Equilibrium Binding of Proteins to F-Actin

  • Joseph M. Chalovich
Part of the Methods in Molecular Biology™ book series (MIMB, volume 392)


This chapter reviews some of the many available methods for measuring the binding of myosin and other proteins to actin. Binding to actin has special considerations because actin is a long lattice and the binding site of many of its binding partners consists of multiple actin protomers. The analysis of binding to a lattice cannot be done by standard methods such as a Scatchard plot. Rational methods of analysis are described.

Key Words

Myosin S1 actin G-actin F-actin cooperativity kinetics binding binding analysis equilibrium association constant parking problem ATPase rate equations sedimentation ATP 


  1. 1.
    Criddle, A.H., Geeves, M.A., and Jeffries, T. (1985) The use of actin labelled with N-(1-pyrenyl)iodoacetamide to study the interaction of actin with myosin subfragments and troponin/tropomyosin. Biochem. J. 232, 343–349.PubMedGoogle Scholar
  2. 2.
    Sen, A., Chen, Y.D., Yan, B., and Chalovich, J.M. (2001) Caldesmon reduces the apparent rate of binding of myosin S1 to actin-tropomyosin. Biochemistry 40, 5757–5764.CrossRefPubMedGoogle Scholar
  3. 3.
    Pollard, T.D. and Cooper, J.A. (1982) Methods to characterize actin filament networks. Methods Enzymol. 85, 211–233.CrossRefPubMedGoogle Scholar
  4. 4.
    Faulstich, H., Schafer, A.J., and Weckauf, M. (1977) The dissociation of the phalloidin-actin complex. Hoppe-Seylers Z. Physiol. Chem. 358, 181–184.PubMedGoogle Scholar
  5. 5.
    Dancker, P., Low, I., Hasselbach, W., and Wieland, T. (1975) Interaction of actin with phalloidin: polymerization and stabilization of F-actin. Biochim. Biophys. Acta 400, 407–414.PubMedGoogle Scholar
  6. 6.
    Kurzawa, S.E. and Geeves, M.A. (1996) A novel stopped-flow method for measuring the affinity of actin for myosin head fragments using μg quantities of protein. J. Muscle Res. Cell Motil. 17, 669–676.CrossRefPubMedGoogle Scholar
  7. 7.
    Leinweber, B.D., Fredricksen, R.S., Hoffman, D.R., and Chalovich, J.M. (1999) Fesselin: a novel synaptopodin-like actin binding protein from muscle tissue. J. Muscle Res. Cell Motil. 20, 539–545.CrossRefPubMedGoogle Scholar
  8. 8.
    Wegner, A. (1979) Equilibrium of the actin-tropomyosin interaction. J. Mol. Biol. 131, 839–853.CrossRefPubMedGoogle Scholar
  9. 9.
    Fredricksen, S., Cai, A., Gafurov, B., Resetar, A., and Chalovich, J.M. (2003) Influence of ionic strength, actin state, and caldesmon construct size on the number of actin monomers in a caldesmon binding site. Biochemistry 42, 6136–6148.CrossRefPubMedGoogle Scholar
  10. 10.
    Lakowicz, J.R. (1999) Principles of Fluorescence Spectroscopy. Kluwer Academic/Plenum Publishers, New York.Google Scholar
  11. 11.
    Tobacman, L.S. and Butters, C.A. (2000) A new model of cooperative myosin-thin filament binding. J. Biol. Chem. 275, 27587–27593.PubMedGoogle Scholar
  12. 12.
    Kouyama, T. and Mihashi, K. (1981) Fluorimetry study of N-(1-pyrenyl)iodoacetamide-labeled F-actin: local structural change of actin protomer both on polymerization and on binding of heavy meromyosin. Eur. J. Biochem. 114, 33–38.CrossRefPubMedGoogle Scholar
  13. 13.
    Brenner, S.L. and Korn, E.D. (1983) On the mechanism of actin monomer-polymer subunit exchange at steady state. J. Biol. Chem. 258, 5013–5020.PubMedGoogle Scholar
  14. 14.
    Chalovich, J.M., Stein, L.A., Greene, L.E., and Eisenberg, E. (1984) Interaction of isozymes of myosin subfragment 1 with actin: effect of ionic strength and nucleotide. Biochemistry 23, 4885–4889.CrossRefPubMedGoogle Scholar
  15. 15.
    Weeds, A.G. and Taylor, R.S. (1975) Separation of subfragment-1 isozymes from rabbit skeletal muscle myosin. Nature 257, 54–56.CrossRefPubMedGoogle Scholar
  16. 16.
    Chalovich, J.M. and Eisenberg, E. (1982) Inhibition of actomyosin ATPase activity by troponin-tropomyosin without blocking the binding of myosin to actin. J. Biol. Chem. 257, 2432–2437.PubMedGoogle Scholar
  17. 17.
    Greene, L.E. and Eisenberg, E. (1980) Cooperative binding of myosin subfragment-1 to the actin-troponin-tropomyosin complex. Proc. Natl. Acad. Sci. USA 77, 2616–2620.CrossRefPubMedGoogle Scholar
  18. 18.
    Velaz, L., Hemric, M.E., Benson, C.E., and Chalovich, J.M. (1989) The binding of caldesmon to actin and its effect on the ATPase activity of soluble myosin subfragments in the presence and absence of tropomyosin. J. Biol. Chem. 264, 9602–9610.PubMedGoogle Scholar
  19. 19.
    Lindberg, O. and Ernster, L. (1955) Determination of organic phosphorus compounds by phosphate analysis. In: Methods of Biochemical Analysis (Glick, D., ed.), vol. 3, pp. 1–22. Interscience, New York.Google Scholar
  20. 20.
    Maytum, R., Lehrer, S.S., and Geeves, M.A. (1999) Cooperativity and switching within the three-state model of muscle regulation. Biochemistry 38, 1102–1110.CrossRefPubMedGoogle Scholar
  21. 21.
    McGhee, J.D. and von Hippel, P.H. (1974) Theoretical aspects of DNA-protein interactions: co-operative and non-co-operative binding of large ligands to a one-dimensional homogeneous lattice. J. Mol. Biol. 86, 469–489.CrossRefPubMedGoogle Scholar
  22. 22.
    Chen, Y.D. and Chalovich, J.M (1992) A mosaic multiple-binding model for the binding of caldesmon and myosin subfragment-1 to actin. Biophys. J. 63, 1063–1070.CrossRefPubMedGoogle Scholar
  23. 23.
    Hill, T.L., Eisenberg, E., and Greene, L.E. (1980) Theoretical model for the cooperative equilibrium binding of myosin subfragment 1 to the actin-troponin-tropomyosin complex. Proc. Natl. Acad. Sci. U.S.A. 77, 3186–3190.CrossRefPubMedGoogle Scholar
  24. 24.
    Gafurov, B., Chen, Y.D., and Chalovich, J.M. (2004) Ca2+ and ionic strength dependencies of S1-ADP binding to actin-tropomyosin-troponin: regulatory implications. Biophys. J. 87, 1825–1835.CrossRefPubMedGoogle Scholar
  25. 25.
    McKillop, D.F.A. and Geeves, M.A. (1993) Regulation of the interaction between actin and myosin subfragment 1: evidence for three states of the thin filament. Biophys. J. 65, 693–701.CrossRefPubMedGoogle Scholar
  26. 26.
    Peterson, G.L. (1983) Determination of total protein. Methods Enzymol. 91, 95–119.CrossRefPubMedGoogle Scholar
  27. 27.
    Wells, J.A. and Yount, R.G. (1982) Chemical modification of myosin by active-site trapping of metal-nucleotides with thiol crosslinking reagents. Methods Enzymol. 85, 93–116.CrossRefPubMedGoogle Scholar
  28. 28.
    Chalovich, J.M., Greene, L.E., and Eisenberg, E. (1983) Crosslinked myosin subfragment 1: a stable analogue of the subfragment-1. ATP complex. Proc. Natl. Acad. Sci. U.S.A. 80, 4909–4913.CrossRefPubMedGoogle Scholar
  29. 29.
    Castaneda-Agullo, M., del Castillo, L.M., Whitaker, J.R., and Tappel, A.L. (1961) Effect of ionic strength on the kinetics of trypsin and alpha chymotrypsin. J. Gen. Physiol. 44, 1103–1120.CrossRefPubMedGoogle Scholar
  30. 30.
    Chalovich, J.M., Chock, P.B., and Eisenberg, E. (1981) Mechnism of action of troponin-tropomyosin: inhibition of actomyosin ATPase activity without inhibition of myosin binding to actin. J. Biol. Chem. 256, 575–578.PubMedGoogle Scholar

Copyright information

© Humana Press Inc., Totowa, NJ 2007

Authors and Affiliations

  • Joseph M. Chalovich
    • 1
  1. 1.Department of Biochemistry and Molecular Biology, Brody School of MedicineEast Carolina UniversityGreenville

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