Calcium Structural Transition of Troponin in the Complexes, on the Thin Filament, and in Muscle Fibres, as Studied By Site-Directed Spin-Labelling EPR

  • Toshiaki Arata
  • Tomoki Aihara
  • Keisuke Ueda
  • Motoyoshi Nakamura
  • Shoji Ueki
Part of the Advances in Experimental Medicine and Biology book series (AEMB, volume 592)

12.1. Abstract

We have measured the intersite distance, side-chain mobility and orientation of specific site(s) of troponin (Tn) complex on the thin filaments or in muscle fibres as well as in solution by means of site-directed spin labeling electron paramagnetic resonance (SDSL-EPR). We have examined the Ca2+-induced movement of the B and C helices relative to the D helix in a human cardiac (hc)TnC monomer state and hcTnC-hcTnI binary complex. An interspin distance between G42C (B helix) and C84 (D helix) was 18.4 Å in the absence of Ca2+. The distance between Q58C (C helix) and C84 (D helix) was 18.3 Å. Distance changes were observed by the addition of Ca2+ and by the formation of a complex with TnI. Both Ca2+ and TnI are essential for the full opening ∼3 Å of the N-domain in cardiac TnC.

We have determined the in situ distances between C35 and C84 by measuring pulsed electron-electron double resonance (PELDOR) spectroscopy. The distances were 26.0 and 27.2 Å in the monomer state and in reconstituted fibres, respectively. The addition of Ca2+ decreased the distance to 23.2 Å in fibres but only slightly in the monomer state, indicating that Ca2+ binding to the N-lobe of hcTnC induced a larger structural change in muscle fibres than in the monomer state.

We also succeeded in synthesizing a new bifunctional spin labels that is firmly fixed on a central E-helix (94C–101C) of skeletal(sk)TnC to examine its orientation in reconstituted muscle fibres. EPR spectrum showed that this helix is disordered with respect to the filament axis.

We have studied the calcium structural transition in skTnI and tropomyosin on the filament by SDSL-EPR. The spin label at a TnI switch segment (C133) showed three motional states depending on Ca2+ and actin. The data suggested that the TnI switch segment binds to TnC N-lobe in +Ca2+ state, and that in −Ca2+ state it is free in TnC-I-T complex alone while fixed to actin in the reconstituted thin filaments. In contrast, the side chain spin labels along the entire tropomyosin molecule showed no Ca2+-induced mobility changes.


Electron Paramagnetic Resonance Electron Paramagnetic Resonance Spectrum Spin Label Rotational Correlation Time Monomer State 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


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12.6. References

  1. 1.
    J. Gergely, Molecular switches in troponin, Adv. Exp. Med. Biol. 453, 169–176 (1998).PubMedGoogle Scholar
  2. 2.
    S. Ebashi, and M. Endo, Calcium ion and muscle contraction, Prog. Biophys. Mol. Biol. 18, 123–183 (1968).PubMedCrossRefGoogle Scholar
  3. 3.
    S. S. Lehrer, and M. A. Geeves, The muscle thin filament as a classical cooperative/allosteric regulatory system, J. Mol. Biol. 277, 1081–1089 (1998).PubMedCrossRefGoogle Scholar
  4. 4.
    A. S. Zot, and J. D. Potter, Reciprocal coupling between troponin C and myosin crossbridge attachment, Annu. Rev. Biophys. Biophys. Chem. 16, 535–559 (1987).PubMedCrossRefGoogle Scholar
  5. 5.
    T. Tao, B. J. Gong, and P. C. Leavis, Calcium-induced movement of troponin-I relative to actin in skeletal muscle thin filaments, Science 247, 1339–1341 (1990).PubMedCrossRefGoogle Scholar
  6. 6.
    S. V. Perry, Troponin I: inhibitor or facilitator, Mol. Cell. Biochem. 190, 9–32 (1999).PubMedCrossRefGoogle Scholar
  7. 7.
    O. Herzberg, and M. N. James, Refined crystal structure of troponin C from turkey skeletal muscle at 2.0 A resolution, J. Mol. Biol. 203, 761–779 (1988).PubMedCrossRefGoogle Scholar
  8. 8.
    D. G. Vassylyev, S. Takeda, S. Wakatsuki, K. Maeda, and Y. Maeda, Crystal structure of troponin C in complex with troponin I fragment at 2.3 Å-resolution, Proc. Natl. Acad. Sci. USA 95, 4849–4852 (1998).CrossRefGoogle Scholar
  9. 9.
    S. M. Gagne, S. Tsuda, M. X. Li, L. B. Smillie, and B. D. Sykes, Structures of the troponin C regulatory domains in the apo and calcium-saturated states, Nat. Struct. Biol. 2, 784–789 (1995).PubMedCrossRefGoogle Scholar
  10. 10.
    C. M. Slupsky, and B. D. Sykes, Solution secondary structure of calcium-saturated troponin C monomer determined by multidimensional heteronuclear NMR spectroscopy, Biochemistry 34, 15953–15964 (1995).PubMedCrossRefGoogle Scholar
  11. 11.
    L. Spyracopoulos, M. X. Li, S. K. Sia, S. M. Gagne, M. Chandra, R. J. Solaro, and B. D. Sykes, Calciuminduced structural transition in the regulatory domain of human cardiac troponin C, Biochemistry 36, 12138–12146 (1997).PubMedCrossRefGoogle Scholar
  12. 12.
    R. T. McKay, J. R. Pearlstone, D. C. Corson, S. M. Gagne, L. B. Smillie, and B. D. Sykes, Structure and interaction site of the regulatory domain of troponin C when complexed with the 96–148 region of troponin-I, Biochemistry 37, 12419–12130 (1998).PubMedCrossRefGoogle Scholar
  13. 13.
    M. X. Li, L. Spyracopoulos, and B. D. Sykes, Binding of cardiac troponin-I 147–163 induces a structural opening in human cardiac troponin C, Biochemistry 38, 8289–8298 (1999).PubMedCrossRefGoogle Scholar
  14. 14.
    X. Wang, M. X. Li, and B. D. Sykes, Structure of the regulatory N-domain of human cardiac troponin C in complex with human cardiac troponin I 147–163 and bepridil, J. Biol. Chem. 277, 31124–31133 (2002).PubMedCrossRefGoogle Scholar
  15. 15.
    S. K. Sia, M. X. Li, L. Spyracopoulos, S. M. Gagne, W. Liu, J. A. Putkey, and B. D. Sykes, Structure of cardiac muscle troponin C unexpectedly reveals a closed regulatory domain, J. Biol. Chem. 272, 18216–18221 (1997).PubMedCrossRefGoogle Scholar
  16. 16.
    W. J. Dong, J. Xing, M. Villain, M. Hellinger, J. M. Robinson, M. Chandra, R. J. Solaro, P. K. Umeda, and H. C. Cheung, Conformation of the regulatory domain of cardiac muscle troponin C in its complex with cardiac troponin I, J. Biol. Chem. 274, 31382–31390 (1999).PubMedCrossRefGoogle Scholar
  17. 17.
    S. Takeda, A. Yamashita, K. Maeda, and Y. Maeda, Structure of the core domain of human cardiac troponin in the Ca2+-saturated form, Nature 424, 35–41 (2003).PubMedCrossRefGoogle Scholar
  18. 18.
    M. V. Vinogradova, D. B. Stone, G. G. Malanina, C. Karatzaferi, R. Cooke, R. A. Mendelson, R. J. Fletterick, Ca2+-regulated structural chanS. ges in troponin, Proc. Natl. Acad. Sci. USA 102, 5038–5043 (2005).PubMedCrossRefGoogle Scholar
  19. 19.
    W. J. Dong, J. M. Robinson, S. Stagg, J. Xing, and H. C. Cheung, Ca2+-induced conformational transition in the inhibitory and regulatory regions of cardiac troponin I, J. Biol. Chem. 278, 8686–8692 (2003).PubMedCrossRefGoogle Scholar
  20. 20.
    M. Miki, T. Kobayashi, H. Kimura, A. Hagiwara, H. Hai, and Y. Maeda, Ca2+-induced distance change between points on actin and troponin in skeletal muscle in filaments estimated by fluorescence energy transfer spectroscopy, J. Biochem. 123, 324–331 (1998).PubMedGoogle Scholar
  21. 21.
    R. Craig, and W. Lehman, Crossbridge and tropomyosin positions observed in native, interacting thick and thin filaments, J. Mol. Biol. 311, 1027–1036 (2001).PubMedCrossRefGoogle Scholar
  22. 22.
    A. Narita, T. Yasunaga, T. Ishikawa, K. Mayanagi, and T. Wakabayashi, Ca2+-induced switching of troponin and tropomyosin on actin filaments as revealed by electron cryo-microscopy, J. Mol. Biol. 308, 241–261 (2001).PubMedCrossRefGoogle Scholar
  23. 23.
    C. Bacchiocchi, and S. S. Lehrer, Ca2+-induced movement of tropomyosin in skeletal muscle thin filaments observed by multi-site FRET, Biophys. J. 82, 1524–1536 (2002).PubMedGoogle Scholar
  24. 24.
    M. Miki, H. Hai, K. Saeki, Y. Shitaka, K. Sano, Y. Maeda, and T. Wakabayashi, Fluorescence resonance energy transfer between points on actin and the C-Terminal region of tropomyosin in skeletal muscle thin filaments, J. Biochem. 136, 39–47 (2004).PubMedCrossRefGoogle Scholar
  25. 25.
    Y. Tonomura, S. Watanabe, and M. Morales, Conformational changes in the molecular control of muscle contraction, Biochemistry 8, 2171–2176 (1969).PubMedCrossRefGoogle Scholar
  26. 26.
    S. Ebashi, S. Onishi, S. Abe, and K. Maruyama, A spin-label study on calcium-induced conformational changes of troponin components, J. Biochem. 75, 211–213 (1974).PubMedGoogle Scholar
  27. 27.
    T. Arata, and H. Shimizu, Spin-label study of actin-myosin-nucleotide interactions in contracting glycerinated muscle fibers. J. Mol. Biol. 151, 411–437 (1981).PubMedCrossRefGoogle Scholar
  28. 28.
    V. A. Barnett, and D. D. Thomas, Resolution of conformational states of spin-labeled myosin during steady-state ATP hydrolysis. Biochemistry 26, 314–323 (1987).PubMedCrossRefGoogle Scholar
  29. 29.
    K. Sugata, M. Nakamura, S. Ueki, P. G. Fajer, and T. Arata, ESR reveals the mobility of the neck linker in dimeric kinesin, Biochem. Biophys. Res. Commun. 314, 447–451 (2004).PubMedCrossRefGoogle Scholar
  30. 30.
    J. D. Potter, J. C. Seidel, P. Leavis, S. S. Lehrer, and J. Gergely, Effect of Ca2+ binding on troponin C. Changes in spin label mobility, extrinsic fluorescence, and sulfhydryl reactivity, J. Biol. Chem. 251, 7551–7556 (1976).PubMedGoogle Scholar
  31. 31.
    H. C. Li, K. Hideg, and P. G. Fajer, The mobility of troponin C and troponin I in muscle, J. Mol. Recognit. 10, 194–201 (1997).PubMedCrossRefGoogle Scholar
  32. 32.
    D. D. Thomas, and R. Cooke, Orientation of spin-labeled myosin heads in glycerinated muscle fibers, Biophys. J. 32, 891–906 (1980).PubMedCrossRefGoogle Scholar
  33. 33.
    T. Arata, Orientation of spin-labeled light chain 2 of myosin heads in muscle fibers, J. Mol. Biol. 214, 471–478 (1990).PubMedCrossRefGoogle Scholar
  34. 34.
    D. Szczesna, and P. G. Fajer, The tropomyosin domain is flexible and disordered in reconstituted thin filaments, Biochemistry 34, 3614–3620 (1995).PubMedCrossRefGoogle Scholar
  35. 35.
    H. C. Li, and P. G. Fajer, Orientational changes of troponin C associated with thin filament activation, Biochemistry 33, 14324–14332 (1994).PubMedCrossRefGoogle Scholar
  36. 36.
    H. C. Li, and P. G. Fajer, Structural coupling of troponin C and actomyosin in muscle fibers, Biochemistry 37, 6628–6635 (1998).PubMedCrossRefGoogle Scholar
  37. 37.
    L. J. Brown, K. L. Sale, R. Hills, C. Rouviere, L. Song, X. Zhang, and P. G. Fajer, Structure of the inhibitory region of troponin by site directed spin labeling electron paramagnetic resonance, Proc. Natl. Acad. Sci. USA 99, 12765–12770 (2002).PubMedCrossRefGoogle Scholar
  38. 38.
    P. P. Borbat, H. S. McHaourab, and J. H. Freed, J. Am. Chem. Soc. 124, 5304–5314 (2002).PubMedCrossRefGoogle Scholar
  39. 39.
    R. L. Moss, G. G. Giulian, and M. L. Greaser, Physiological effects accompanying the removal of myosin LC2 from skinned skeletal muscle fibers, J. Biol. Chem. 257, 8588–8591 (1982).PubMedGoogle Scholar
  40. 40.
    S. Morimoto, and I. Ohtsuki, 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. 101, 291–301 (1987).PubMedGoogle Scholar
  41. 41.
    R. L. Moss, Ca2+ regulation of mechanical properties of striated muscle. Mechanistic studies using extraction and replacement of regulatory proteins, Circulation Research 70, 865–884 (1992).PubMedGoogle Scholar
  42. 42.
    M. Irving, T. St. Claire Allen, C. Sabido-David, J. S. Craik, B. Brandmeier, J. Kendrick-Jones, J. E. Corrie, D. R. Trentham, and Y. E. Goldman, Tilting of the light-chain region of myosin during step length changes and active force generation in skeletal muscle, Nature 375, 688–691 (1995).PubMedCrossRefGoogle Scholar
  43. 43.
    R. E. Ferguson, Y. B. Sun, P. Mercier, A. S. Brack, B. D. Sykes, J. E. Corrie, D. R. Trentham, and M. Irving, In situ orientations of protein domains: troponin C in skeletal muscle fibers, Mol. Cell. 11, 865–874 (2003).PubMedCrossRefGoogle Scholar
  44. 44.
    S. Ueki, M. Nakamura, T. Komori, T. Arata, Site-directed spin labeling electron paramagnetic resonance study of the calcium-induced structural transition in the N-domain of human cardiac troponin C complexed with troponin I, Biochemistry 44, 411–416 (2005).PubMedCrossRefGoogle Scholar
  45. 45.
    M. Nakamura, S. Ueki, H. Hara, and T. Arata, Calcium structural transition of human cardiac troponin C in reconstituted muscle fibres as studied by site-directed spin labelling, J. Mol. Biol. 348, 127–137 (2005).PubMedCrossRefGoogle Scholar
  46. 46.
    S. Chatani, M. Nakamura, H. Akahane, N. Kohyama, M. Taki, T. Arata, and Y. Yamamoto, Synthesis of C 2-chiral bifunctional spin labels and their application to troponin C, Chem. Commun. 1880–1882 (2005).Google Scholar
  47. 47.
    T. Aihara, S. Ueki, M. Nakamura, and T. Arata, Calcium-dependent movement of troponin I between troponin C and actin as revealed by spin-labeling EPR, Biochem. Biophys. Res. Commun. 349, 449–456 (2006).CrossRefGoogle Scholar
  48. 48.
    K. Murakami, F. Yumoto, S. Y. Ohki, T. Yasunaga, M. Tanokura, and T. Wakabayashi, Structural basis for Ca2+-regulated muscle relaxation at interaction sites of troponin with actin and tropomyosin, J. Mol. Biol. 352, 178–201 (2005).PubMedCrossRefGoogle Scholar

Copyright information

© Springer 2007

Authors and Affiliations

  • Toshiaki Arata
    • 1
  • Tomoki Aihara
    • 1
  • Keisuke Ueda
    • 1
  • Motoyoshi Nakamura
    • 1
  • Shoji Ueki
    • 1
  1. 1.Department of Biological Sciences, Graduate School of ScienceOsaka University and CREST/JSTToyonaka, OsakaJapan

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