Structural analysis of the interaction between spinophilin, a GPCR-signaling modulator, with GPCR
| Project/Area Number |
17K07301
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Multi-year Fund |
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| Section | 一般 |
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| Research Field |
Structural biochemistry
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| Research Institution | Gunma 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 |
¥4,940,000 (Direct Cost: ¥3,800,000、Indirect Cost: ¥1,140,000)
Fiscal Year 2019: ¥1,690,000 (Direct Cost: ¥1,300,000、Indirect Cost: ¥390,000)
Fiscal Year 2018: ¥1,690,000 (Direct Cost: ¥1,300,000、Indirect Cost: ¥390,000)
Fiscal Year 2017: ¥1,560,000 (Direct Cost: ¥1,200,000、Indirect Cost: ¥360,000)
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| Keywords | 凝集 / アミロイド / オスモライト / NMR / スピノフィリン / α2A-AR / 細胞内第三ループ / 複合体形成 / α2Aアドレナリンレセプター / HSQC / titration / 脳神経疾患 / 生体分子 / 蛋白質 / 生物物理 / 構造生物学 |
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| Outline of Final Research Achievements |
By pull-down and NMR analyses, we determined the segments in spinophilin (SPL) and alpha2-adrenergic receptor (ADR) molecules that are involved in mutual interaction: 74 and 19 amino acid residues for SPL and ADR, respectively. Although ADR peptide exhibited strong tendency to form amyloid fibrils, we succeeded in preventing the amyloid formation by employing tagged ADR peptide, by adding osmolyte during concentration and titration, and by paying various precautions. [15N]SPL was titrated with non-labeled ADR and 15N-1H HSQC spectra were recorded successfully. Small but significant signal changes were observed on addition of ADR, indicating that the structural changes in the main chain of SPL is small. We are now analyzing the side chain signals of SPL by using 13C-labeled SPL, because the interaction between the two molecules is expected to be mainly of ionic nature. The strategy we developed in this study would be widely applicable for proteins that tend to form amyloid fibrils.
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| Academic Significance and Societal Importance of the Research Achievements |
ガンや精神疾患に関与するタンパク質は他のタンパク質と相互作用することでその機能を発揮することが多い，そこでタンパク質どうしの相互作用を解析することは医学的・薬学的に重要であるが，相互作用する2種のタンパク質を混合した時に凝集したり，アミロイドを形成してしまい，相互作用を解析できないことがしばしば起こる．本研究で開発した方法はこのような問題を回避できるので，創薬の基礎的研究を推進すると期待される．
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Report
(4 results)
Research Products
(11 results)
