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. 1985 Nov;48(5):727–739. doi: 10.1016/S0006-3495(85)83831-5

Energetics of the binding of calcium and troponin I to troponin C from rabbit skeletal muscle.

C K Wang, H C Cheung
PMCID: PMC1329398  PMID: 4074834

Abstract

We determined the free energy of interaction between rabbit skeletal troponin I (TNI) and troponin C (TNC) at 10 degrees and 20 degrees C with fluorescently labeled proteins. The sulfhydryl probe 5-iodoacetamidoeosin (IAE) was attached to cysteine (Cys)-98 of TNC and to Cys-133 of TNI, and each of the labeled proteins was titrated with the other unlabeled protein. The association constant for formation of the complex between labeled TNC (TNC*) and TNI was 6.67 X 10(5) M-1 in 0.3 M KCl, and pH 7.5 at 20 degrees C. In the presence of bound Mg2+, the binding constant increased to 4.58 X 10(7) M-1 and in the presence of excess of Ca2+, the association constant was 5.58 X 10(9) M-1. Very similar association constants were obtained when labeled TNI was titrated with unlabeled TNC. The energetics of Ca2+ binding to TNC* and the complex TNI X TNC* were also determined at 20 degrees C. The two sets of results were used to separately determine the coupling free energy for binding TNI and Mg2+, or Ca2+ to TNC. The results yielded a total coupling free energy of -5.4 kcal. This free energy appeared evenly partitioned into the two species: TNI X TNC(Mg)2 or TNI X TNC(Ca)2, and TNI X TNC(Ca)4. The first two species were each stabilized by -2.6 kcal, with respect to the Ca2+ free TNI X TNC complex, and TNI X TNC(Ca)4 was stabilized by -2.8 kcal, respect to TNI X TNC(Ca)2 or TNI X TNC(Mg)2. The coupling free energy was shown to produce cooperatively complexes formed between TNI and TNC in which the high affinity sites were initially saturated as a function of free Ca2+ to yield TNI X TNC(Ca)4. This saturation occurred in the free Ca2+ concentration range 10(-7) to 10(-5) M. The cooperative strengthening of the linkage between TNI and TNC induced by Ca2+ binding to the Ca2+-specific sites of TNC may have a direct relationship to activation of actomyosin ATPase. The nature of the forces involved in the Ca2+-induced strengthening of the complex is discussed.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Brzeska H., Szynkiewicz J., Drabikowski W. Localization of hydrophobic sites in calmodulin and skeletal muscle troponin C studied using tryptic fragments: a simple method of their preparation. Biochem Biophys Res Commun. 1983 Aug 30;115(1):87–93. doi: 10.1016/0006-291x(83)90972-5. [DOI] [PubMed] [Google Scholar]
  2. Cachia P. J., Sykes B. D., Hodges R. S. Calcium-dependent inhibitory region of troponin: a proton nuclear magnetic resonance study on the interaction between troponin C and the synthetic peptide N alpha-acetyl[FPhe106]TnI-(104-115) amide. Biochemistry. 1983 Aug 16;22(17):4145–4152. doi: 10.1021/bi00286a024. [DOI] [PubMed] [Google Scholar]
  3. Cheung H. C., Wang C. K., Garland F. Fluorescence energy transfer studies of skeletal troponin C proximity between methionine-25 and cysteine-98. Biochemistry. 1982 Oct 12;21(21):5135–5142. doi: 10.1021/bi00264a005. [DOI] [PubMed] [Google Scholar]
  4. Chong P. C., Hodges R. S. Proximity of sulfhydryl groups to the sites of interaction between components of the troponin complex from rabbit skeletal muscle. J Biol Chem. 1982 Mar 10;257(5):2549–2555. [PubMed] [Google Scholar]
  5. Chothia C., Janin J. Principles of protein-protein recognition. Nature. 1975 Aug 28;256(5520):705–708. doi: 10.1038/256705a0. [DOI] [PubMed] [Google Scholar]
  6. ELLMAN G. L. A colorimetric method for determining low concentrations of mercaptans. Arch Biochem Biophys. 1958 Apr;74(2):443–450. doi: 10.1016/0003-9861(58)90014-6. [DOI] [PubMed] [Google Scholar]
  7. Gariépy J., Sykes B. D., Reid R. E., Hodges R. S. Proton nuclear magnetic resonance investigation of synthetic calcium-binding peptides. Biochemistry. 1982 Mar 30;21(7):1506–1512. doi: 10.1021/bi00536a007. [DOI] [PubMed] [Google Scholar]
  8. Grabarek Z., Drabikowski W., Leavis P. C., Rosenfeld S. S., Gergely J. Proteolytic fragments of troponin C. Interactions with the other troponin subunits and biological activity. J Biol Chem. 1981 Dec 25;256(24):13121–13127. [PubMed] [Google Scholar]
  9. Grabarek Z., Grabarek J., Leavis P. C., Gergely J. Cooperative binding to the Ca2+-specific sites of troponin C in regulated actin and actomyosin. J Biol Chem. 1983 Dec 10;258(23):14098–14102. [PubMed] [Google Scholar]
  10. Grand R. J., Levine B. A., Perry S. V. Proton-magnetic-resonance studies on the interaction of rabbit skeletal-muscle troponin I with troponin C and actin. Biochem J. 1982 Apr 1;203(1):61–68. doi: 10.1042/bj2030061. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Iio T., Kondo H. Fluorescence titration and fluorescence stopped-flow studies on skeletal troponin C labeled with fluorescent maleimide reagent or dansylaziridine. J Biochem. 1981 Jul;90(1):163–175. doi: 10.1093/oxfordjournals.jbchem.a133446. [DOI] [PubMed] [Google Scholar]
  12. Janin J., Chothia C. Role of hydrophobicity in the binding of coenzymes. Appendix. Translational and rotational contribution to the free energy of dissociation. Biochemistry. 1978 Jul 25;17(15):2943–2948. doi: 10.1021/bi00608a001. [DOI] [PubMed] [Google Scholar]
  13. Johnson J. D., Charlton S. C., Potter J. D. A fluorescence stopped flow analysis of Ca2+ exchange with troponin C. J Biol Chem. 1979 May 10;254(9):3497–3502. [PubMed] [Google Scholar]
  14. KAUZMANN W. Some factors in the interpretation of protein denaturation. Adv Protein Chem. 1959;14:1–63. doi: 10.1016/s0065-3233(08)60608-7. [DOI] [PubMed] [Google Scholar]
  15. Leavis P. C., Rosenfeld S. S., Gergely J., Grabarek Z., Drabikowski W. Proteolytic fragments of troponin C. Localization of high and low affinity Ca2+ binding sites and interactions with troponin I and troponin T. J Biol Chem. 1978 Aug 10;253(15):5452–5459. [PubMed] [Google Scholar]
  16. Levine B. A., Coffman D. M., Thornton J. M. Calcium binding by troponin-C. A proton magnetic resonance study. J Mol Biol. 1977 Oct 5;115(4):743–760. doi: 10.1016/0022-2836(77)90113-9. [DOI] [PubMed] [Google Scholar]
  17. Murray A. C., Kay C. M. Hydrodynamic and optical properties of troponin A. Demonstration of a conformational change upon binding calcium ion. Biochemistry. 1972 Jul 4;11(14):2622–2627. doi: 10.1021/bi00764a012. [DOI] [PubMed] [Google Scholar]
  18. Nagy B., Potter J. D., Gergely J. Calcium-induced conformational changes in a cyanogen bromide fragment of troponin C that contains one of the binding sites. J Biol Chem. 1978 Sep 10;253(17):5971–5974. [PubMed] [Google Scholar]
  19. Perry S. V., Cole H. A. Phosphorylation of troponin and the effects of interactions between the components of the complex. Biochem J. 1974 Sep;141(3):733–743. doi: 10.1042/bj1410733. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Perry S. V. The regulation of contractile activity in muscle. Biochem Soc Trans. 1979 Aug;7(4):593–617. doi: 10.1042/bst0070593. [DOI] [PubMed] [Google Scholar]
  21. Potter J. D., Gergely J. The calcium and magnesium binding sites on troponin and their role in the regulation of myofibrillar adenosine triphosphatase. J Biol Chem. 1975 Jun 25;250(12):4628–4633. [PubMed] [Google Scholar]
  22. Reid R. E., Gariépy J., Saund A. K., Hodges R. S. Calcium-induced protein folding. Structure-affinity relationships in synthetic analogs of the helix-loop-helix calcium binding unit. J Biol Chem. 1981 Mar 25;256(6):2742–2751. [PubMed] [Google Scholar]
  23. Talbot J. A., Hodges R. S. Synthetic studies on the inhibitory region of rabbit skeletal troponin I. Relationship of amino acid sequence to biological activity. J Biol Chem. 1981 Mar 25;256(6):2798–2802. [PubMed] [Google Scholar]
  24. Weber G. Energetics of ligand binding to proteins. Adv Protein Chem. 1975;29:1–83. doi: 10.1016/s0065-3233(08)60410-6. [DOI] [PubMed] [Google Scholar]
  25. Wilkinson J. M. The preparation and properties of the components of troponin B. Biochim Biophys Acta. 1974 Aug 8;359(2):379–388. doi: 10.1016/0005-2795(74)90238-4. [DOI] [PubMed] [Google Scholar]

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