Challenges to NMR structural analysis of higher molecular weight proteins by direct measurement of carbon nuclei

2019 Fiscal Year Final Research Report

• Project/Area Number: 16K14683
• Research Category: Grant-in-Aid for Challenging Exploratory Research
• Allocation Type: Multi-year Fund
• Research Field: Structural biochemistry
• Research Institution: Yokohama City University
• Principal Investigator: Ikegami Takahisa 横浜市立大学, 生命医科学研究科, 教授 (20283939)
• Project Period (FY): 2016-04-01 – 2020-03-31
• Keywords: 核磁気共鳴 / NMR / 帰属 / TROSY / 13C / 酵素

Outline of Final Research Achievements

Analyses of proteins by nuclear magnetic resonance normally require assignment that determines the origins of signal peaks. However, the assignment becomes more difficult to achieve at higher pH conditions and for higher molecular weight proteins. In order to overcome this problem, the author tried shifting the pivot of sequential resonance assignment from the amide group 1H/15N to 1H/13C. The results showed that even with the most advanced NMR equipment, the 13C-FID sensitivity was still low and a concentration as high as 1 mM was required for analysis of high molecular weight proteins. However, 13C measurement is effective for samples with paramagnetic centers, such as metalloproteins, and intrinsically disordered proteins with no particular structure. Further progress is expected in the future with the development of pulse programs and hardware.

Free Research Field

核磁気共鳴

Academic Significance and Societal Importance of the Research Achievements

核磁気共鳴（NMR）は、病院で使われる MRI と同じ原理で作動する装置である。原子核と原子核の間の距離を見積もることができるため、この情報から例えば蛋白質の立体構造を決定することができる。これまでは蛋白質のアミド基 1H/15N を中心に解析が進められてきたが、当該研究ではこれを 13C に置き換えた。結果として 13C は 1H よりも感度が低く、完全な結果を得るまでには至らなかったが、シミュレーションなども通して、何が問題で将来に向けて改善していけばよいかという指標を得ることができた。