Skip to main content
SpringerLink
Log in

Deconvolution of Calcium-Binding Curves

Facts and Fantasies

  • Protocol
Calcium-Binding Protein Protocols: Volume 2: Methods and Techniques

Part of the book series: Methods in Molecular Biology™ ((MIMB,volume 173))

  • 1310 Accesses

Abstract

Ca2+signaling is of paramount importance in intracellular communication of eukaryotic cells. Many external stimuli trigger a transient change in the cytosolic-free Ca2+concentration (in the form of a Ca2+wave or Ca2+oscillations). The internal Ca2+modulation is deciphered by Ca2+-binding proteins, which undergo conformational changes upon Ca2+-binding allowing them to act as enzymatic or protein modulators. These Ca2+-binding proteins have been well described in the past three decades (1–6). Calmodulin, an ubiquitous and multifunctional protein, is considered as the prototype of the Ca2+-binding protein family containing EF-hand domains (7–16). Because of its pivotal role in many Ca2+-dependent cellular events, the understanding of the mechanism of action of this protein at the molecular level has been the aim of several research groups. For such a study, four main points have to be tackled:

  • Description of the mechanism of Ca2+-binding to calmodulin;

  • Understanding of the conformational changes induced by Ca2+-binding;

  • Analysis of the interaction of calmodulin with the different targets; and

  • Deciphering the activation or the modulation of the calmodulin/target protein complexes.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 84.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 109.00
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 109.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Gerke, V., Heizmann, C. W., and Krebs J. (1998) Special Issue. 5th European symposium on calcium binding proteins in normal and transformed cells, in Biochem. Biophys. Acta. Molecular Cell Research, vol.1148(2) (Avruch, J., ed.), Elsevier, Amsterdam.

    Google Scholar 

  2. Wasserman, R. H., Corradino, R. A., Carafoli, E., Kretsinger, R. H., MacLennan, D. H., and Siegel, F. L. (1977) Calcium-Binding Proteins and Calcium Function, North Holland, New York.

    Google Scholar 

  3. Heizmann, C. W. (1991) Novel calcium binding proteins, in Fundamentals and Clinical Implications, Springer-Verlag, Berlin, Heidelberg.

    Google Scholar 

  4. Kretsinger, R. H. (1975) Hypothesis: calcium modulated proteins contain EF hands, in Calcium Transport in Contraction and Secretion (Carafoli, E., et al., eds.), Amsterdam, North-Holland, pp. 469–78.

    Google Scholar 

  5. Pochet, R., Lawson, D. E. M., and Heizmann, C. W. (1989) Calcium binding proteins in normal and transformed cells, in Advances in Experimental Medicine and Biology, vol. 269, Plenum, New York.

    Google Scholar 

  6. Goodman, M., Pechere, J. F., Haiech, J., and Demaille, J. G. (1979) Evolutionary diversification of structure and function in the family of intracellular calcium-binding proteins. J. Mol. Evol. 13, 331–352.

    Article  PubMed  CAS  Google Scholar 

  7. Cheung, W. Y. (1970) Cyclic 3′,5′-nucleotide phosphodiesterase. Demonstration of an activator. Biochem. Biophys. Res. Commun. 38, 533–538.

    Article  PubMed  CAS  Google Scholar 

  8. Cheung, W. Y. (1980) Calmodulin plays a pivotal role in cellular regulation. Science 207, 19–27.

    Article  PubMed  CAS  Google Scholar 

  9. Kakiuchi, S. and Yamazaki, R. (1970) Calcium dependent phosphodiesterase activity and its activating factor (PAF) from brain studies on cyclic 3′,5′-nucleotide phosphodiesterase (3). Biochem. Biophys. Res. Commun. 41, 1104–1110.

    Article  PubMed  CAS  Google Scholar 

  10. Klee, C. B., Crouch, T. H., and Richman, P. G. (1980) Calmodulin. Annu. Rev. Biochem. 49, 489–515.

    Article  PubMed  CAS  Google Scholar 

  11. Klee, C. B. and Vanaman, T. C. (1982) Calmodulin. Adv. Prot. Chem. 35, 213–321.

    Article  CAS  Google Scholar 

  12. Watterson, D. M. (1980) Calmodulin and Cell Functions, vol. 356, The New York Academy of Sciences, New York.

    Google Scholar 

  13. Marx, J. L. (1980) Calmodulin: a protein for all seasons. Science 208, 274–276.

    Article  PubMed  CAS  Google Scholar 

  14. Cox, J. A., Comte, M., Malnoe, A., and Stein, E. A. (1984) Mode of action of the regulatory protein calmodulin, in Metal Ions in Biological Systems, vol. 17, (Sigel, H., ed.), Marcel Dekker, New York, pp. 215–273.

    Google Scholar 

  15. Watterson, D. M., Burgess, W. H., Lukas, T. J., Iverson, D., Marshak, D. R., Schleicher, M., et al. (1984) Towards a molecular and atomic anatomy of calmodulin and calmodulin-binding proteins, in Advances in Cyclic Nucleotide and Protein Phosphorylation Research (Strada, S. J., ed.), Raven, New York, pp. 205–226.

    Google Scholar 

  16. Cohen, P. (1988) Calmodulin, in Molecular Aspects of Cellular Regulation, vol. 5 (Cohen, P., ed.), Elsevier, Amsterdam.

    Google Scholar 

  17. Klotz, I. M. (1997) Ligand-Receptor Energetics: A Guide for the Perplexed, Wiley, New York.

    Google Scholar 

  18. Moncrief, N. D., Kretsinger, R. H., and Goodman, M. (1990) Evolution of EF-hand calcium-modulated proteins. I. Relationships based on amino acid sequences. J. Mol. Evol. 30, 522–562.

    Article  PubMed  CAS  Google Scholar 

  19. Kretsinger, R. H. and Nockolds, C. E. (1973) Carp muscle calcium-binding protein. II. Structure determination and general description. J. Biol. Chem. 248, 3313–3326.

    PubMed  CAS  Google Scholar 

  20. Kretsinger, R. H. (1979) The informational role of calcium in the cytosol. Adv. Cyclic Nucleotide Res. 11, 1–26.

    PubMed  CAS  Google Scholar 

  21. Kawasaki, H. and Kretsinger, R. H. (1995) Calcium-binding proteins 1: EF-hands. Protein Profile 2, 297–490.

    PubMed  CAS  Google Scholar 

  22. Anderson, J. M. and Cormier, M. J. (1978) Calcium-dependent regulation of NAD kinase. Biochem. Biophys. Res. Commun. 84 595–602.

    Article  PubMed  CAS  Google Scholar 

  23. Brostrom, C. O., Huang, Y. C., Breckenridge, B. M., and Wolff, D. J. (1975) Identification of a calcium-binding protein as a calcium-dependent regulator of brain adenylate cyclase. Proc. Natl. Acad. Sci. USA 72, 64–68.

    Article  PubMed  CAS  Google Scholar 

  24. Cohen, P., Burchell, A., Foulkes, J. G., and Cohen, P. T. (1978) Identification of the Ca2+-dependent modulator protein as the fourth subunit of rabbit skeletal muscle phosphorylase kinase. FEBS Lett. 92, 287–293.

    Article  PubMed  CAS  Google Scholar 

  25. Dabrowska, R., Aromatorio, D., Sherry, J. M., and Hartshorne, D. J. (1977) Composition of the myosin light chain kinase from chicken gizzard. Biochem. Biophys. Res. Commun. 78, 1263–1272.

    Article  PubMed  CAS  Google Scholar 

  26. Gopinath, R. M. and Vincenzi, F. F. (1977) Phosphodiesterase protein activator mimics red blood cell cytoplasmic activator of (Ca2+-Mg2+)ATPase. Biochem.Biophys. Res. Commun. 77, 1203–1209.

    Article  PubMed  CAS  Google Scholar 

  27. Jarrett, H. W. and Penniston, J. T. (1977) Partial purification of the Ca2+-Mg2+ATPase activator from human erythrocytes: its similarity to the activator of 3′:5′-cyclic nucleotide phosphodiesterase. Biochem. Biophys. Res. Commun. 77, 1210–1216.

    Article  PubMed  CAS  Google Scholar 

  28. Schulman, H. and Greengard, P. (1978) Stimulation of brain membrane protein phosphorylation by calcium and an endogenous heat-stable protein. Nature 271, 478–479.

    Article  PubMed  CAS  Google Scholar 

  29. Stewart, A. A., Ingebritsen, T. S., Manalan, A., Klee, C. B., and Cohen, P. (1982) Discovery of a Ca2+-and calmodulin-dependent protein phosphatase: probable identity with calcineurin (CaM-BP80). FEBS Lett. 137, 80–84.

    Article  PubMed  CAS  Google Scholar 

  30. Yazawa, M. and Yagi, K. (1977) Calcium-binding subunit of myosin light chain kinase. J. Biochem. (Tokyo) 82, 287–289.

    CAS  Google Scholar 

  31. Watterson, D. M., Sharief, F., and Vanaman, T. C. (1980) The complete amino acid sequence of the Ca2+-dependent modulator protein (calmodulin) of bovine brain. J. Biol. Chem. 255, 962–975.

    PubMed  CAS  Google Scholar 

  32. Watterson, D. M., Iverson, D. B., and Van Eldik, L. J. (1980) Spinach calmodulin: isolation, characterization, and comparison with vertebrate calmodulins. Biochemistry 19, 5762–5768.

    Article  PubMed  CAS  Google Scholar 

  33. Babu, Y. S., Sack, J. S., Greenhough, T. J., Bugg, C. E., Means, A. R., and Cook, W. J. (1985) Three-dimensional structure of calmodulin. Nature 315, 37–40.

    Article  PubMed  CAS  Google Scholar 

  34. Babu, Y. S., Bugg, C. E., and Cook, W. J. (1988) Structure of calmodulin refined at 2.2 Å resolution. J. Mol. Biol. 204, 191–204.

    Article  PubMed  CAS  Google Scholar 

  35. Teo, T. S. and Wang, J. H. (1973) Mechanism of activation of a cyclic adenosine 3′:5′-monophosphate phosphodiesterase from bovine heart by calcium ions. Identification of the protein activator as a Ca2+binding protein. J. Biol. Chem. 248, 5950–5955.

    PubMed  CAS  Google Scholar 

  36. Crouch, T. H. and Klee, C. B. (1980) Positive cooperative binding of calcium to bovine brain calmodulin. Biochemistry 19, 3692–3698.

    Article  PubMed  CAS  Google Scholar 

  37. Dedman, J. R., Potter, J. D., Jackson, R. L., Johnson, J. D., and Means, A. R. (1977) Physicochemical properties of rat testis Ca2+-dependent regulator protein of cyclic nucleotide phosphodiesterase. Relationship of Ca2+-binding, conformational changes, and phosphodiesterase activity. J. Biol. Chem. 252, 8415–8422.

    PubMed  CAS  Google Scholar 

  38. Haiech, J., Klee, C. B., and Demaille, J. G. (1981) Effects of cations on affinity of calmodulin for calcium: ordered binding of calcium ions allows the specific activation of calmodulin-stimulated enzymes. Biochemistry 20, 3890–3897.

    Article  PubMed  CAS  Google Scholar 

  39. Jarrett, H. W. and Kyte, J. (1979) Human erythrocyte calmodulin. Further chemical characterization and the site of its interaction with the membrane. J. Biol. Chem. 254, 8237–8244.

    PubMed  CAS  Google Scholar 

  40. Keller, C. H., Olwin, B. B., LaPorte, D. C., and Storm, D. R. (1982) Determination of the free-energy coupling for binding of calcium ions and troponin I to calmodulin. Biochemistry 21, 156–162.

    Article  PubMed  CAS  Google Scholar 

  41. Wolff, D. J., Poirier, P. G., Brostrom, C. O., and Brostrom, M. A. (1977) Divalent cation binding properties of bovine brain Ca2+-dependent regulator protein. J. Biol. Chem. 252, 4108–4117.

    PubMed  CAS  Google Scholar 

  42. Burger, D., Cox, J. A., Comte, M., and Stein, E. A. (1984) Sequential conformational changes in calmodulin upon binding of calcium. Biochemistry 23, 1966–1971.

    Article  CAS  Google Scholar 

  43. Huang, C. Y., Chau, V., Chock, P. B., Wang, J. H., and Sharma, R. K. (1981) Mechanism of activation of cyclic nucleotide phosphodiesterase: requirement of the binding of four Ca2+to calmodulin for activation. Proc. Natl. Acad. Sci. USA 78, 871–874.

    Article  PubMed  CAS  Google Scholar 

  44. Ogawa, Y. and Tanokura, M. (1984) Calcium binding to calmodulin: effects of ionic strength, Mg2+, pH and temperature. J. Biochem. (Tokyo) 95, 19–28.

    CAS  Google Scholar 

  45. Forsen, S., Thulin, E., Drakenberg, T., Krebs, J., and Seamon, K. (1980) A 113Cd NMR study of calmodulin and its interaction with calcium, magnesium and triflu-operazine. FEBS Lett. 117, 189–194.

    Article  PubMed  CAS  Google Scholar 

  46. Seamon, K. (1980) NMR studies on tyrosine-138 of calmodulin. Ann. NY Acad. Sci. 356, 433–434.

    Article  PubMed  CAS  Google Scholar 

  47. Krebs, J., Carafoli, E., Thulin, E., and Forsen, S. (1980) 1H-and 113Cd-NMR studies of calmodulin. Ann. NY Acad. Sci. 356, 397–398.

    Article  PubMed  CAS  Google Scholar 

  48. Andersson, T., Drakenberg, T., Forsen, S., and Thulin, E. (1982) Characterization of the Ca2+binding sites of calmodulin from bovine testis using 43Ca and 113Cd NMR. Eur. J. Biochem. 126, 501–505.

    Article  PubMed  CAS  Google Scholar 

  49. Teleman, A., Drakenberg, T., and Forsen, S. (1986) Kinetics of Ca2+binding to calmodulin and its tryptic fragments studied by 43Ca-NMR. Biochim. Biophys. Acta. 873, 204–213.

    Article  PubMed  CAS  Google Scholar 

  50. Yazawa, M., Kawamura, E., Minowa, O., Yagi, K., Ikura, M., and Hikichi, K. (1984) N-terminal region (domain 1) of calmodulin is the low affinity site for Ca2+. A 13C NMR study of S-cyanocalmodulin. J. Biochem. (Tokyo) 95, 443–446.

    CAS  Google Scholar 

  51. Drabikowski, W., Kuznicki, J., and Grabarek, Z. (1977) Similarity in Ca2+-induced changes between troponic-C and protein activator of 3′:5′-cyclic nucleotide phos-phodiesterase and their tryptic fragments. Biochim. Biophys. Acta. 485, 124–133.

    PubMed  CAS  Google Scholar 

  52. Klee, C. B. (1977) Conformational transition accompanying the binding of Ca2+to the protein activator of 3′,5′-cyclic adenosine monophosphate phosphodiesterase. Biochemistry 16, 1017–1024.

    Article  PubMed  CAS  Google Scholar 

  53. Richman, P. G. and Klee, C. B. (1979) Specific perturbation by Ca2+of tyrosyl residue 138 of calmodulin. J. Biol. Chem. 254, 5372–5376.

    PubMed  CAS  Google Scholar 

  54. Drabikowski, W., Brzeska, H., Kuznicki, J., and Grabarek, Z. (1980) Studies on structure and function of calmodulin. Ann. NY Acad. Sci. 356, 374–375.

    Article  PubMed  CAS  Google Scholar 

  55. Kilhoffer, M. C., Demaille, J. G., and Gerard, D. (1981) Tyrosine fluorescence of ram testis and octopus calmodulins. Effects of calcium, magnesium, and ionic strength. Biochemistry 20, 4407–4414.

    Article  PubMed  CAS  Google Scholar 

  56. Wang, C. L. (1985) A note on Ca2+binding to calmodulin. Biochem. Biophys. Res. Commun. 130, 426–430.

    Article  PubMed  CAS  Google Scholar 

  57. Drabikowski, W., Brzeska, H., and Venyaminov, S. (1982) Tryptic fragments of calmodulin. Ca2+-and Mg2+-induced conformational changes. J. Biol. Chem. 257, 11,584–11,590.

    PubMed  CAS  Google Scholar 

  58. Minowa, O. and Yagi, K. (1984) Calcium binding to tryptic fragments of calmodulin. J. Biochem. (Tokyo) 96, 1175–1182.

    CAS  Google Scholar 

  59. Dalgarno, D. C., Klevit, R. E., Levine, B. A., Williams, R. J., Dobrowolski, Z., and Drabikowski, W. (1984) 1H NMR studies of calmodulin. Resonance assignments by use of tryptic fragments. Eur. J. Biochem. 138, 281–289.

    Article  PubMed  CAS  Google Scholar 

  60. Aulabaugh, A., Niemczura, W. P., and Gibbons, W. A. (1984) High field proton NMR studies of tryptic fragments of calmodulin: a comparison with the native protein. Biochem. Biophys. Res. Commun. 118, 225–232.

    Article  PubMed  CAS  Google Scholar 

  61. Maune, J. F., Klee, C. B., and Beckingham, K. (1992) Ca2+binding and conformational change in two series of point mutations to the individual Ca(2+)-binding sites of calmodulin. J. Biol. Chem. 267, 5286–5295.

    PubMed  CAS  Google Scholar 

  62. Beckingham, K. (1991) Use of site-directed mutations in the individual Ca2(+)-binding sites of calmodulin to examine Ca2(+)-induced conformational changes. J. Biol. Chem. 266, 6027–6030.

    PubMed  CAS  Google Scholar 

  63. Kilhoffer, M. C., Roberts, D. M., Adibi, A. O., Watterson, D. M., and Haiech, J. (1988) Investigation of the mechanism of calcium binding to calmodulin. Use of an isofunctional mutant with a tryptophan introduced by site-directed mutagenesis. J. Biol. Chem. 263, 17,023–17,029.

    PubMed  CAS  Google Scholar 

  64. Kilhoffer, M. C., Kubina, M., Travers, F., and Haiech, J. (1992) Use of engineered proteins with internal tryptophan reporter groups and pertubation techniques to probe the mechanism of ligand-protein interactions: investigation of the mechanism of calcium binding to calmodulin. Biochemistry 31, 8098–8106.

    Article  PubMed  CAS  Google Scholar 

  65. Pedigo, S. and Shea, M. A. (1995) Discontinuous equilibrium titrations of cooperative calcium binding to calmodulin monitored by 1-D 1H-nuclear magnetic resonance spectroscopy. Biochemistry 34, 10,676–10,689.

    Article  PubMed  CAS  Google Scholar 

  66. Pedigo, S. and Shea, M. A. (1995) Quantitative endoproteinase GluC footprinting of cooperative Ca2+binding to calmodulin: proteolytic susceptibility of E31 and E87 indicates interdomain interactions. Biochemistry 34, 1179–1196.

    Article  PubMed  CAS  Google Scholar 

  67. Shea, M. A., Verhoeven, A. S., and Pedigo, S. (1996) Calcium-induced interactions of calmodulin domains revealed by quantitative thrombin footprinting of Arg37 and Arg106. Biochemistry 35, 2943–2957.

    Article  PubMed  CAS  Google Scholar 

  68. Sorensen, B. R. and Shea, M. A. (1998) Interactions between domains of apo calmodulin alter calcium binding and stability. Biochemistry 37, 4244–4253.

    Article  PubMed  CAS  Google Scholar 

  69. Kilhoffer, M. C., Roberts, D. M., Adibi, A., Watterson, D. M., and Haiech, J. (1989) Fluorescence characterization of VU-9 calmodulin, an engineered calmodulin with one tryptophan in calcium binding domain III. Biochemistry 28, 6086–6092.

    Article  PubMed  CAS  Google Scholar 

  70. Haiech, J., Kilhoffer, M. C., Craig, T. A., Lukas, T. J., Wilson, E., Guerra-Santos, L., and Watterson, D. M. (1990) Mutant analysis approaches to understanding calcium signal transduction through calmodulin and calmodulin regulated enzymes. Adv. Exp. Med. Biol. 269, 43–56.

    PubMed  CAS  Google Scholar 

  71. Haiech, J., Derancourt, J., Pechere, J. F., and Demaille, J. G. (1979) Magnesium and calcium binding to parvalbumins: evidence for differences between parvalbumins and an explanation of their relaxing function. Biochemistry 18, 2752–2758.

    Article  PubMed  CAS  Google Scholar 

  72. Lafitte, D., Capony, J. P., Grassy, G., Haiech, J., and Calas, B. (1995) Analysis of the ion binding sites of calmodulin by electrospray ionization mass spectrometry. Biochemistry 34, 13,825–13,832.

    Article  PubMed  CAS  Google Scholar 

  73. Heizmann, C. W. and Cox, J. A. (1998) New perspectives on S100 proteins: a multi-functional Ca(2+)-, Zn(2+)-and Cu(2+)-binding protein family. Biometals 11, 383–397.

    Article  PubMed  CAS  Google Scholar 

  74. Declercq, J. P., Tinant, B., Parello, J., and Rambaud, J. (1991) Ionic interactions with parvalbumins. Crystal structure determination of pike 4. 10 parvalbumin in four different ionic environments. J. Mol. Biol. 220, 1017–1039.

    Article  PubMed  CAS  Google Scholar 

  75. Gilli, R., Lafitte, D., Lopez, C., Kilhoffer, M., Makarov, A., Briand, C., and Haiech, J. (1998) Thermodynamic analysis of calcium and magnesium binding to calmodulin. Biochemistry 37, 5450–5456.

    Article  PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Pharmacologie et Physico-Chimie des Interactions Cellulaires et Moléculaires, Faculté de Pharmacie, Université Louis Pasteur, Strasbourg, Illkirch, France

    Jacques Haiech & Marie-Claude Kilhoffer

Authors
  1. Jacques Haiech
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Marie-Claude Kilhoffer
    View author publications

    You can also search for this author in PubMed Google Scholar

Editor information

Editors and Affiliations

  1. Department of Biological Sciences, University of Calgary Calgary, AB, Canada

    Hans J. Vogel

Rights and permissions

Reprints and permissions

Copyright information

© 2002 Humana Press Inc.

About this protocol

Cite this protocol

Haiech, J., Kilhoffer, MC. (2002). Deconvolution of Calcium-Binding Curves. In: Vogel, H.J. (eds) Calcium-Binding Protein Protocols: Volume 2: Methods and Techniques. Methods in Molecular Biology™, vol 173. Springer, Totowa, NJ. https://doi.org/10.1385/1-59259-184-1:025

Download citation

  • DOI: https://doi.org/10.1385/1-59259-184-1:025

  • Publisher Name: Springer, Totowa, NJ

  • Print ISBN: 978-0-89603-689-5

  • Online ISBN: 978-1-59259-184-8

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation