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Simultaneous Observation of Chemomechanical Coupling of a Molecular Motor

  • Takayuki NishizakaEmail author
  • Yuh Hasimoto
  • Tomoko Masaike
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 778)

Abstract

F1-ATPase is the smallest rotary molecular motor ever found. Unidirectional rotation of the γ-shaft is driven by precisely coordinated sequential ATP hydrolysis reactions in three catalytic sites arranged 120° apart in the cylinder. Single-molecule observation allows us to directly watch the rotation of the shaft using micron-sized plastic beads. Additionally, an advanced version of “total internal reflection fluorescence microscope (TIRFM)” enables us to detect binding and release of energy currency through fluorescently labeled ATP. In this chapter, we describe how to set up the system for simultaneous observation of these two critical events. This specialized optical setup is applicable to a variety of research, not only molecular motors but also other single-molecule topics.

Key words

Molecular motor F1-ATPase TIRFM Single-molecule observation Cy3-ATP Simulta­neous observation Chemomechanical coupling Rotation assay 

Notes

Acknowledgments

Authors would like to thank Drs. Kazuhiro Oiwa, Kazuhiko Kinosita Jr., Kengo Adachi, and Mitsuhiro Sugawa for their advice and encouragement, Ayako Tatsuguchi for technical assistance, and Junya Okawa and Takayuki Hirakawa for helping in the initial development of the advanced TIRFM. This work was supported in part by a Grant-in-Aid for Scientific Research on Priority Areas (No. 18074008) from the Ministry of Education, Culture, Sports, Science and Technology of Japan and a grant from the New Energy and Industrial Technology Development Organization to T.N.

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Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • Takayuki Nishizaka
    • 1
    Email author
  • Yuh Hasimoto
    • 1
  • Tomoko Masaike
    • 1
  1. 1.Department of PhysicsGakushuin UniversityTokyoJapan

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