Rapid freezing quench and ESR studies of biological molecules in transient states

2019 Fiscal Year Final Research Report

• Project/Area Number: 17K05758
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Multi-year Fund
• Section: 一般
• Research Field: Physical chemistry
• Research Institution: Osaka City University
• Principal Investigator: Matsuoka Hideto 大阪市立大学, 大学院理学研究科, 特任准教授 (90414002)
• Project Period (FY): 2017-04-01 – 2020-03-31
• Keywords: ESR / スピンラベル / タンパク質 / 核酸 / 構造変化

Outline of Final Research Achievements

Pulsed ESR dipolar spectroscopy (PDS) methodology was extended to cases where the high-field approximation breaks down. Equations for the dipolar coupling constant was derived. It was shown on a heme protein that a PDS method called relaxation-induced dipolar modulation enhancement is well-suited to measuring such spectra and that the experimentally obtained dipolar spectra are in full agreement with the derived equations. We introduced a spin labeling strategy based on click chemistry in solution that, in combination with enzymatic ligation, allows highly efficient labeling of complex. Conformations of the preQ1 aptamer and its dynamics were monitored by ESR and PDS in the frozen state. A rapid freezing equipment was successfully constructed, and applied to the reaction of metmyoglobin with sodium azide, which was monitored by ESR.

Free Research Field

物理化学

Academic Significance and Societal Importance of the Research Achievements

本研究では、ESRを用いた構造解析のための、新しい解析手法やスピンラベル化法の提唱、ならびに急速凍結システムの構築を行った。対象とした系は、リボスイッチやイオンチャンネルなど、その構造変化の観測が実際に様々な分野で注目されているものであり、本研究成果は、関連分野にインパクトを与えるものであった。また、本研究で報告した研究手法は、構造変化を伴う生体反応の分子レベルでの追跡に有用なものとして、今後も活用されるものと期待できる。