The Use of FRET in the Analysis of Motor Protein Structure

  • Andrzej A. Kasprzak
Part of the Methods in Molecular Biology™ book series (MIMB, volume 392)


Fluorescence resonance energy transfer (FRET) is a spectroscopic phenomenon that consists of long-range dipole-dipole interaction between two chromophores. This method can be employed to gain quantitative distance information on macromolecules. FRET is particularly useful to characterize structural states of motor proteins, because the spatial relationship between various mechanical elements of the motor undergoing its mechanical cycle is essential to understand how force and movement are generated. In this chapter, we describe the technique, including the equations, methods of introducing fluorescence probes in specific loci of the protein, and data analysis. Practical guidelines and hints are also provided for protein preparation, labeling, and measuring FRET efficiency. The protocol is presented for interhead distance measurements in the dimeric kinesin-like motor, Ncd. However, it can easily be adapted to many other motor proteins.

Key Words

Fluorescence resonance energy transfer (FRET) motor proteins the Förster equation myosin kinesin fluorescence probes 


  1. 1.
    Shih, W.M., Gryczynski, Z., Lakowicz, J.R., and Spudich, J.A. (2000) A FRET-based sensor reveals large ATP hydrolysis-induced conformational changes and three distinct states of the molecular motor myosin. Cell 102, 683–694.CrossRefPubMedGoogle Scholar
  2. 2.
    Xu, J. and Root, D.D. (2000) Conformational selection during weak binding at the actin and myosin interface. Biophys. J. 79, 1498–1510.CrossRefPubMedGoogle Scholar
  3. 3.
    Palm, T., Sale, K., Brown, L., Li, H., Hambly, B., and Fajer, P.G. (1999) Intradomain distances in the regulatory domain of the myosin head in prepower and postpower stroke states: fluorescence energy transfer. Biochemistry 38, 13026–13034.CrossRefPubMedGoogle Scholar
  4. 4.
    Smyczynski, C. and Kasprzak, A.A. (1997) Effect of nucleotides and actin on the orientation of the light chain-binding domain in myosin subfragment 1. Biochemistry 36, 13201–13207.CrossRefPubMedGoogle Scholar
  5. 5.
    Rice, S., Lin, A.W., Safer, D., Hart, C.L., Naber, N., Carragher, B.O., Cain, S.M., Pechatnikova, E., Wilson-Kubalek, E.M., Whittaker, M., Pate, E., Cooke, R., Taylor, E.W., Milligan, R.A., and Vale, R.D. (1999) A structural change in the kinesin motor protein that drives motility. Nature 402, 778–784.CrossRefPubMedGoogle Scholar
  6. 6.
    Geeves, M.A. and Holmes, K.C. (1999) Structural mechanism of muscle contraction. Annu. Rev. Biochem. 68, 687–728.CrossRefPubMedGoogle Scholar
  7. 7.
    Selvin, P.R. (2000) The renaissance of fluorescence resonance energy transfer. Nat. Struct. Biol. 7, 730–734.CrossRefPubMedGoogle Scholar
  8. 8.
    Heyduk, T. (2002) Measuring protein conformational changes by FRET/LRET. Curr. Opin. Biotechnol. 13, 292–296.CrossRefPubMedGoogle Scholar
  9. 9.
    Heyduk, T. (2001) Luminescence resonance energy transfer analysis of RNA polymerase complexes. Methods 25, 44–53.CrossRefPubMedGoogle Scholar
  10. 10.
    Xiao, M., Reifenberger, J.G., Wells, A.L., Baldacchino, C., Chen, L.Q., Ge, P., Sweeney, H.L., and Selvin, P.R. (2003) An actin-dependent conformational change in myosin. Nat. Struct. Biol. 10, 402–408.CrossRefPubMedGoogle Scholar
  11. 11.
    Rosenfeld, S.S., Xing, J., Jefferson, G.M., and King, P.H. (2005) Docking and rolling, a model how the mitotic motor Eg5 works. J. Biol. Chem. 280, 35684–35695.CrossRefPubMedGoogle Scholar
  12. 12.
    Yasuda, R., Masaike, T., Adachi, K., Noji, H., Itoh, H., and Kinoshita, K., Jr. (2003)The ATP-waiting conformation of rotating F1-ATPase revealed by single-pair fluorescence resonance energy transfer. Proc. Natl. Acad. Sci. USA 100, 9314–9318.CrossRefPubMedGoogle Scholar
  13. 13.
    dos Remedios, C.G., Miki, M., and Barden, J.A. (1987) Fluorescence resonance energy transfer measurements of distances in actin and myosin. A critical evaluation. J. Muscle Res. Cell Motil. 8, 97–117.CrossRefPubMedGoogle Scholar
  14. 14.
    Botts, J., Thomason, J.F., and Morales, M.F. (1989) On the origin and transmission of force in actomyosin subfragment 1. Proc. Natl. Acad. Sci. USA 86, 2204–2208.CrossRefPubMedGoogle Scholar
  15. 15.
    Bevington, P.R. (1969) Data Reduction and Error Analysis for Physical Sciences. McGraw-Hill, New York.Google Scholar
  16. 16.
    Dale, R.E., Eisinger, J., and Blumberg, W.E. (1979) The orientation freedom of molecular probes. The orientation factor in intramolecular energy transfer. Biophys. J. 26, 161–194.CrossRefPubMedGoogle Scholar
  17. 17.
    Mandelkow, E.M., Herrmann, M., and Ruhl, U. (1985) Tubulin domains probed by limited proteolysis and subunit-specific antibodies. J. Mol. Biol. 185, 311–327.CrossRefPubMedGoogle Scholar
  18. 18.
    Haran, G., Haas, E., Szpikowska, B.K., and Mas, M.T. (1992) Domain motions in phosphoglycerate kinase: determination of interdomain distance distributions by site-specific labeling and time-resolved fluorescence energy transfer. Proc. Natl. Acad. Sci. USA 89, 11764–11768.CrossRefPubMedGoogle Scholar
  19. 19.
    Marsh, D.J. and Lowey, S. (1980) Fluorescence energy transfer in myosin subfragment 1. Biochemistry 19, 774–784.CrossRefPubMedGoogle Scholar
  20. 20.
    Takashi, R. and Kasprzak, A.A. (1987) Measurement of interprotein distances in the acto-subfragment 1 rigor complex. Biochemistry 26, 7471–7477.CrossRefPubMedGoogle Scholar
  21. 21.
    Cheng, J.-Q., Jiang, W., and Hackney, D.D. (1998) Interaction of mantadenosine nucleotides and magnesium with kinesin. Biochemistry 37, 5288–5295.CrossRefPubMedGoogle Scholar
  22. 22.
    Hiratsuka, T. (2003) Fluorescent and colored trinitrophenylated analogs of ATP and GTP. Eur. J. Biochem. 270, 3479–3485.CrossRefPubMedGoogle Scholar
  23. 23.
    Grazi, E., Cintio, O., Magri, E., and Trombetta, G. (2001) A possible solvent effect of adenosine diphosphate influences the binding of 1,N 6-ethenoadenosine diphosphate to myosin from skeletal muscle. Biochim. Biophys. Acta 1525, 130–135.PubMedGoogle Scholar
  24. 24.
    Suzuki, Y., Yasunaga, T., Ohkura, R., Wakabayashi, T., and Sutoh, K. (1998) Swing of the lever arm of a myosin motor at the isomerization and phosphate-release steps. Nature 396, 380–383.CrossRefPubMedGoogle Scholar
  25. 25.
    Hajdo, L., Skowronek, K., and Kasprzak, A.A. (2004) Spatial relationship between heads of dimeric Ncd in the presence of nucleotides and microtubules. Arch. Biochem. Biophys. 421, 217–226.CrossRefPubMedGoogle Scholar
  26. 26.
    Kasprzak, A.A., Takashi, R., and Morales, M.F. (1988) Orientation of actin monomer in the F-actin filament: radial coordinate of glutamine-41 and effect of myosin subfragment 1 binding on the monomer orientation. Biochemistry 27, 4512–4522.CrossRefPubMedGoogle Scholar
  27. 27.
    Lakowicz, J. (1999) Principles of the Fluorescence Spectroscopy. Kluwer Academic/Plenum, New York.Google Scholar
  28. 28.
    Root, D.D., Shangguan, X., Xu, J., and McAllister, M.A. (1999) Determination of fluorescent probe orientations on biomolecules by conformational searching: algorithm testing and applications to the atomic model of myosin. J. Struct. Biol. 127, 22–34.CrossRefPubMedGoogle Scholar
  29. 29.
    Yun, M., Bronner, C.E., Park, C.-G., Cha, S.-S., Park, H.-W., and Endow, S.A. (2003) Rotation of the stalk/neck and one head in a new crystal structure of the kinesin motor protein, Ncd. EMBO J. 22, 5382–5389.CrossRefPubMedGoogle Scholar
  30. 30.
    Endres, N.F., Yoshioka, C., Milligan, R.A., and Vale, R.D. (2006) A leverarm rotation drives motility of the minus-end-directed kinesin Ncd. Nature 439, 875–878.CrossRefPubMedGoogle Scholar
  31. 31.
    Hyman, A.A., Salser, S., Drechsel, D., Unwin, N.N., and Mitchison, T.J. (1992) Role of GTP hydrolysis in microtubule dynamics: information from a slowly hydrolysable analogue, GMPCPP. Mol. Biol. Cell 3, 1155–1167.PubMedGoogle Scholar
  32. 32.
    Read, S.M. and Northcote, D.H. (1981) Minimization of variation in the response to different proteins of the Coomassie blue G dye-binding assay for protein. Anal. Biochem. 116, 53–64.CrossRefPubMedGoogle Scholar
  33. 33.
    Sablin, E., Case, R.B., Dai, S.C., Hart, C.L., Ruby, A., Vale, R.D., and Fletterick, R.J. (1998) Direction determination in the minus-end-directed kinesin motor ncd. Nature 395, 813–816.CrossRefPubMedGoogle Scholar

Copyright information

© Humana Press Inc., Totowa, NJ 2007

Authors and Affiliations

  • Andrzej A. Kasprzak
    • 1
  1. 1.Motor Proteins Laboratory, Department of Muscle BiochemistryNencki Institute of Experimental BiologyWarsawPoland

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