Skip to main content
PMC home page
Protein Science : A Publication of the Protein Society logoLink to Protein Science : A Publication of the Protein Society
. 1997 Apr;6(4):916–918. doi: 10.1002/pro.5560060420

Production, crystallization, and preliminary X-ray analysis of rabbit skeletal muscle troponin complex consisting of troponin C and fragment (1-47) of troponin I.

Y Saijo 1, S Takeda 1, A Scherer 1, T Kobayashi 1, Y Maéda 1, H Taniguchi 1, M Yao 1, S Wakatsuki 1
PMCID: PMC2144766  PMID: 9098903

Abstract

Troponin is a ternary protein complex consisting of subunits TnC. TnI, and TnT, and plays a key role in calcium regulation of the skeletal and cardiac muscle contraction. In the present study, a partial complex (CI47) was prepared from Escherichia coli-expressed rabbit skeletal muscle TnC and fragment 1-47 of TnI, which is obtained by chemical cleavage of an E. coli-expressed mutant of rabbit skeletal muscle TnI. Within the ternary troponin complex, CI47 is thought to form a core that is resistant to proteolytic digestion, and the interaction within CI47 likely maintains the integrity of the troponin complex. Complex CI47 was crystallized in the presence of sodium citrate. The addition of trehalose improved the diffraction pattern of the crystals substantially. The crystal lattice belongs to the space group P3(1)(2)21, with unit cell dimensions a = b = 48.2 A, c = 162 A. The asymmetric unit presumably contains one CI47 complex. Soaking with p-chloromercuribenzenesulfonate (PCMBS) resulted in loss of isomorphism, but enhanced the quality of the crystals. The crystals diffracted up to 2.3 A resolution, with completeness of 91% and R(merge) = 6.4%. The crystals of PCMBS-derivative should be suitable for X-ray studies using the multiple-wavelength anomalous diffraction technique. This is the first step for elucidating the structure of the full troponin complex.

Full Text

The Full Text of this article is available as a PDF (320.2 KB).

Selected References

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

  1. Ebashi S., Endo M. Calcium ion and muscle contraction. Prog Biophys Mol Biol. 1968;18:123–183. doi: 10.1016/0079-6107(68)90023-0. [DOI] [PubMed] [Google Scholar]
  2. Farah C. S., Miyamoto C. A., Ramos C. H., da Silva A. C., Quaggio R. B., Fujimori K., Smillie L. B., Reinach F. C. Structural and regulatory functions of the NH2- and COOH-terminal regions of skeletal muscle troponin I. J Biol Chem. 1994 Feb 18;269(7):5230–5240. [PubMed] [Google Scholar]
  3. Fujita-Becker S., Kluwe L., Miegel A., Maeda K., Maéda Y. Reconstitution of rabbit skeletal muscle troponin from the recombinant subunits all expressed in and purified from E. coli. J Biochem. 1993 Sep;114(3):438–444. doi: 10.1093/oxfordjournals.jbchem.a124194. [DOI] [PubMed] [Google Scholar]
  4. Fujita S., Maéda K., Maéda Y. Complete coding sequences of cDNAs of four variants of rabbit skeletal muscle troponin T. J Muscle Res Cell Motil. 1991 Dec;12(6):560–565. doi: 10.1007/BF01738444. [DOI] [PubMed] [Google Scholar]
  5. 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]
  6. Kluwe L., Maeda K., Maéda Y. E. coli expression and characterization of a mutant troponin I with the three cysteine residues substituted. FEBS Lett. 1993 May 24;323(1-2):83–88. doi: 10.1016/0014-5793(93)81453-7. [DOI] [PubMed] [Google Scholar]
  7. Kobayashi T., Tao T., Gergely J., Collins J. H. Structure of the troponin complex. Implications of photocross-linking of troponin I to troponin C thiol mutants. J Biol Chem. 1994 Feb 25;269(8):5725–5729. [PubMed] [Google Scholar]
  8. Krudy G. A., Kleerekoper Q., Guo X., Howarth J. W., Solaro R. J., Rosevear P. R. NMR studies delineating spatial relationships within the cardiac troponin I-troponin C complex. J Biol Chem. 1994 Sep 23;269(38):23731–23735. [PubMed] [Google Scholar]
  9. Lehrer S. S. The regulatory switch of the muscle thin filament: Ca2+ or myosin heads? J Muscle Res Cell Motil. 1994 Jun;15(3):232–236. doi: 10.1007/BF00123476. [DOI] [PubMed] [Google Scholar]
  10. Lischwe M. A., Newton R. C., Huang J. J., Yates R. A., Breth L. A., Larsen B. S. Escherichia coli: derived murine interleukin-1 beta with N-terminus partially N alpha-acetylated. Protein Expr Purif. 1993 Oct;4(5):499–502. doi: 10.1006/prep.1993.1065. [DOI] [PubMed] [Google Scholar]
  11. Ngai S. M., Hodges R. S. Biologically important interactions between synthetic peptides of the N-terminal region of troponin I and troponin C. J Biol Chem. 1992 Aug 5;267(22):15715–15720. [PubMed] [Google Scholar]
  12. Schick B., Jurnak F. Extension of the diffraction resolution of crystals. Acta Crystallogr D Biol Crystallogr. 1994 Jul 1;50(Pt 4):563–568. doi: 10.1107/S0907444994001976. [DOI] [PubMed] [Google Scholar]
  13. Sheng Z., Pan B. S., Miller T. E., Potter J. D. Isolation, expression, and mutation of a rabbit skeletal muscle cDNA clone for troponin I. The role of the NH2 terminus of fast skeletal muscle troponin I in its biological activity. J Biol Chem. 1992 Dec 15;267(35):25407–25413. [PubMed] [Google Scholar]
  14. Slupsky C. M., Sykes B. D. NMR solution structure of calcium-saturated skeletal muscle troponin C. Biochemistry. 1995 Dec 12;34(49):15953–15964. doi: 10.1021/bi00049a010. [DOI] [PubMed] [Google Scholar]
  15. Smillie L. B., Golosinska K., Reinach F. C. Sequences of complete cDNAs encoding four variants of chicken skeletal muscle troponin T. J Biol Chem. 1988 Dec 15;263(35):18816–18820. [PubMed] [Google Scholar]
  16. Swenson C. A., Fredricksen R. S. Interaction of troponin C and troponin C fragments with troponin I and the troponin I inhibitory peptide. Biochemistry. 1992 Apr 7;31(13):3420–3429. doi: 10.1021/bi00128a017. [DOI] [PubMed] [Google Scholar]
  17. Syska H., Wilkinson J. M., Grand R. J., Perry S. V. The relationship between biological activity and primary structure of troponin I from white skeletal muscle of the rabbit. Biochem J. 1976 Feb 1;153(2):375–387. doi: 10.1042/bj1530375. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Taniguchi H., Suzuki M., Manenti S., Titani K. A mass spectrometric study on the in vivo posttranslational modification of GAP-43. J Biol Chem. 1994 Sep 9;269(36):22481–22484. [PubMed] [Google Scholar]

Articles from Protein Science : A Publication of the Protein Society are provided here courtesy of The Protein Society

RESOURCES