Skip to main content
NCBI home page
Biophysical Journal logoLink to Biophysical Journal
. 2000 Feb;78(2):908–917. doi: 10.1016/S0006-3495(00)76648-3

Three-dimensional reconstruction of thin filaments containing mutant tropomyosin.

M Rosol 1, W Lehman 1, R Craig 1, C Landis 1, C Butters 1, L S Tobacman 1
PMCID: PMC1300693  PMID: 10653803

Abstract

Interactions of the components of reconstituted thin filaments were investigated using a tropomyosin internal deletion mutant, D234, in which actin-binding pseudo-repeats 2, 3, and 4 are missing. D234 retains regions of tropomyosin that bind troponin and form end-to-end tropomyosin bonds, but has a length to span only four instead of seven actin monomers. It inhibits acto-myosin subfragment 1 ATPase (acto-S-1 ATPase) and filament sliding in vitro in both the presence and absence of Ca(2+) (, J. Biol. Chem. 272:14051-14056) and lowers the affinity of S-1.ADP for actin while increasing its cooperative binding. Electron microscopy and three-dimensional reconstruction of reconstituted thin filaments containing actin, troponin, and wild-type or D234 tropomyosin were carried out to determine if Ca(2+)-induced movement of D234 occurred in the filaments. In the presence and absence of Ca(2+), the D234 position was indistinguishable from that of the wild-type tropomyosin, demonstrating that the mutation did not affect normal tropomyosin movement induced by Ca(2+) and troponin. These results suggested that, in the presence of Ca(2+) and troponin, D234 tropomyosin was trapped on filaments in the Ca(2+)-induced position and was unable to undergo a transition to a completely activated position. By adding small amounts of rigor-bonded N-ethyl-maleimide-treated S-1 to mutant thin filaments, thus mimicking the myosin-induced "open" state, inhibition could be overcome and full activation restored. This myosin requirement for full activation provides support for the existence of three functionally distinct thin filament states (off, Ca(2+)-induced, myosin-induced; cf.;, J. Mol. Biol. 266:8-14). We propose a further refinement of the three-state model in which the binding of myosin to actin causes allosteric changes in actin that promote the binding of tropomyosin in an otherwise energetically unfavorable "open" state.

Full Text

The Full Text of this article is available as a PDF (474.8 KB).

Selected References

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

  1. Amos L. A., Klug A. Three-dimensional image reconstructions of the contractile tail of T4 bacteriophage. J Mol Biol. 1975 Nov 25;99(1):51–64. doi: 10.1016/s0022-2836(75)80158-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Cassell M., Tobacman L. S. Opposite effects of myosin subfragment 1 on binding of cardiac troponin and tropomyosin to the thin filament. J Biol Chem. 1996 May 31;271(22):12867–12872. doi: 10.1074/jbc.271.22.12867. [DOI] [PubMed] [Google Scholar]
  3. Coates J. H., Criddle A. H., Geeves M. A. Pressure-relaxation studies of pyrene-labelled actin and myosin subfragment 1 from rabbit skeletal muscle. Evidence for two states of acto-subfragment 1. Biochem J. 1985 Dec 1;232(2):351–356. doi: 10.1042/bj2320351. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Cohen I., Cohen C. A tropomyosin-like protein from human platelets. J Mol Biol. 1972 Jul 21;68(2):383–387. doi: 10.1016/0022-2836(72)90220-3. [DOI] [PubMed] [Google Scholar]
  5. Criddle A. H., Geeves M. A., Jeffries T. The use of actin labelled with N-(1-pyrenyl)iodoacetamide to study the interaction of actin with myosin subfragments and troponin/tropomyosin. Biochem J. 1985 Dec 1;232(2):343–349. doi: 10.1042/bj2320343. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. DeRosier D. J., Moore P. B. Reconstruction of three-dimensional images from electron micrographs of structures with helical symmetry. J Mol Biol. 1970 Sep 14;52(2):355–369. doi: 10.1016/0022-2836(70)90036-7. [DOI] [PubMed] [Google Scholar]
  7. Eaton B. L. Tropomyosin binding to F-actin induced by myosin heads. Science. 1976 Jun 25;192(4246):1337–1339. doi: 10.1126/science.131972. [DOI] [PubMed] [Google Scholar]
  8. Egelman E. H. An algorithm for straightening images of curved filamentous structures. Ultramicroscopy. 1986;19(4):367–373. doi: 10.1016/0304-3991(86)90096-3. [DOI] [PubMed] [Google Scholar]
  9. Flicker P. F., Phillips G. N., Jr, Cohen C. Troponin and its interactions with tropomyosin. An electron microscope study. J Mol Biol. 1982 Dec 5;162(2):495–501. doi: 10.1016/0022-2836(82)90540-x. [DOI] [PubMed] [Google Scholar]
  10. Geeves M. A., Halsall D. J. The dynamics of the interaction between myosin subfragment 1 and pyrene-labelled thin filaments, from rabbit skeletal muscle. Proc R Soc Lond B Biol Sci. 1986 Oct 22;229(1254):85–95. doi: 10.1098/rspb.1986.0076. [DOI] [PubMed] [Google Scholar]
  11. Hill L. E., Mehegan J. P., Butters C. A., Tobacman L. S. Analysis of troponin-tropomyosin binding to actin. Troponin does not promote interactions between tropomyosin molecules. J Biol Chem. 1992 Aug 15;267(23):16106–16113. [PubMed] [Google Scholar]
  12. Hill T. L., Eisenberg E., Greene L. Theoretical model for the cooperative equilibrium binding of myosin subfragment 1 to the actin-troponin-tropomyosin complex. Proc Natl Acad Sci U S A. 1980 Jun;77(6):3186–3190. doi: 10.1073/pnas.77.6.3186. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Hitchcock-DeGregori S. E., An Y. Integral repeats and a continuous coiled coil are required for binding of striated muscle tropomyosin to the regulated actin filament. J Biol Chem. 1996 Feb 16;271(7):3600–3603. doi: 10.1074/jbc.271.7.3600. [DOI] [PubMed] [Google Scholar]
  14. Hitchcock-DeGregori S. E., Varnell T. A. Tropomyosin has discrete actin-binding sites with sevenfold and fourteenfold periodicities. J Mol Biol. 1990 Aug 20;214(4):885–896. doi: 10.1016/0022-2836(90)90343-K. [DOI] [PubMed] [Google Scholar]
  15. Jones T. A., Zou J. Y., Cowan S. W., Kjeldgaard M. Improved methods for building protein models in electron density maps and the location of errors in these models. Acta Crystallogr A. 1991 Mar 1;47(Pt 2):110–119. doi: 10.1107/s0108767390010224. [DOI] [PubMed] [Google Scholar]
  16. Landis C. A., Bobkova A., Homsher E., Tobacman L. S. The active state of the thin filament is destabilized by an internal deletion in tropomyosin. J Biol Chem. 1997 May 30;272(22):14051–14056. doi: 10.1074/jbc.272.22.14051. [DOI] [PubMed] [Google Scholar]
  17. Landis C., Back N., Homsher E., Tobacman L. S. Effects of tropomyosin internal deletions on thin filament function. J Biol Chem. 1999 Oct 29;274(44):31279–31285. doi: 10.1074/jbc.274.44.31279. [DOI] [PubMed] [Google Scholar]
  18. Lehman W., Craig R., Vibert P. Ca(2+)-induced tropomyosin movement in Limulus thin filaments revealed by three-dimensional reconstruction. Nature. 1994 Mar 3;368(6466):65–67. doi: 10.1038/368065a0. [DOI] [PubMed] [Google Scholar]
  19. Lehman W., Vibert P., Uman P., Craig R. Steric-blocking by tropomyosin visualized in relaxed vertebrate muscle thin filaments. J Mol Biol. 1995 Aug 11;251(2):191–196. doi: 10.1006/jmbi.1995.0425. [DOI] [PubMed] [Google Scholar]
  20. Lehrer S. S., Geeves M. A. The muscle thin filament as a classical cooperative/allosteric regulatory system. J Mol Biol. 1998 Apr 17;277(5):1081–1089. doi: 10.1006/jmbi.1998.1654. [DOI] [PubMed] [Google Scholar]
  21. Lehrer S. S., Morris E. P. Dual effects of tropomyosin and troponin-tropomyosin on actomyosin subfragment 1 ATPase. J Biol Chem. 1982 Jul 25;257(14):8073–8080. [PubMed] [Google Scholar]
  22. 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]
  23. Lin D., Bobkova A., Homsher E., Tobacman L. S. Altered cardiac troponin T in vitro function in the presence of a mutation implicated in familial hypertrophic cardiomyopathy. J Clin Invest. 1996 Jun 15;97(12):2842–2848. doi: 10.1172/JCI118740. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Lorenz M., Poole K. J., Popp D., Rosenbaum G., Holmes K. C. An atomic model of the unregulated thin filament obtained by X-ray fiber diffraction on oriented actin-tropomyosin gels. J Mol Biol. 1995 Feb 10;246(1):108–119. doi: 10.1006/jmbi.1994.0070. [DOI] [PubMed] [Google Scholar]
  25. Lorenz M., Popp D., Holmes K. C. Refinement of the F-actin model against X-ray fiber diffraction data by the use of a directed mutation algorithm. J Mol Biol. 1993 Dec 5;234(3):826–836. doi: 10.1006/jmbi.1993.1628. [DOI] [PubMed] [Google Scholar]
  26. McGough A., Way M. Molecular model of an actin filament capped by a severing protein. J Struct Biol. 1995 Sep-Oct;115(2):144–150. doi: 10.1006/jsbi.1995.1038. [DOI] [PubMed] [Google Scholar]
  27. McKillop D. F., Geeves M. A. Regulation of the interaction between actin and myosin subfragment 1: evidence for three states of the thin filament. Biophys J. 1993 Aug;65(2):693–701. doi: 10.1016/S0006-3495(93)81110-X. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. McLachlan A. D., Stewart M. The 14-fold periodicity in alpha-tropomyosin and the interaction with actin. J Mol Biol. 1976 May 15;103(2):271–298. doi: 10.1016/0022-2836(76)90313-2. [DOI] [PubMed] [Google Scholar]
  29. Milligan R. A., Flicker P. F. Structural relationships of actin, myosin, and tropomyosin revealed by cryo-electron microscopy. J Cell Biol. 1987 Jul;105(1):29–39. doi: 10.1083/jcb.105.1.29. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Milligan R. A., Whittaker M., Safer D. Molecular structure of F-actin and location of surface binding sites. Nature. 1990 Nov 15;348(6298):217–221. doi: 10.1038/348217a0. [DOI] [PubMed] [Google Scholar]
  31. Monteiro P. B., Lataro R. C., Ferro J. A., Reinach F. de C. Functional alpha-tropomyosin produced in Escherichia coli. A dipeptide extension can substitute the amino-terminal acetyl group. J Biol Chem. 1994 Apr 8;269(14):10461–10466. [PubMed] [Google Scholar]
  32. Moody C., Lehman W., Craig R. Caldesmon and the structure of smooth muscle thin filaments: electron microscopy of isolated thin filaments. J Muscle Res Cell Motil. 1990 Apr;11(2):176–185. doi: 10.1007/BF01766496. [DOI] [PubMed] [Google Scholar]
  33. Murray J. M., Knox M. K., Trueblood C. E., Weber A. Potentiated state of the tropomyosin actin filament and nucleotide-containing myosin subfragment 1. Biochemistry. 1982 Mar 2;21(5):906–915. doi: 10.1021/bi00534a015. [DOI] [PubMed] [Google Scholar]
  34. Owen C. H., Morgan D. G., DeRosier D. J. Image analysis of helical objects: the Brandeis Helical Package. J Struct Biol. 1996 Jan-Feb;116(1):167–175. doi: 10.1006/jsbi.1996.0027. [DOI] [PubMed] [Google Scholar]
  35. Parry D. A., Squire J. M. Structural role of tropomyosin in muscle regulation: analysis of the x-ray diffraction patterns from relaxed and contracting muscles. J Mol Biol. 1973 Mar 25;75(1):33–55. doi: 10.1016/0022-2836(73)90527-5. [DOI] [PubMed] [Google Scholar]
  36. Pemrick S., Weber A. Mechanism of inhibition of relaxation by N-ethylmaleimide treatment of myosin. Biochemistry. 1976 Nov 16;15(23):5193–5198. doi: 10.1021/bi00668a038. [DOI] [PubMed] [Google Scholar]
  37. Phillips G. N., Jr, Fillers J. P., Cohen C. Tropomyosin crystal structure and muscle regulation. J Mol Biol. 1986 Nov 5;192(1):111–131. doi: 10.1016/0022-2836(86)90468-7. [DOI] [PubMed] [Google Scholar]
  38. Rayment I., Holden H. M., Whittaker M., Yohn C. B., Lorenz M., Holmes K. C., Milligan R. A. Structure of the actin-myosin complex and its implications for muscle contraction. Science. 1993 Jul 2;261(5117):58–65. doi: 10.1126/science.8316858. [DOI] [PubMed] [Google Scholar]
  39. Spudich J. A., Watt S. The regulation of rabbit skeletal muscle contraction. I. Biochemical studies of the interaction of the tropomyosin-troponin complex with actin and the proteolytic fragments of myosin. J Biol Chem. 1971 Aug 10;246(15):4866–4871. [PubMed] [Google Scholar]
  40. Szczesna D., Borovikov Y. S., Kakol I., Sobieszek A. Interaction of tropomyosin with F-actin-heavy meromyosin complex. Biol Chem Hoppe Seyler. 1989 May;370(5):399–407. doi: 10.1515/bchm3.1989.370.1.399. [DOI] [PubMed] [Google Scholar]
  41. Tobacman L. S., Adelstein R. S. Mechanism of regulation of cardiac actin-myosin subfragment 1 by troponin-tropomyosin. Biochemistry. 1986 Feb 25;25(4):798–802. doi: 10.1021/bi00352a010. [DOI] [PubMed] [Google Scholar]
  42. Trachtenberg S., DeRosier D. J. Three-dimensional structure of the frozen-hydrated flagellar filament. The left-handed filament of Salmonella typhimurium. J Mol Biol. 1987 Jun 5;195(3):581–601. doi: 10.1016/0022-2836(87)90184-7. [DOI] [PubMed] [Google Scholar]
  43. Vibert P., Craig R., Lehman W. Steric-model for activation of muscle thin filaments. J Mol Biol. 1997 Feb 14;266(1):8–14. doi: 10.1006/jmbi.1996.0800. [DOI] [PubMed] [Google Scholar]
  44. Vibert P., Craig R., Lehman W. Three-dimensional reconstruction of caldesmon-containing smooth muscle thin filaments. J Cell Biol. 1993 Oct;123(2):313–321. doi: 10.1083/jcb.123.2.313. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Weber A., Murray J. M. Molecular control mechanisms in muscle contraction. Physiol Rev. 1973 Jul;53(3):612–673. doi: 10.1152/physrev.1973.53.3.612. [DOI] [PubMed] [Google Scholar]
  46. Weeds A. G., Taylor R. S. Separation of subfragment-1 isoenzymes from rabbit skeletal muscle myosin. Nature. 1975 Sep 4;257(5521):54–56. doi: 10.1038/257054a0. [DOI] [PubMed] [Google Scholar]
  47. Willadsen K. A., Butters C. A., Hill L. E., Tobacman L. S. Effects of the amino-terminal regions of tropomyosin and troponin T on thin filament assembly. J Biol Chem. 1992 Nov 25;267(33):23746–23752. [PubMed] [Google Scholar]
  48. Williams D. L., Jr, Greene L. E. Comparison of the effects of tropomyosin and troponin-tropomyosin on the binding of myosin subfragment 1 to actin. Biochemistry. 1983 May 24;22(11):2770–2774. doi: 10.1021/bi00280a027. [DOI] [PubMed] [Google Scholar]
  49. Williams D. L., Jr, Greene L. E., Eisenberg E. Cooperative turning on of myosin subfragment 1 adenosinetriphosphatase activity by the troponin-tropomyosin-actin complex. Biochemistry. 1988 Sep 6;27(18):6987–6993. doi: 10.1021/bi00418a048. [DOI] [PubMed] [Google Scholar]
  50. Xu C., Craig R., Tobacman L., Horowitz R., Lehman W. Tropomyosin positions in regulated thin filaments revealed by cryoelectron microscopy. Biophys J. 1999 Aug;77(2):985–992. doi: 10.1016/S0006-3495(99)76949-3. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Biophysical Journal are provided here courtesy of The Biophysical Society

RESOURCES