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プレスチン結晶化の試み〜聴覚を鋭敏にしているモーター蛋白質の構造解明を目指して〜

Research Project

Project/Area Number 18659495
Research Category

Grant-in-Aid for Exploratory Research

Allocation TypeSingle-year Grants
Research Field Otorhinolaryngology
Research InstitutionTohoku University

Principal Investigator

和田 仁  Tohoku University, 大学院・工学研究科, 教授 (30111264)

Co-Investigator(Kenkyū-buntansha) 小林 俊光  東北大学, 大学院・医学系研究科, 教授 (80133958)
熊谷 泉  東北大学, 大学院・工学研究科, 教授 (10161689)
Project Period (FY) 2006 – 2007
Project Status Completed (Fiscal Year 2007)
Budget Amount *help
¥3,300,000 (Direct Cost: ¥3,300,000)
Fiscal Year 2007: ¥1,600,000 (Direct Cost: ¥1,600,000)
Fiscal Year 2006: ¥1,700,000 (Direct Cost: ¥1,700,000)
Keywords分子モータ / 膜タンパク質 / Prestin / 2次元結晶 / 原子間力顕微鏡 / 国際研究者交流 / スイス
Research Abstract

哺乳類の聴覚は内耳外有毛細胞の伸縮運動により鋭敏なものになっている.その伸縮運動の源は細胞側壁に高密度に発現しているタンパク質モータprestinの構造変化であると考えられている.Prestinについてその同定以来様々な研究が行われてきたが,prestinの形状や構造変化メカニズムは解明されていない.そこで本研究では,prestinの2次元結晶を作製し,その結晶を用いて,原子間力顕微鏡によるprestinの原子レベルでの可視化を目指した.本研究では,まず,これまでに構築したFLAGタグ融合型prestinを安定発現する細胞からprestinを精製した.精製したサンプルをSDS-PAGEで評価し,以降の実験に十分な純度まで精製できていることを確認した.また,細胞上のprestinの構造の安定性を検討した結果,サリチル酸ナトリウムが存在するとprestinの構造が安定することが明らかになった.これはprestinの結晶を作製する際にサリチル酸ナトリウムを添加すると良いことを示唆している.さらに,FLAGタグ融合型prestinを安定発現する細胞の細胞膜を単離し,細胞膜中のFLAGタグ融合型prestinをQ dotで標識した.そして,それを原子間力顕微鏡を用いて,Q dotを示す粒子の周辺の構造物の詳細な形状を観察した.その結果,prestinを示すと考えられる4つの突起を有し直径10nm前後の構造体を観察することが出来た.

Report

(2 results)
  • 2007 Annual Research Report
  • 2006 Annual Research Report
  • Research Products

    (16 results)

All 2008 2007 2006

All Journal Article (6 results) (of which Peer Reviewed: 2 results) Presentation (9 results) Book (1 results)

  • [Journal Article] Effects of heat stress on filamentous actin and prestin of outer haircells in mice2007

    • Author(s)
      Yoko Kitsunai
    • Journal Title

      Brain Research 1177

      Pages: 47-58

    • Related Report
      2007 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Recent findings on our auditory system: it is highly sensitiveowing to the motility of sensory cells2007

    • Author(s)
      Hiroshi Wada
    • Journal Title

      International Journal of Acoustics and Vibration 12

      Pages: 75-82

    • Related Report
      2007 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Local mechanical properties of mouse outer hair cells : Atomic force microscopic study2006

    • Author(s)
      Michio Murakoshi
    • Journal Title

      Auris Nasus Larynx 33

      Pages: 149-157

    • Related Report
      2006 Annual Research Report
  • [Journal Article] Effects of heat stress on Young's modulus of outer hair cells in mice2006

    • Author(s)
      Michio Murakoshi
    • Journal Title

      Brain Research 1107

      Pages: 121-130

    • Related Report
      2006 Annual Research Report
  • [Journal Article] Imaging by atomic force microscopy of the plasma membrane of prestin-transfected Chinese hamster ovary cells2006

    • Author(s)
      Michio Murakoshi
    • Journal Title

      Journal of the Association for Research in Otolaryngology 7

      Pages: 267-278

    • Related Report
      2006 Annual Research Report
  • [Journal Article] Analysis of the dynamic behavior of the inner hair cell stereocilia by the finite element method2006

    • Author(s)
      Toshihiro Matsui
    • Journal Title

      JSME International Journal 49C

      Pages: 828-836

    • Related Report
      2006 Annual Research Report
  • [Presentation] Immune atomic force microscopy of the plasma membrane of prestin-transfected Chinese hamster ovary cells using quantum dots2008

    • Author(s)
      Michio Murakoshi
    • Organizer
      The 31st Association for Research in Otolaryngology Midwinter meeting
    • Place of Presentation
      Phoenix
    • Related Report
      2007 Annual Research Report
  • [Presentation] Contribution of the GTSRH sequence to the folding of prestin: refolding of transport-defective prestin mutants by salicylate2008

    • Author(s)
      Shun Kumano
    • Organizer
      The 31st Association for Research in Otolaryngology Midwinter meeting
    • Place of Presentation
      Phoenix
    • Related Report
      2007 Annual Research Report
  • [Presentation] 特異的なアミノ酸への変異導入によるpresttnの活性増加2007

    • Author(s)
      和田 仁
    • Organizer
      第17回日本耳科学会総会
    • Place of Presentation
      福岡
    • Related Report
      2007 Annual Research Report
  • [Presentation] タンパク質モータprestinの精製2007

    • Author(s)
      飯田 浩司
    • Organizer
      第17回日本耳科学会総会
    • Place of Presentation
      福岡
    • Related Report
      2007 Annual Research Report
  • [Presentation] Structure and function of human auditory system: its high sensitivity is achieved by cochlear amplification.2007

    • Author(s)
      Hiroshi Wada
    • Organizer
      The 3rd Tbhoku-NUS Joint Symposium on Nano-Biomedical Engineering in the East Asian-Pacific Rim Region. Nano-Biomedlcal Engineering in the East Asian-Pacific Rim Region
    • Place of Presentation
      Singapore
    • Related Report
      2007 Annual Research Report
  • [Presentation] Structural analysis of the motor protein prestin.2007

    • Author(s)
      Hiroshi Wada
    • Organizer
      Symposium on Biomechanics at Micro-and Nanoscale Levels
    • Place of Presentation
      Tokyo
    • Related Report
      2007 Annual Research Report
  • [Presentation] Misfolding and mislocalization by mutations of the motor protein prestin.2007

    • Author(s)
      Shun Kumano
    • Organizer
      Third Asian Pacific Conference on Biomechanics
    • Place of Presentation
      Tokyo
    • Related Report
      2007 Annual Research Report
  • [Presentation] Improper membrane-targeting of prestin by mutations in the GTSRH sequence2007

    • Author(s)
      Shun Kumano
    • Organizer
      6th Molecular Biology Hearing & Deafness Conference
    • Place of Presentation
      Hinxton
    • Related Report
      2007 Annual Research Report
  • [Presentation] Mutational analysis of the GTSRH sequence of prestin, the motor protein of outer hair cells.2007

    • Author(s)
      Shun Kumano
    • Organizer
      JCA2007 The Japan-China Joint Confbrence on Acoustics
    • Place of Presentation
      Sendai
    • Related Report
      2007 Annual Research Report
  • [Book] Handbook of Noise and Vibration Control2007

    • Author(s)
      Croker, M. J.
    • Publisher
      John Wiley & Sons Inc
    • Related Report
      2007 Annual Research Report

URL: 

Published: 2006-04-01   Modified: 2016-04-21  

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