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. 1992 Apr;61(4):892–901. doi: 10.1016/S0006-3495(92)81896-9

Pyrene-labeled cardiac troponin C. Effect of Ca2+ on monomer and excimer fluorescence in solution and in myofibrils.

Y M Liou 1, F Fuchs 1
PMCID: PMC1260348  PMID: 1581502

Abstract

The two cysteine residues (Cys-35 and Cys-84) of bovine cardiac troponin C (cTnC) were labeled with the pyrene-containing SH-reactive compounds, N-(1-pyrene) maleimide, and N-(1-pyrene)iodoacetamide in order to study conformational changes in the regulatory domain of cTnC associated with cation binding and cross-bridge attachment. The labeled cTnC exhibits the characteristic fluorescence spectrum of pyrene with two sharp monomer fluorescence peaks and one broad excimer fluorescence peak. The excimer fluorescence results from dimerization of adjacent pyrene groups. With metal binding (Mg2+ or Ca2+) to the high affinity sites of cTnC (sites III and IV), there is a small decrease in monomer fluorescence but no effect on excimer fluorescence. In contrast, Ca2+ binding to the low affinity regulatory (site II) site elicits an increase in monomer fluorescence and a reduction in excimer fluorescence. These results can be accounted for by assuming that the pyrene attached to Cys-84 is drawn into a hydrophobic pocket formed by the binding of Ca2+ to site II. When the labeled cTnC is incorporated into the troponin complex or substituted into cardiac myofibrils the monomer fluorescence is enhanced while the excimer fluorescence is reduced. This suggests that the association with other regulatory components in the thin filament might influence the proximity (or mobility) of the two pyrene groups in a way similar to that of Ca2+ binding. With the binding of Ca2+ to site II the excimer fluorescence is further reduced while the monomer fluorescence is not changed significantly. In myofibrils, cross-bridge detachment (5 mM MgATP, pCa 8.0) causes a reduction in monomer fluorescence but has no effect on excimer fluorescence. However, saturation of the cTnC with Ca2+ reduces excimer fluorescence but causes no further change in monomer fluorescence. Thus, the pyrene fluorescence spectra define the different conformations of cTnC associated with weak-binding, cycling, and rigor cross-bridges.

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Selected References

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

  1. Betcher-Lange S. L., Lehrer S. S. Pyrene excimer fluorescence in rabbit skeletal alphaalphatropomyosin labeled with N-(1-pyrene)maleimide. A probe of sulfhydryl proximity and local chain separation. J Biol Chem. 1978 Jun 10;253(11):3757–3760. [PubMed] [Google Scholar]
  2. Blanchard E. M., Solaro R. J. Inhibition of the activation and troponin calcium binding of dog cardiac myofibrils by acidic pH. Circ Res. 1984 Sep;55(3):382–391. doi: 10.1161/01.res.55.3.382. [DOI] [PubMed] [Google Scholar]
  3. Fabiato A., Fabiato F. Calculator programs for computing the composition of the solutions containing multiple metals and ligands used for experiments in skinned muscle cells. J Physiol (Paris) 1979;75(5):463–505. [PubMed] [Google Scholar]
  4. Fuchs F., Liou Y. M., Grabarek Z. The reactivity of sulfhydryl groups of bovine cardiac troponin C. J Biol Chem. 1989 Dec 5;264(34):20344–20349. [PubMed] [Google Scholar]
  5. Fujimori K., Sorenson M., Herzberg O., Moult J., Reinach F. C. Probing the calcium-induced conformational transition of troponin C with site-directed mutants. Nature. 1990 May 10;345(6271):182–184. doi: 10.1038/345182a0. [DOI] [PubMed] [Google Scholar]
  6. Grabarek Z., Tan R. Y., Wang J., Tao T., Gergely J. Inhibition of mutant troponin C activity by an intra-domain disulphide bond. Nature. 1990 May 10;345(6271):132–135. doi: 10.1038/345132a0. [DOI] [PubMed] [Google Scholar]
  7. Güth K., Potter J. D. Effect of rigor and cycling cross-bridges on the structure of troponin C and on the Ca2+ affinity of the Ca2+-specific regulatory sites in skinned rabbit psoas fibers. J Biol Chem. 1987 Oct 5;262(28):13627–13635. [PubMed] [Google Scholar]
  8. Herzberg O., James M. N. Structure of the calcium regulatory muscle protein troponin-C at 2.8 A resolution. Nature. 1985 Feb 21;313(6004):653–659. doi: 10.1038/313653a0. [DOI] [PubMed] [Google Scholar]
  9. Herzberg O., Moult J., James M. N. A model for the Ca2+-induced conformational transition of troponin C. A trigger for muscle contraction. J Biol Chem. 1986 Feb 25;261(6):2638–2644. [PubMed] [Google Scholar]
  10. Hitchcock S. E. Regulation of muscle contraction: bindings of troponin and its components to actin and tropomyosin. Eur J Biochem. 1975 Mar 17;52(2):255–263. doi: 10.1111/j.1432-1033.1975.tb03993.x. [DOI] [PubMed] [Google Scholar]
  11. Hoar P. E., Potter J. D., Kerrick W. G. Skinned ventricular fibres: troponin C extraction is species-dependent and its replacement with skeletal troponin C changes Sr2+ activation properties. J Muscle Res Cell Motil. 1988 Apr;9(2):165–173. doi: 10.1007/BF01773738. [DOI] [PubMed] [Google Scholar]
  12. Hofmann P. A., Fuchs F. Effect of length and cross-bridge attachment on Ca2+ binding to cardiac troponin C. Am J Physiol. 1987 Jul;253(1 Pt 1):C90–C96. doi: 10.1152/ajpcell.1987.253.1.C90. [DOI] [PubMed] [Google Scholar]
  13. Hofmann P. A., Fuchs F. Evidence for a force-dependent component of calcium binding to cardiac troponin C. Am J Physiol. 1987 Oct;253(4 Pt 1):C541–C546. doi: 10.1152/ajpcell.1987.253.4.C541. [DOI] [PubMed] [Google Scholar]
  14. Holroyde M. J., Robertson S. P., Johnson J. D., Solaro R. J., Potter J. D. The calcium and magnesium binding sites on cardiac troponin and their role in the regulation of myofibrillar adenosine triphosphatase. J Biol Chem. 1980 Dec 25;255(24):11688–11693. [PubMed] [Google Scholar]
  15. Ingraham R. H., Hodges R. S. Effects of Ca2+ and subunit interactions on surface accessibility of cysteine residues in cardiac troponin. Biochemistry. 1988 Aug 9;27(16):5891–5898. doi: 10.1021/bi00416a011. [DOI] [PubMed] [Google Scholar]
  16. Ishii Y., Lehrer S. S. Excimer fluorescence of pyrenyliodoacetamide-labeled tropomyosin: a probe of the state of tropomyosin in reconstituted muscle thin filaments. Biochemistry. 1990 Feb 6;29(5):1160–1166. doi: 10.1021/bi00457a010. [DOI] [PubMed] [Google Scholar]
  17. 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]
  18. Johnson J. D., Collins J. H., Robertson S. P., Potter J. D. A fluorescent probe study of Ca2+ binding to the Ca2+-specific sites of cardiac troponin and troponin C. J Biol Chem. 1980 Oct 25;255(20):9635–9640. [PubMed] [Google Scholar]
  19. Leavis P. C., Gergely J. Thin filament proteins and thin filament-linked regulation of vertebrate muscle contraction. CRC Crit Rev Biochem. 1984;16(3):235–305. doi: 10.3109/10409238409108717. [DOI] [PubMed] [Google Scholar]
  20. 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]
  21. Morano I., Rüegg J. C. What does TnCDANZ fluorescence reveal about the thin filament state? Pflugers Arch. 1991 May;418(4):333–337. doi: 10.1007/BF00550870. [DOI] [PubMed] [Google Scholar]
  22. Morimoto S., Ohtsuki I. Ca2+- and Sr2+-sensitivity of the ATPase activity of rabbit skeletal myofibrils: effect of the complete substitution of troponin C with cardiac troponin C, calmodulin, and parvalbumins. J Biochem. 1987 Feb;101(2):291–301. doi: 10.1093/oxfordjournals.jbchem.a121913. [DOI] [PubMed] [Google Scholar]
  23. Morimoto S., Ohtsuki I. Effect of substitution of troponin C in cardiac myofibrils with skeletal troponin C or calmodulin on the Ca2+- and Sr2+-sensitive ATPase activity. J Biochem. 1988 Jul;104(1):149–154. doi: 10.1093/oxfordjournals.jbchem.a122412. [DOI] [PubMed] [Google Scholar]
  24. 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]
  25. Potter J. D., Gergely J. Troponin, tropomyosin, and actin interactions in the Ca2+ regulation of muscle contraction. Biochemistry. 1974 Jun 18;13(13):2697–2703. doi: 10.1021/bi00710a007. [DOI] [PubMed] [Google Scholar]
  26. Potter J. D. Preparation of troponin and its subunits. Methods Enzymol. 1982;85(Pt B):241–263. doi: 10.1016/0076-6879(82)85024-6. [DOI] [PubMed] [Google Scholar]
  27. Putkey J. A., Sweeney H. L., Campbell S. T. Site-directed mutation of the trigger calcium-binding sites in cardiac troponin C. J Biol Chem. 1989 Jul 25;264(21):12370–12378. [PubMed] [Google Scholar]
  28. Smith P. K., Krohn R. I., Hermanson G. T., Mallia A. K., Gartner F. H., Provenzano M. D., Fujimoto E. K., Goeke N. M., Olson B. J., Klenk D. C. Measurement of protein using bicinchoninic acid. Anal Biochem. 1985 Oct;150(1):76–85. doi: 10.1016/0003-2697(85)90442-7. [DOI] [PubMed] [Google Scholar]
  29. Solaro R. J., Pang D. C., Briggs F. N. The purification of cardiac myofibrils with Triton X-100. Biochim Biophys Acta. 1971 Aug 6;245(1):259–262. doi: 10.1016/0005-2728(71)90033-8. [DOI] [PubMed] [Google Scholar]
  30. Tao T., Gong B. J., Leavis P. C. Calcium-induced movement of troponin-I relative to actin in skeletal muscle thin filaments. Science. 1990 Mar 16;247(4948):1339–1341. doi: 10.1126/science.2138356. [DOI] [PubMed] [Google Scholar]
  31. Verin A. D., Gusev N. B. Ca2+-induced conformational changes in cardiac troponin C as measured by N-(1-pyrene)maleimide fluorescence. Biochim Biophys Acta. 1988 Sep 21;956(2):197–208. doi: 10.1016/0167-4838(88)90266-x. [DOI] [PubMed] [Google Scholar]
  32. Wang C. L., Leavis P. C. Distance measurements in cardiac troponin C. Arch Biochem Biophys. 1990 Jan;276(1):236–241. doi: 10.1016/0003-9861(90)90032-t. [DOI] [PubMed] [Google Scholar]
  33. Zehr B. D., Savin T. J., Hall R. E. A one-step, low background coomassie staining procedure for polyacrylamide gels. Anal Biochem. 1989 Oct;182(1):157–159. doi: 10.1016/0003-2697(89)90734-3. [DOI] [PubMed] [Google Scholar]
  34. Zot A. S., Potter J. D. Reciprocal coupling between troponin C and myosin crossbridge attachment. Biochemistry. 1989 Aug 8;28(16):6751–6756. doi: 10.1021/bi00442a031. [DOI] [PubMed] [Google Scholar]
  35. Zot A. S., Potter J. D. Structural aspects of troponin-tropomyosin regulation of skeletal muscle contraction. Annu Rev Biophys Biophys Chem. 1987;16:535–559. doi: 10.1146/annurev.bb.16.060187.002535. [DOI] [PubMed] [Google Scholar]
  36. Zot H. G., Potter J. D. A structural role for the Ca2+-Mg2+ sites on troponin C in the regulation of muscle contraction. Preparation and properties of troponin C depleted myofibrils. J Biol Chem. 1982 Jul 10;257(13):7678–7683. [PubMed] [Google Scholar]
  37. van Eerd J. P., Takahshi K. Determination of the complete amino acid sequence of bovine cardiac troponin C. Biochemistry. 1976 Mar 9;15(5):1171–1180. doi: 10.1021/bi00650a033. [DOI] [PubMed] [Google Scholar]

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