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

• 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
• Project Status: Completed (Fiscal Year 2021)
• Budget Amount: ¥4,810,000 (Direct Cost: ¥3,700,000、Indirect Cost: ¥1,110,000); Fiscal Year 2019: ¥1,300,000 (Direct Cost: ¥1,000,000、Indirect Cost: ¥300,000); Fiscal Year 2018: ¥1,170,000 (Direct Cost: ¥900,000、Indirect Cost: ¥270,000); Fiscal Year 2017: ¥1,170,000 (Direct Cost: ¥900,000、Indirect Cost: ¥270,000); Fiscal Year 2016: ¥1,170,000 (Direct Cost: ¥900,000、Indirect Cost: ¥270,000)
• 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 / ｔRNA / メチル基転位酵素 / 高分子構造・物性 / ミトコンドリアｔＲＮＡ

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.

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。

Research Products

• [Int'l Joint Research] Vilnius University(リトアニア)
• [Int'l Joint Research] Vilnius University(リトアニア)
• [Int'l Joint Research] Vilnius University(リトアニア)
• [Int'l Joint Research] Vilnius University(リトアニア)
• [Int'l Joint Research] Academia Sinica(台湾)
• [Int'l Joint Research] Vilnius University(リトアニア)
• [Int'l Joint Research] Academia Sinica(台湾)
• [Journal Article] Serial crystallography captures dynamic control of sequential electron and proton transfer events in a flavoenzyme (2022)
  • Author(s): Maestre-Reyna M, Yang CH, Nango E, Huang WC, Putu EPGN, Wu WJ, Wang PH, Franz-Badur S, Saft M, Emmerich HJ, WHY, LCC, HKF, CYK, LJH, WJH, GW, CCW, PAH, SM, OS, HY, JY, YA, TR, TT, LF, TK, HKC, KS, Schapiro I, Spadaccini R, Royant A, Yamamoto J, Iwata S, Essen LO*, Bessho Y*, Tsai MD*
  • Journal Title: Nature Chemistry Volume: － Issue: 6 Pages: 677-685
  • DOI: 10.1038/s41557-022-00922-3
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Micro-liquid enclosure array and its semi-automated assembling system for x-ray free-electron laser diffractive imaging of samples in solution (2020)
  • Author(s): Takashi Kimura, Akihiro Suzuki, Ying Yang, Yoshiya Niida, Akiko Nishioka, Masashi Takei, Jinjian Wei, Hideyuki Mitomo, Yasutaka Matsuo, Kenichi Niikura, Kuniharu Ijiro, Kensuke Tono, Makina Yabashi, Tetsuya Ishikawa, Tairo Oshima, Yoshitaka Bessho Yasumasa Joti, and Yoshinori Nishino
  • Journal Title: Review of Scientific Instruments Volume: 91 Issue: 8 Pages: 083706-083706
  • DOI: 10.1063/5.0008398
  • NAID: 120007053062
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Design of a liquid cell toward three-dimensional imaging of unidirectionally-aligned particles in solution using X-ray free-electron lasers (2020)
  • Author(s): Suzuki Akihiro、Kimura Takashi、Yang Ying、Niida Yoshiya、Nishioka Akiko、Tachibana Tatsuro、Takei Masashi、Tono Kensuke、Yabashi Makina、Ishikawa Tetsuya、Oshima Tairo、Bessho Yoshitaka、Joti Yasumasa、Nishino Yoshinori
  • Journal Title: Physical Chemistry Chemical Physics Volume: 22 Issue: 5 Pages: 2622-2628
  • DOI: 10.1039/c9cp03658j
  • NAID: 120006957264
  • Peer Reviewed / Int'l Joint Research
• [Journal Article] Ribosomal protein L31 in Escherichia coli contributes to ribosome subunit association and translation, whereas short L31 cleaved by protease 7 reduces both activities (2017)
  • Author(s): Masami Ueta, Chieko Wada, Yoshitaka Bessho, Maki Maeda, Akira Wada
  • Journal Title: Genes to Cells Volume: 22 Issue: 5 Pages: 452-471
  • DOI: 10.1111/gtc.12488
  • Peer Reviewed / Acknowledgement Compliant
• [Presentation] 高度好熱菌 Thermus thermophilus HB27のミニセルの作製 (2019)
  • Author(s): 楊影、森屋利幸、別所義隆、西野吉則、大島泰郎
  • Organizer: 第92回日本生化学会大会
• [Presentation] XFEL Coherent Diffractive Bio-Imaging at SACLA/SPring-8 (2019)
  • Author(s): Yoshitaka Bessho, Toshiyuki Moriya, Ying Yang, Akihiro Suzuki, Yasumasa Joti, Yoshinori Nishino, Tairo Oshima
  • Organizer: 15th International Congress on Thermophiles
  • Int'l Joint Research
• [Presentation] ThermusDB: Renewal to a Comprehensive Knowledge Simulator for the Biological Information of Thermus thermophilus (2019)
  • Author(s): Kei Yura, Yoshitaka Bessho, Kohsuke Honda, Gota Kawai, Toshiyuki Moriya, Naoki Nemoto, Makoto Nishiyama, Gen-ichi Sampei, Tairo Oshima
  • Organizer: 15th International Congress on Thermophiles
  • Int'l Joint Research
• [Presentation] 光修復酵素ＤＮＡフォトリアーゼのＸＦＥＬ結晶構造解析 (2018)
  • Author(s): 別所義隆、山元淳平、田中里枝、南後恵理子、岩田想、Ming-Daw Tsai、Lars-Oliver Essen、Manuel Maestre-Reyna
  • Organizer: 第18回日本蛋白質科学会年会
  • Invited
• [Presentation] SACLAでの生体溶液試料XFEL回折イメージング手法の開発 (2018)
  • Author(s): 別所義隆
  • Organizer: 第5回RIBOSOME MEETING
• [Presentation] 大腸菌リボソーム蛋白 L31 と L36 およびそれらのパラログ(YkgM, YkgO)の機能解析 (2018)
  • Author(s): 上田雅美、和田千惠子、別所義隆、和田明
  • Organizer: 第5回RIBOSOME MEETING
• [Presentation] XFEL analysis of light-mediated pyrimidine dimer repair by DNA photolyase (2018)
  • Author(s): 別所義隆
  • Organizer: 第56回日本生物物理学会
• [Presentation] XFEL Diffractive Bio-Imaging at SACLA/SPring-8 (2018)
  • Author(s): 別所義隆
  • Organizer: International workshop on “50th anniversary of Thermus thermophilus discovery”
  • Int'l Joint Research
• [Presentation] Advanced X-ray bioimaging technology for structural genomics (2017)
  • Author(s): Yoshitaka Bessho
  • Organizer: 8th International Conference on Proteomics and Bioinformatics
  • Int'l Joint Research / Invited
• [Presentation] 遺伝暗号の初期進化とtRNA修飾酵素の役割 (2017)
  • Author(s): 別所義隆
  • Organizer: 2017年度生命科学系学会合同年次大会 (ConBio2017)
  • Invited
• [Presentation] 大腸菌リボソーム蛋白L31, L36パラログはzur変異によって恒常的に発現する (2017)
  • Author(s): 上田雅美、和田千惠子、別所義隆、和田明
  • Organizer: 2017年度生命科学系学会合同年次大会 (ConBio2017)
• [Presentation] タンパク質生合成系遺伝子群の成立と進化 (2016)
  • Author(s): 別所義隆
  • Organizer: 第39回日本分子生物学会年会
  • Place of Presentation: パシフィコ横浜（神奈川県横浜市）
  • Year and Date: 2016-12-02
  • Invited
• [Presentation] 大腸菌リボソームの翻訳活性は従来の測定値より40％高い：L31蛋白損傷の影響を排除する (2016)
  • Author(s): 上田雅美、和田千惠子、別所義隆、和田明
  • Organizer: 第39回日本分子生物学会年会
  • Place of Presentation: パシフィコ横浜（神奈川県横浜市）
  • Year and Date: 2016-12-02
• [Remarks] 中央研究院・生物化學研究所・Structure-based Protein Functions
  • URL: http://www.ibc.sinica.edu.tw/Visit_Bessho_C.asp