Single-molecule FRET measurement of structural dynamics of intrinsically disordered domain in EGFR

• Project/Area Number: 26440088
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Multi-year Fund
• Section: 一般
• Research Field: Biophysics
• Research Institution: Institute of Physical and Chemical Research
• Principal Investigator: Okamoto Kenji 国立研究開発法人理化学研究所, 佐甲細胞情報研究室, 専任研究員 (40402763)
• Project Period (FY): 2014-04-01 – 2018-03-31
• Project Status: Completed (Fiscal Year 2017)
• Budget Amount: ¥5,070,000 (Direct Cost: ¥3,900,000、Indirect Cost: ¥1,170,000); Fiscal Year 2016: ¥1,430,000 (Direct Cost: ¥1,100,000、Indirect Cost: ¥330,000); Fiscal Year 2015: ¥1,560,000 (Direct Cost: ¥1,200,000、Indirect Cost: ¥360,000); Fiscal Year 2014: ¥2,080,000 (Direct Cost: ¥1,600,000、Indirect Cost: ¥480,000)
• Keywords: １分子計測 / FRET / 天然変性蛋白質 / 分子構造変化ダイナミクス / 上皮成長因子受容体分子 / 分子構造ダイナミクス / EGFR / １分子計測 (SMD) / 天然変性タンパク質 / １分子計測(SMD)

Outline of Final Research Achievements

Structural dynamics of the intrinsically disordered C-tail domain of the epidermal growth factor receptor (EGFR-CT) was investigated by in vitro single-molecule Foerster resonance energy transfer (FRET) measurements using fragment molecules of EGFR-CT. Although imaging experiments could not directly capture very fast structural fluctuation of the intrinsically disordered protein, it detected changes of the FRET distribution dependent on solution condition (ionic strength). Then FRET distributions of diffusing molecules were acquired by burst measurements to investigate dependence on solution conditions. It was found that FRET was dependent rather on denaturant concentration than on ionic strength. The results suggest that the structure of EGFR-CT is principally determined by locally formed secondary structures while structures of intrinsically disordered proteins are generally thought to be affected by charge interactions.

Research Products

• [Journal Article] State transition analysis of spontaneous branch migration of the Holliday junction by photon-based single-molecule fluorescence resonance energy transfer. (2016)
  • Author(s): Okamoto, K. and Sako, Y.
  • Journal Title: Biophys. Chem. Volume: 209 Pages: 21-27
  • DOI: 10.1016/j.bpc.2015.11.004
  • Peer Reviewed / Open Access
• [Presentation] EGF受容体C-末端天然変性ドメインの1分子FRET計測 (2017)
  • Author(s): 岡本憲二
  • Organizer: 第55回日本生物物理学会年会
• [Presentation] Single-molecule FRET measurement for conformational dynamics and in-cell structural states of biomolecules (2017)
  • Author(s): Kenji Okamoto
  • Organizer: International Nanophotonics Symposium
  • Int'l Joint Research
• [Presentation] 単一分子計測で明らかにする細胞中の分子動態 (2017)
  • Author(s): 岡本憲二、廣島通夫、日比野佳代、佐甲靖志
  • Organizer: レーザー学会学術講演会第37回年次大会
  • Place of Presentation: 徳島大学常三島キャンパス（徳島県徳島市）
  • Invited
• [Presentation] In-cell Single-molecule FRET Measurement of Cytosolic Proteins (2017)
  • Author(s): Kenji Okamoto
  • Organizer: Protein & Peptide Conference-2017
  • Place of Presentation: ヒルトン福岡シーホーク（福岡県福岡市）
  • Int'l Joint Research / Invited
• [Presentation] Raf の生細胞内構造分布を明らかにする１分子 FRET 計測 (2015)
  • Author(s): 岡本 憲二、日比野 佳代、佐甲 靖志
  • Organizer: 第53回 日本生物物理学会年会
  • Place of Presentation: 金沢大学（石川県・金沢市）
  • Year and Date: 2015-09-13
• [Presentation] 上皮成長因子受容体(EGFR)の膜貫通および膜近傍ドメインの構造ダイナミクス解析 (2015)
  • Author(s): 前田 亮、佐藤 毅、岡本 憲二、稲葉 岳彦、佐甲 靖志
  • Organizer: 第53回 日本生物物理学会年会
  • Place of Presentation: 金沢大学（石川県・金沢市）
  • Year and Date: 2015-09-13
• [Presentation] Holliday junction DNA の自発的 branch migration 過程の FRET による解析 (2014)
  • Author(s): 岡本憲二、佐甲靖志
  • Organizer: 第52回日本生物物理学会年会
  • Place of Presentation: 札幌コンベンションセンター（北海道札幌市）
  • Year and Date: 2014-09-25 – 2014-09-27
• [Presentation] 生体分子の１分子ＦＲＥＴ計測:分子構造の分布とダイナミクス (2014)
  • Author(s): 岡本憲二、日比野佳代、佐甲靖志
  • Organizer: 第８回分子科学討論会
  • Place of Presentation: 広島大学（広島県東広島市）
  • Year and Date: 2014-09-21 – 2014-09-24