| 研究課題/領域番号 |
20K15738
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| 研究機関 | 国立研究開発法人理化学研究所 |
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研究代表者 |
Oktaviani NA 国立研究開発法人理化学研究所, 環境資源科学研究センター, 基礎科学特別研究員 (70753838)
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| 研究期間 (年度) |
2020-04-01 – 2023-03-31
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| キーワード | タンパク質NMR / タンパク質の構造とダイナミクス / 生体高分子 |
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| 研究実績の概要 |
In this fiscal year, I successfully establish the expression, labeling (13C, 15N) and purification of several recombinant spider silk proteins (spidroin) major ampullate spidroin 2 (MaSp2), which are C-terminal domain, repetitive domain (Rep6), N-terminal domain, Rep6-CTD (repetitive domain connected with c-terminal domain), NTD-Rep6 (N-terminal domain connected with repetitive domain) and NTD-Rep6-CTD (N-terminal domain, repetitive domain and C-terminal domain connected together). I also have performed NMR backbone chemical shift assignment of CTD, Rep6, and Rep6-CTD at pH 7 in the absence of liquid-liquid phase separation(LLPS). Backbone chemical shift assignment had been translated into secondary structure of CTD, Rep6 and Rep6-CTD. Dynamics experiment ({1H}-15N heteronuclear NOE) have been recorded to measure the local dynamics of each protein constructs.
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| 現在までの達成度 (区分) |
現在までの達成度 (区分)
2: おおむね順調に進展している
理由
Even though for several months of fiscal year 2020, I could not do any experiments because of COVID-19 outbreak, the research still progress rather smoothly. For the first few months, I initially had problem with the expression and purification of CTD, but after that the problem can be solved and the protocol for expression and purification can be established. Once the protocol established, I started NMR experiments to assign the backbone chemical shift of CTD and Rep6. Fortunately, the CTD and Rep6 NMR 2D NMR 1H-15N HSQC spectra overlaid very well with Rep6-CTD NMR 2D NMR 1H-15N HSQC spectra, which allow me to transfer the chemical shift assignment from individual domain CTD and Rep6 to Rep6-CTD and verify the assignment using few 3D NMR experiments. Therefore, once NMR assignment can be completed, I can use those information to interpret the dynamics of CTD, Rep6 and Rep6-CTD.
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| 今後の研究の推進方策 |
In the fiscal year 2021, I plan to investigate conformation and dynamics of NTD, NTD-Rep6, NTD-Rep6-CTD in the absence of LLPS. According to a previous study (Malay, AD et al, Sci Adv, 2020), CTD and Rep6 are responsible to the LLPS formation. Therefore, I would like to use NTD and NTD-Rep6 as control experiments since those constructs does not trigger the LLPS formation. Furthermore, I would like to investigate the conformation and dynamics of Rep6-CTD in the presence of LLPS. The key residues which are responsible to the LLPS formation will be elucidated as well. To monitor the sequential step for conformational changes upon LLPS formation, I would like to perform phosphate ion titrations on CTD and Rep6-CTD.
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