Tracking of protein folding transition path by single-molecule fluorescence measurements

2018 Fiscal Year Final Research Report

• Project/Area Number: 17K17608
• Research Category: Grant-in-Aid for Young Scientists (B)
• Allocation Type: Multi-year Fund
• Research Field: Physical chemistry; Biophysics
• Research Institution: Tohoku University
• Principal Investigator: Oikawa Hiroyuki 東北大学, 多元物質科学研究所, 助教 (40536778)
• Research Collaborator: Takahashi Satoshi
• Project Period (FY): 2017-04-01 – 2019-03-31
• Keywords: タンパク質 / フォールディング / 遷移経路 / 一分子蛍光測定

Outline of Final Research Achievements

We aimed to track transition paths on protein folding events by single-molecule fluorescence resonance energy transfer (FRET) measurements. The time resolution of our existing line-confocal microscope combined with microfluidic chip was insufficient for tracking the transition events. To improve the time resolution, we built a new system based on hybrid photo detectors (HPD). By introducing HPD to the line-confocal microscope, we could obtain the single-molecule FRET traces with the time resolution of ten microsecond and the observation time of more than ten millisecond. As the target for the tracking of the transition path by using the new system, we tried to observe the fast conformational changes of F1-ATPase induced by the ATP hydrolysis. Unfortunately, although the conformational changes of F1-ATPase could not be observed, the method of single-molecule fluorescence measurement for tracking transition paths of biopolymer conformational changes was established.

Free Research Field

生物物理

Academic Significance and Societal Importance of the Research Achievements

タンパク質などの生体高分子は機能発現時に多くの場合立体構造が変化し、構造変化時にたどる遷移経路は酵素反応や分子生物学的過程と深く関わる。遷移経路の理解は、タンパク質が関わる疾病の治療や、人工タンパク質の創製などに有益であり、社会的に重要である。近年、分子動力学計算によって、計算機では生体分子の構造転移を追跡できるようになったが、実験的手法はほとんどない。本研究で開発した一分子の蛍光共鳴エネルギー移動(FRET)効率を10マイクロ秒分解能で追跡する手法はその有力な候補である。この手法は広範な生体高分子の構造変化の遷移経路追跡に適用可能であるため、学術的にも重要である。