Simultaneous Observation of Chemomechanical Coupling of a Molecular Motor

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


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 



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.


  1. 1.
    Svoboda, K., Schmidt, C. F., Schnapp, B. J., and Block, S. M. (1993) Direct observation of kinesin stepping by optical trapping interferometry. Nature 365, 721–727.PubMedCrossRefGoogle Scholar
  2. 2.
    Miyata, H., Hakozaki, H., Yoshikawa, H., Suzuki, N., Kinosita, K., Jr., Nishizaka, T., and Ishiwata, S. (1994) Stepwise motion of an actin filament over a small number of heavy meromyosin molecules is revealed in an in vitro motility assay. J. Biochem. 115, 644–647.PubMedGoogle Scholar
  3. 3.
    Finer, J. T., Simmons, R. M., and Spudich, J. A. (1994) Single myosin molecule mechanics: piconewton forces and nanometre steps. Nature 368, 113–119.PubMedCrossRefGoogle Scholar
  4. 4.
    Mehta, A. D., Rock, R. S., Rief, M., Spudich, J. A., Mooseker, M. S., and Cheney, R. E. (1999) Myosin-V is a processive actin-based motor. Nature 400, 590–593.PubMedCrossRefGoogle Scholar
  5. 5.
    Noji, H., Yasuda, R., Yoshida, M., and Kinosita, K., Jr. (1997) Direct observation of the rotation of F1-ATPase. Nature 386, 299–302.PubMedCrossRefGoogle Scholar
  6. 6.
    Yasuda, R., Noji, H., Kinosita, K., Jr., and Yoshida, M. (1998) F1-ATPase is a highly efficient molecular motor that rotates with discrete 120° steps. Cell 93, 1117–1124.PubMedCrossRefGoogle Scholar
  7. 7.
    Ishijima, A., Kojima, H., Funatsu, T., Tokunaga, M., Higuchi, H., Tanaka, H., and Yanagida, T. (1998) Simultaneous observation of individual ATPase and mechanical events by a single myosin molecule during interaction with actin. Cell 92, 161–171.PubMedCrossRefGoogle Scholar
  8. 8.
    Nishizaka, T., Oiwa, K., Noji, H., Kimura, S., Muneyuki, E., Yoshida, M., and Kinosita, K., Jr. (2004) Chemomechanical coupling in F1-ATPase revealed by simultaneous observation of nucleotide kinetics and rotation. Nat. Struct. Mol. Biol. 11, 142–148.PubMedCrossRefGoogle Scholar
  9. 9.
    Adachi, K., Kinosita Jr, K., and Ando, T. (1999) Single-fluorophore imaging with an unmodified epifluorescence microscope and conventional video camera. J. Microsc. 195, 125–132.PubMedCrossRefGoogle Scholar
  10. 10.
    Sakamoto, T., Webb, M. R., Forgacs, E., White, H. D., and Sellers, J. R. (2008) Direct observation of the mechanochemical coupling in myosin Va during processive movement. Nature 455, 128–132.PubMedCrossRefGoogle Scholar
  11. 11.
    Ohtsubo, M., Yoshida, M., Ohta, S., Kagawa, Y., Yohda, M., and Date, T. (1987) In vitro mutated beta subunits from the F1-ATPase of the thermophilic bacterium, PS3, containing glutamine in place of glutamic acid in positions 190 or 201 assembles with the alpha and gamma subunits to produce inactive complexes. Biochem. Biophys. Res. Commun. 146, 705–710.PubMedCrossRefGoogle Scholar
  12. 12.
    Masaike, T., Koyama-Horibe, F., Oiwa, K., Yoshida, M., and Nishizaka, T. (2008) Cooperative three-step motions in catalytic subunits of F1-ATPase correlate with 80° and 40° substep rotations. Nat. Struct. Mol. Biol. 15, 1326–1333.PubMedCrossRefGoogle Scholar
  13. 13.
    Matsui, T. and Yoshida, M. (1995) Expression of the wild-type and the Cys-/Trp-less a3b3g complex of thermophilic F1-ATPase in Escherichia coli. Biochim. Biophys. Acta 1231, 139–146.PubMedCrossRefGoogle Scholar
  14. 14.
    Adachi, K., Yasuda, R., Noji, H., Itoh, H., Harada, Y., Yoshida, M., and Kinosita Jr., K. (2000) Stepping rotation of F1-ATPase visualized through angle-resolved single-fluorophore imaging. Proc. Natl. Acad. Sci. USA 97, 7243–7247.PubMedCrossRefGoogle Scholar
  15. 15.
    Yasuda, R., Noji, H., Yoshida, M., Kinosita, K., Jr., and Itoh, H. (2001) Resolution of distinct rotational substeps by submillisecond kinetic analysis of F1-ATPase. Nature 410, 898–904.PubMedCrossRefGoogle Scholar
  16. 16.
    Rondelez, Y., Tresset, G., Nakashima, T., Kato-Yamada, Y., Fujita, H., Takeuchi, S., and Noji, H. (2005) Highly coupled ATP synthesis by F1-ATPase single molecules. Nature 433, 773–777.PubMedCrossRefGoogle Scholar
  17. 17.
    Funatsu, T., Harada, Y., Tokunaga, M., Saito, K., and Yanagida, T. (1995) Imaging of single fluorescent molecules and individual ATP turnovers by single myosin molecules in aqueous solution. Nature 374, 555–559.PubMedCrossRefGoogle Scholar
  18. 18.
    Adachi, K., Oiwa, K., Nishizaka, T., Furuike, S., Noji, H., Itoh, H., Yoshida, M., and Kinosita, K., Jr. (2007) Coupling of rotation and catalysis in F1-ATPase revealed by single-molecule imaging and manipulation. Cell 130, 309–321.PubMedCrossRefGoogle Scholar
  19. 19.
    Stout, A. L. and Axelrod, D. (1989) Evanescent field excitation of fluorescence by epi-illumination microscopy. Appl. Opt. 28, 5237–5242.PubMedCrossRefGoogle Scholar
  20. 20.
    Oiwa, K., Eccleston, J. F., Anson, M., Kikumoto, M., Davis, C. T., Reid, G. P., Ferenczi, M. A., Corrie, J. E., Yamada, A., Nakayama, H., and Trentham, D. R. (2000) Comparative single-molecule and ensemble myosin enzymology: sulfoindocyanine ATP and ADP derivatives. Biophys. J. 78, 3048–3071.PubMedCrossRefGoogle Scholar
  21. 21.
    Nishizaka, T., Mizutani, K., and Masaike, T. (2007) Single-Molecule Observation of Rotation of F1-ATPase Through Microbeads. Methods Mol. Biol. 392, 171–182.PubMedCrossRefGoogle Scholar

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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