XFEL-SFXによる化学反応解明を目指した「反応追跡結晶」の構築

• Project/Area Number: 22H00347
• Research Category: Grant-in-Aid for Scientific Research (A)
• Allocation Type: Single-year Grants
• Section: 一般
• Review Section: Medium-sized Section 37:Biomolecular chemistry and related fields
• Research Institution: Institute of Science Tokyo
• Principal Investigator: 上野 隆史 東京科学大学, 生命理工学院, 教授 (70332179)
• Co-Investigator(Kenkyū-buntansha): 庄司 光男 筑波大学, 計算科学研究センター, 教授 (00593550) ; 南後 恵理子 東北大学, 多元物質科学研究所, 教授 (90376947)
• Project Period (FY): 2022-04-01 – 2026-03-31
• Project Status: Granted (Fiscal Year 2025)
• Budget Amount: ¥42,250,000 (Direct Cost: ¥32,500,000、Indirect Cost: ¥9,750,000); Fiscal Year 2025: ¥9,360,000 (Direct Cost: ¥7,200,000、Indirect Cost: ¥2,160,000); Fiscal Year 2024: ¥10,010,000 (Direct Cost: ¥7,700,000、Indirect Cost: ¥2,310,000); Fiscal Year 2023: ¥9,360,000 (Direct Cost: ¥7,200,000、Indirect Cost: ¥2,160,000); Fiscal Year 2022: ¥13,520,000 (Direct Cost: ¥10,400,000、Indirect Cost: ¥3,120,000)
• Keywords: 化学反応 / 構造解析 / タンパク質結晶 / XFEL / X線自由電子レーザー / 有機金属錯体 / カルボニル配位子 / 自由電子レーザー / 結晶構造解析 / タンパク質ケージ / フェリチン / シリアルフェムト秒結晶解析

Outline of Research at the Start

近年の結晶構造解析技術の進歩により、天然タンパク質の反応過程を原子分解能で実時間追跡することが可能となってきた。しかし、その手法を様々な化学反応へと展開するには多くの課題が残されている。本研究では人工反応の高分解能実時間構造追跡に向けた汎用性の高い分子システムの確立に向け、タンパク質ケージであるフェリチンを用いた「反応観察結晶」の構築を目指す。

Outline of Annual Research Achievements

近年の結晶構造解析技術の進歩により、天然タンパク質の反応過程を原子分解能で実時間追跡することが可能となってきた。しかし、その手法を様々な化学反応へと展開するには多くの課題が残されている。本研究では人工反応の高分解能実時間構造追跡に向けた汎用性の高い分子システムの確立に向け、タンパク質結晶を用いた「反応観察結晶」の構築を目指し、結晶中に標的分子を特異的に導入し、結晶内部に一分子反応の追跡を可能とする活性部位を構築した。エックス線自由電子レーザーシリアルフェムト秒結晶解析により、反応過程で刻々と変化する構造を原子レベルで追跡し、正確な中間構造を用いた理論計算によって真の反応の追跡を達成した。

Current Status of Research Progress

2: Research has progressed on the whole more than it was originally planned.

Reason

前年度に得られた光トリガーにより金属ー配位子結合の開裂を生じるカルボニル（CO）錯体を固定し、サブピコーナノ秒で生じる反応を追跡し、マンガンから配位
乖離の変化をナノ秒スケールで追跡した構造変化の結果を、QM/MMにより解析を行った。

Strategy for Future Research Activity

新たなタンパク質結晶による金属配位構造を実現し、すでにXFELによる追跡が完了しているHEWLと比較

Research Products

• [Int'l Joint Research] 清華大学(中国)
• [Int'l Joint Research] TATA Institute of Fundamental Research(インド)
• [Int'l Joint Research] ナポリ大学(イタリア)
• [Journal Article] Cross-Linked Crystals of Dirhodium Tetraacetate/RNase A Adduct Can Be Used as Heterogeneous Catalysts (2023)
  • Author(s): Loreto Domenico、Maity Basudev、Morita Taiki、Nakamura Hiroyuki、Merlino Antonello、Ueno Takafumi
  • Journal Title: Inorganic Chemistry Volume: 62 Issue: 19 Pages: 7515-7524
  • DOI: 10.1021/acs.inorgchem.3c00852
• [Journal Article] Elucidating Conformational Dynamics and Thermostability of Designed Aromatic Clusters by Using Protein Cages (2023)
  • Author(s): Hishikawa Yuki、Noya Hiroki、Nagatoishi Satoru、Yoshidome Takashi、Maity Basudev、Tsumoto Kouhei、Abe Satoshi、Ueno Takafumi
  • Journal Title: Chemistry - A European Journal Volume: 29 Issue: 34
  • DOI: 10.1002/chem.202300488
• [Journal Article] A Generalized Method for Metal Fixation in Horse Spleen L-Ferritin Cage (2023)
  • Author(s): Maity Basudev、Ueno Takafumi
  • Journal Title: Meth. Mol. Biol. Volume: 2671 Pages: 135-145
  • DOI: 10.1007/978-1-0716-3222-2_8
  • ISBN: 9781071632215, 9781071632222
• [Journal Article] Apo-ferritin-Caged Pt Nanoparticles for Selective Hydrogenation of Chloronitrobenzene (2023)
  • Author(s): Zhou Yinhuan、Zheng Yilin、Lu Chenlin、Maity Basudev、Chen Yakui、Ueno Takafumi、Liu Zheng、Lu Diannan
  • Journal Title: ACS Applied Nano Materials Volume: in press Issue: 7 Pages: 5835-5843
  • DOI: 10.1021/acsanm.3c00231
  • Peer Reviewed / Int'l Joint Research
• [Journal Article] Atomic-Level Insights into a Unique Semi-Clathrate Hydrate Formed in a Confined Environment of Porous Protein Crystal (2023)
  • Author(s): B. Maity, J. Tian, T. Furuta, S. Abe, T. Ueno
  • Journal Title: Cryst. Growth Des. Volume: 23 Issue: 10 Pages: 7448-7458
  • DOI: 10.1021/acs.cgd.3c00880
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Crystal structure of the in-cell Cry1Aa purified from Bacillus thuringiensis (2023)
  • Author(s): Tanaka Junko、Abe Satoshi、Hayakawa Tohru、Kojima Mariko、Yamashita Keitaro、Hirata Kunio、Ueno Takafumi
  • Journal Title: Biochemical and Biophysical Research Communications Volume: 685 Pages: 149144-149144
  • DOI: 10.1016/j.bbrc.2023.149144
  • Peer Reviewed / Open Access
• [Journal Article] Engineering of an in-cell protein crystal for fastening a metastable conformation of a target miniprotein (2023)
  • Author(s): Mariko Kojima, Satoshi Abe, Tadaomi Furuta, Duy Phuoc Tran, Kunio Hirata, Keitaro Yamashita, Yuki Hishikawa, Akio Kitao, Takafumi Ueno
  • Journal Title: Biomaterial Science Volume: 11 Issue: 4 Pages: 1350-1357
  • DOI: 10.1039/d2bm01759h
  • Peer Reviewed
• [Journal Article] Design of a Hierarchical Assembly at a Solid Liquid Interface Using an Asymmetric Protein Needle (2023)
  • Author(s): Kikuchi Kosuke、Date Koki、Ueno Takafumi
  • Journal Title: Langmuir Volume: 39 Issue: 6 Pages: 2389-2397
  • DOI: 10.1021/acs.langmuir.2c03146
  • Peer Reviewed
• [Journal Article] Cell-free protein crystallization for nanocrystal structure determination (2022)
  • Author(s): Abe Satoshi、Tanaka Junko、Kojima Mariko、Kanamaru Shuji、Hirata Kunio、Yamashita Keitaro、Kobayashi Ayako、Ueno Takafumi
  • Journal Title: Scientific Reports Volume: 12 Issue: 1 Pages: 16031-16031
  • DOI: 10.1038/s41598-022-19681-9
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Artificial metalloenzymes based on protein assembly (2022)
  • Author(s): Maity Basudev、Taher Mohd、Mazumdar Shyamalava、Ueno Takafumi
  • Journal Title: Coordination Chemistry Reviews Volume: 469 Pages: 214593-214593
  • DOI: 10.1016/j.ccr.2022.214593
  • Peer Reviewed / Int'l Joint Research
• [Journal Article] Design of a gold clustering site in an engineered apo-ferritin cage (2022)
  • Author(s): Lu Chenlin、Maity Basudev、Peng Xue、Ito Nozomi、Abe Satoshi、Sheng Xiang、Ueno Takafumi、Lu Diannan
  • Journal Title: Communications Chemistry Volume: 5 Issue: 1 Pages: 1-11
  • DOI: 10.1038/s42004-022-00651-1
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Controlled Uptake of an Iridium Complex inside Engineered apo‐Ferritin Nanocages: Study of Structure and Catalysis** (2022)
  • Author(s): Taher Mohd、Maity Basudev、Nakane Taiki、Abe Satoshi、Ueno Takafumi、Mazumdar Shyamalava
  • Journal Title: Angewandte Chemie International Edition Volume: 61 Issue: 13
  • DOI: 10.1002/anie.202116623
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Presentation] Crystal of Mn-carbonyl/RNaseA adduct as a template for tracking metal complex reaction. (2023)
  • Author(s): B. Maity and T. Ueno
  • Organizer: The 73rd JSCC conference
• [Presentation] Crystallization of ferritin directly from E. Coli cell lysate and structure determinatio (2023)
  • Author(s): B. Maity and T. Ueno
  • Organizer: The 103rd CSJ Annual Meeting
• [Presentation] 生体材料の自律的合成 -静から働の機能へ向けて- (2023)
  • Author(s): Takafumi Ueno
  • Organizer: The 103rd CSJ Annual Meeting
  • Invited
• [Presentation] 発動分子システムを基盤とする 生体分子集合体材料の創成 (2023)
  • Author(s): Takafumi Ueno
  • Organizer: The 103rd CSJ Annual Meeting
  • Invited
• [Presentation] Encapsulation of α-Synuclein in Ferritin cage (2023)
  • Author(s): Riwa Hao, Basudev Maity, Takafumi Ueno
  • Organizer: The 103rd CSJ Annual Meeting
• [Presentation] Crystallization of ferritin directly from E. Coli cell lysate and structure determination (2023)
  • Author(s): Basudev Maity, Date Koki, Satoshi Abe, Takafumi Ueno
  • Organizer: The 103rd CSJ Annual Meeting
• [Presentation] Molecular design for immobilizing foreign molecules into porous protein crystals (2023)
  • Author(s): Yao Xinchen, Yuki Kojima, Satoshi Abe, Takafumi Ueno
  • Organizer: The 103rd CSJ Annual Meeting
• [Presentation] In-cell protein crystal engineering for structure determination of intrinsically disordered protein (2023)
  • Author(s): Mariko Kojima, Satoshi Abe, Takafumi Ueno
  • Organizer: The 103nd CSJ Annual Meeting (2023)
• [Presentation] Stabilization of an in-cell protein crystal by engineering the molecular interface (2023)
  • Author(s): Misaki Nagama, Satoshi Abe, Takafumi Ueno
  • Organizer: The 103rd CSJ Annual Meeting
• [Presentation] Structure analysis of pH-responsive piston dynamics of R-body (2023)
  • Author(s): DATE Koki, Thuc Toan PHAM, Hyeonho LEE, UCHIHASHI Takayuki, KAMIKUBO Hironari, SONG Chihong, MURATA Kazuyoshi, UENO Takafumi
  • Organizer: The 103rd CSJ Annual Meeting (2023)
• [Presentation] Engineering of in-cell Protein Crystals for the Construction of Solid Materials (2023)
  • Author(s): satoshi Abe, Junko Tanaka, Yuto Nakasuji, Takafumi Ueno
  • Organizer: The 103rd CSJ Annual Meeting
• [Presentation] Accumulation of Protein Cage into in-cell Protein Crystal (2023)
  • Author(s): Toan Thuc Pham, Koki Date, Satoshi Abe, Takafumi Ueno
  • Organizer: The 103rd CSJ Annual Meeting
• [Presentation] Construction of antifreeze protein like semi-clathrate hydrate in the ferritin cage (2023)
  • Author(s): Jiaxin Tian, Basudev Maity, Satoshi Abe, Takafumi Ueno
  • Organizer: The 103rd CSJ Annual Meeting
• [Presentation] Developing protein-cellulose nanocrystal hybrid films for structural coloration (2023)
  • Author(s): Kosuke Kikuchi, Bruno Frka-Petesic, Silvia Vignolini, Takafumi Ueno
  • Organizer: 103rd CSJAnnual Meeting
• [Presentation] タンパク質結晶化を促進する細胞内鋳型結晶設計 (2022)
  • Author(s): Satoshi Abe, Yuki Kojima, Takafumi Ueno
  • Organizer: 日本結晶学会年会
• [Presentation] Nanocrystal Structure Determination Using Cell-free Protein Crystallization (2022)
  • Author(s): Takafumi Ueno
  • Organizer: AsCA2022
  • Int'l Joint Research / Invited
• [Presentation] フェリチンを用いたトリプトファン集積構造の構築 (2022)
  • Author(s): Asuka Asanuma, Yuki Hishikawa, Hiroki Noya, Takafumi Ueno
  • Organizer: 12th CSJ Chemistry Festa
• [Presentation] pH応答性伸縮タンパク質集合体の細胞内形成メカニズム (2022)
  • Author(s): Koki Date, Toan Thuc Pham, Hyeonho Lee, Kosuke Kikuchi, Takafumi Ueno
  • Organizer: 12th CSJ Chemistry Festa
• [Presentation] タンパク質集合材料の 構造・機能・動き (2022)
  • Author(s): Takafumi Ueno
  • Organizer: 第71回高分子討論会
  • Invited
• [Presentation] Comprehensive structure analysis of intrinsically disordered protein using in-cell protein crystal (2022)
  • Author(s): Mariko Kojima, Satoshi Abe, Takafumi Ueno
  • Organizer: 第60回日本生物物理学会年会
• [Presentation] Full-length structure analysis of Cry1Aa from Bt bacteria for designing in-cell protein crystal template (2022)
  • Author(s): Junko Tanaka, Satoshi Abe, Tohru Hayakawa, Takafumi Ueno
  • Organizer: The 16th Symposium on Biorelevant Chemistry
• [Presentation] Cell-free protein crystallization for rapid structure analysis (2022)
  • Author(s): Junko Tanaka, Satoshi Abe, Takafumi Ueno
  • Organizer: 71st Symposium on Macromolecules
• [Presentation] Structural analysis of pH-responsive piston protein assembly (2022)
  • Author(s): DATE Koki, Thuc Toan PHAM, UCHIHASHI Takayuki, KAMIKUBO Hironari, SONG Chihong, MURATA Kazuyoshi, UENO Takafumi
  • Organizer: The 16th Symposium on Biorelevant Chemistry
• [Presentation] Protein crystallization and structure analysis using cell-free protein synthesis (2022)
  • Author(s): Satoshi Abe, Junko Tanaka, Yuki Kojima, Takafumi Ueno
  • Organizer: 第60回日本生物物理学会年会
• [Presentation] 人工タンパク質針を内包した細胞内タンパク質結晶材料の創成 (2022)
  • Author(s): Satoshi Abe, Toan Thuc Pham, Takafumi Ueno
  • Organizer: 第16回バイオ関連化学シンポジウム
• [Presentation] Construction of histidine clusters in a ferritin cage for the oxidase-mimetic catalysis (2022)
  • Author(s): Jiaxin Tian, Basudev Maity, Abe Satoshi, Takafumi Ueno
  • Organizer: The 71st Symposium on Macromolecules
• [Presentation] Head-to-head and Tail-to-tail Dual Interactions by Head-tail Asymmetric Protein Needles for Designing Hierarchical Two-dimensional Assembly Networks Head-tail (2022)
  • Author(s): Kosuke Kikuchi, Koki Date, Takafumi Ueno
  • Organizer: 71st Symposium on Macromolecules
• [Presentation] Engineering of Protein Cage for Coordination Nanomaterials (2022)
  • Author(s): Takafumi Ueno
  • Organizer: XXX International Materials Research Congress
  • Int'l Joint Research / Invited
• [Presentation] 無細胞タンパク質結晶化を用いたナノ結晶構造解析 (2022)
  • Author(s): Satoshi Abe, Junko Tanaka, Yuki Kojima, Takafumi Ueno
  • Organizer: 第32回バイオ・高分子シンポジウム
• [Presentation] In-cell Synthesis of Cage-Based Protein Solid Material (2022)
  • Author(s): Yuto Nakasuji, Satoshi Abe, Hironari Kamikubo, Takafumi Ueno
  • Organizer: 第32回バイオ・高分子シンポジウム
• [Presentation] Reaction mechanism of pH responsive piston protein assembly (2022)
  • Author(s): Koki Date, Thuc Toan Pham, Tatsuya Niwa, Taguchi Hideki, Takafumi Ueno
  • Organizer: 71st SPSJ Annual Meeting
• [Presentation] In-situ analysis of in-cell protein crystallization (2022)
  • Author(s): Yuto Nakasuji, Satoshi Abe, Takafumi Ueno
  • Organizer: 71st SPSJ Annual Meeting
• [Presentation] Construction of Antifreeze protein like semi-clathrate hydrate in the ferritin cage (2022)
  • Author(s): Jiaxin Tian, Kenji Yasuda, Basudev Maity, Satoshi Abe, Takeshi Murata, Takafumi Ueno
  • Organizer: The 71st SPSJ annual meeting
• [Remarks] Ueno Lab
  • URL: https://www.ueno.bio.titech.ac.jp/