Diffusion measurement on proteins in liquid-liquid phase separation samples

2021 Fiscal Year Final Research Report

• Project/Area Number: 19K06591
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Multi-year Fund
• Section: 一般
• Review Section: Basic Section 43040:Biophysics-related
• Research Institution: National Institute of Advanced Industrial Science and Technology
• Principal Investigator: Yamamoto Johtaro 国立研究開発法人産業技術総合研究所, 生命工学領域, 研究員 (20585088)
• Project Period (FY): 2019-04-01 – 2022-03-31
• Keywords: 動態計測 / 蛍光相関分光法 / 動的光散乱 / 並進拡散 / 回転拡散

Outline of Final Research Achievements

In this study, I aimed to clarify molecular dynamics of proteins in liquid-liquid phase separation (LLPS) state by developing a system which simultaneously provides a polarization-dependent fluorescence correlation spectroscopy (Pol-FCS) measurement and a dynamic light scattering (DLS) measurement.
The measurement system was successfully developed and demonstrated, however, preparation of samples in LLPS state with a concentration of fluorescent probe proteins suitable for FCS measurement was difficult. Next, I tried to quantitatively evaluate crowding state in macromolecular crowding (MMC) samples like LLPS samples, in which macromolecules are highly condensed, using the developed measurement system. As a result, a quantitative evaluation of MMC state by comparing translational diffusion and rotational diffusion was successfully demonstrated.

Free Research Field

生物物理学

Academic Significance and Societal Importance of the Research Achievements

蛍光プローブ分子の並進拡散と回転拡散を同時測定して比較することで、高分子混雑（MMC）状態にある溶液、生細胞の核・細胞質、ゲルの混雑状態を定量評価する手法を実証した。特に細胞内でのMMCについては、細胞内の実験結果と溶液内の実験結果の齟齬の原因とも言われ、近年注目されている現象である。これまでPol-FCSのように並進拡散と回転拡散を全く同一地点かつ同時に測定可能で、かつ生きた細胞を生かしたまま、部位選択的に測定可能な手法は存在していなかった。本研究成果は、今後のMMCや液-液相分離の研究の発展に大きく寄与可能であると期待している。