to investigate conformation change dynamics of proteins during reactions. For this purpose, luminescence from a metal complex that has a long lifetime was used. Some mutants of Lysozume were prepared and the probe molecule was bound to some sites of that protein. It was confirmed that the secondary structure does not change by the probe molecule. From the decay rate of the polarization of the luminescence, the rotational diffusion coefficient was determined as a function of the concentration of a denaturant. It is interesting to find that the rotational diffusion rate depends on the position of the probe molecule. This result indicates that the unfolding process should not be described by the two-state model, which has been reported by many researchers so far. A new stopped flow system was also developed to be used for the time-resolved rotational diffusion coefficient method.
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| Academic Significance and Societal Importance of the Research Achievements |
タンパク質の機能発現を分子レベルで解明することは、生命科学や医学や薬学においても非常に重要なテーマである。このために、X線結晶解析を主とする構造解析が国家レベルの巨大予算で行われているが、そうした静的構造だけでなく動的特性の解明が反応機構を明らかにするために不可欠である。このために、種々の分光法が開発されてきたが、そうした分光法でも検出できない暗い過程が多く存在する。この難点を克服するために、ここでは時間分解並進拡散係数法とともに時間分解回転拡散係数法を提案し、そのシステム開発を行った。この研究は、多くの学術分野で役に立つ方法というだけでなく、医療や薬開発などの社会的に重要な分野に応用できる。
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Report
(4 results)
Research Products
(62 results)
