Development of protein microcrystallography applied by an effective post-labeling method, using methyltransferases and synthetic AdoMet-derivatives

2021 Fiscal Year Final Research Report

• Project/Area Number: 16K01942
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Multi-year Fund
• Section: 一般
• Research Field: Chemical biology
• Research Institution: Institute of Physical and Chemical Research
• Principal Investigator: Bessho Yoshitaka 国立研究開発法人理化学研究所, 放射光科学研究センター, 客員研究員 (70242815)
• Project Period (FY): 2016-10-21 – 2022-03-31
• Keywords: 生物・生体工学 / バイオテクノロジー / 結晶構造 / タンパク質 / メチル基転移酵素 / tRNA / 人工補酵素 / S-アデノシルメチオニン

Outline of Final Research Achievements

We utilized a chemical biology approach to develop a site-specific DNA/RNA labeling method, in which nucleic acids can be covalently modified at nucleotide precision using engineered methyltransferases and synthetic analogs of their cofactor, AdoMet. By conjugating fluorescent probes such as Cye3, Cy5, and rhodamine into the extended group of the synthetic AdoMet, we successfully developed the core technologies to incorporate fluorescent labels into microcrystals of complexes of tRNA/DNA and their related protein enzymes. The fluorescence and laser system were installed at the microfocus beamline BL32XU of the SPring-8 synchrotron, and the fluorescence was actually detected from the crystal of the complex on the BL32XU gonio stage. The X-ray phase recovery for structural analysis was successfully achieved.

Free Research Field

構造生物学

Academic Significance and Societal Importance of the Research Achievements

最適化された非天然官能基を有するAdoMet類似体が多くのメチル基転移酵素に適用できるならば、汎用的な生体分子工学に応用できる。