最高速AFMが解き明かす生物分子モーターのナノ構造ダイナミクス

• 資料種別: 図書
• タイトル: 最高速AFMが解き明かす生物分子モーターのナノ構造ダイナミクス
• 著者・編者: 安藤, 敏夫; Ando, Toshio
• 著者標目: 安藤, 敏夫; Ando, Toshio
• 出版事項: 金沢大学自然科学研究科
• 出版年月日等: 2008-03-01
• 出版年（W3CDTF）: 2008-03-01
• 並列タイトル等: Development of a highest-speed atomic force microscope and elucidation of the nano-structural dynamics of biological Molecular motors
• タイトル（掲載誌）: 平成19(2007)年度科学研究費補助金 基盤研究(S) 研究成果報告書 = 2007 Fiscal Year Final Research Report
• 巻号年月日等（掲載誌）: 2003-2007
• 掲載巻: 2003-2007
• 掲載ページ: 32p.-
• 本文の言語コード: jpn
• 件名標目: 原子間力顕微鏡; AFM; 高速AFM; バイオイメージング; ナノ動態; 生物分子モーター; ブラウン運動; ミオシン; Motor Proteins; Dynamic; Biomolecular Processes; Myosin; Actin; High-speed AFM; Dynein
• 対象利用者: 一般
• 一般注記: 出版タイプ： AM; ダイナミクスはタンパク質の重要な属性のひとつである。タンパク質分子機械の機能は、ダイナミックな構造変化と他の分子とのダイナミックな相互作用によって生ずる。様々なタンパク質がある中で、力と運動の発生を担うモータタンパク質は、ダイナミクスそのものが機能とも言えるタンパク質であり、ダイナミクスの解明が機能メカニズムに直結する。一方、超えがたい技術的困難により、タンパク質の機能メカニズムの解明において最も不足している重要な情報もダイナミクスである。その技術的困難を打破して、高い空間分解能と有効な時間軸を併せ持つ新しい顕微鏡を開発する必要がある。本研究の目標は、第一に、我々が世界に先駆けて開発した第一世代高速AFMの性能をタンパク質のナノ機能動態を捉えられるレベルに向上させること、第二に、その性能向上により、モータタンパク質を中心にいくつかのタンパク質の機能解明に直結する映像データ得て、この新しい手法の有効性を実証することであった。この5年間にわたる研究により、上記の目標は十分に達成された。当初目指した装置性能の向上を果たすとともに、装置のオペレーションや試料側の工夫により、ミオシンVの歩行運動、GroEL-GroES間の反協同的相互作用といった生理機能動態を捉えることに成功し、それらの分子メカニズム解明に迫ることができた。また、タンパク質の機能動態ばかりでなく、タンパク質2次元結晶中の点欠陥の運動やタンパク質の非構造領域の可視化・同定といった従来の手法では扱えない問題にも適用できることを実証した。; Instrumentation: We developed various devices contained in the tapping mode atomic farce microscope (AFM) to attain a high-speed roan capability as well as low invasiveness to the sample. The small cantilevers developed collaborating with Olympus have a resonant frequency of 1.2 MHz in water and a spring constant of 02 N/m. The bandwidth of the z-scanner has reached an unprecedented bandwidth beyond 500 kHz. Active damping techniques for suppressing the scanner's mechanical vibrations, including an electric circuit which could automatically produce an inverse transfer function of a given transfer function, were developed L The bandwidth of a position sensor for detecting the cantilever deflection has reached 20 MHz The dynamic PID controller, whose gain parameters can be automatically changed depending on the cantilever oscillation amplitude, enables the use of an amplitude set point very close to the free oscillation amplitude of a cantilever This capability ensures a very small force loaded onto the sample from the oscillating cantilever tip and can avoid 'parachuting' of the tip even when the sample is scanned very fast A compensator for drift in the cantilever excitation efficiency allows this small force to be maintained for a long time. The high-speed AFM equipped with these devices can capture an image at an imaging rate of 30-60 ma/frame without damaging the fragile samples. 'Mane as mentioned below various dynamic biomolecular processes has successfully been captured on video. In addition, we developed a fast phase detector for phase contrast imaging. This device can detect the phase change in the cantilever oscillation at every oscillation cycle and at an arbitrary timing with in a cycle. This capability allows us to distinguish the energy conservative and dissipative tip-sample interactions. Therefore, it can simultaneously image heterogeneity of material properties and the topography. Bioimaging: (1) Myosin V The nucleotide-dependent association of double-headed myosin V to actin filaments was first anamyzed by high-speed MM imaging In the rigor state and in the presence of ADP only one of the two heads was bound to F-actin. From the arrow-head structure of the bound head, the bound head was identified to the trailing bead. In the presence of a medium concentration of AMP-PNP which mimics ADP-Pi, the both heads were bound to the same actin filament. Therefore, the binding of AMP-PNP changes the leading bead configuration so that its actin binding site can face an actin filament. In the presence of ATP, the wanting myosin V was captured on video; the two lever arms change the leading and trailing positions alternately (ie., hand-overhand movement). Before the trailing head detached from actin, the leading lever arm bent frontward This bending results in the trailing lever arm being pulled frontward, which accelerates the ADP dissociation from the trailing head and facilitates ATP binding to the trailing bead leading to the dissociation of the trailing head from the actin. Thus, these imaging studies elucidated the molecular mechanism for the processive movement of myosin V on actin track. (2) Dynein. Single-headed dynein C was in the presence of ATP. The stem moiety moved periodically between two distinct two positions, while no apparent movement was detected in the stalk and the main body of denein C. The processive of yeast cytoplasmic dynein (two-headed) along microtubules was successfully imaged. (3) Chaperonin switching the GroES bound and unbound states, as expected from the negative cooperativity (regarding the ATPase reaction) between the two rings of GroEL. However, a GroES-GroEL-GroES complex appeared before the switching. This complex formation had been controversial for a long time. High-speed AFM imaging quickly solved this controversial issue instantly. (4)Defect in 2D protein crystal. Moving point defects in streptavidin 2D crystal on biotin containing lipid bilayers was imaged. Its analysis elucidated the mechanism of defect-free protein 2D crystallization.; 研究課題/領域番号:15101005, 研究期間(年度):2003– 2007, 平成19(2007)年度科学研究費補助金 基盤研究(S) 研究成果報告書の一部(概要)を掲載.; 出典：「最高速AFMが解き明かす生物分子モーターのナノ構造ダイナミクス」研究成果報告書 課題番号15101005 (KAKEN：科学研究費助成事業データベース（国立情報学研究所）) 本文データは著者版報告書より作成
• DOI: 10.24517/00034716; https://doi.org/10.24517/00034716
• 著作権情報: CC BY-NC-ND
• 関連情報: https://kaken.nii.ac.jp/search/?qm=50184320; https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-15101005/; https://kaken.nii.ac.jp/report/KAKENHI-PROJECT-15101005/151010052007kenkyu_seika_hokoku_gaiyo/
• 連携機関・データベース: 国立情報学研究所 : 学術機関リポジトリデータベース（IRDB）（機関リポジトリ）; https://irdb.nii.ac.jp
• 提供元機関・データベース: 金沢大学 : 金沢大学学術情報リポジトリKURA