Skip to main content
PMC home page
The EMBO Journal logoLink to The EMBO Journal
. 1994 Apr 15;13(8):1782–1789. doi: 10.1002/j.1460-2075.1994.tb06446.x

Nebulin, a helical actin binding protein.

M Pfuhl 1, S J Winder 1, A Pastore 1
PMCID: PMC395017  PMID: 8168478

Abstract

Nebulin, a giant protein (molecular mass 800 kDa) specific for the skeletal muscle of vertebrates, has been suggested to be involved in the length regulation of the thin filament as a 'molecular ruler'. Despite its size, nebulin appears to be composed mainly of small repeats of approximately 35 amino acids. We have characterized in this study the conformational and functional properties of single repeats. Complete repeats were found to bind to F-actin while a truncated one did not. One repeat is therefore the smallest unit for nebulin--actin interaction. Circular dichroism and nuclear magnetic resonance spectra measured for the peptides in water indicated a transient helical conformation. The folded region is located for them all around the conserved sequence SDxxYK. The helical conformation is strongly stabilized by anionic detergents and trifluoroethanol while uncharged or positively charged detergents have no effect. Since the surface of the actin filament is known to contain clusters of negative charges, anionic detergents may mimic the effect of an actin environment. 3D structures were calculated for three representative peptides in SDS. In vivo, the nebulin helices should form a complex with the actin filament. Based on the assumed importance of charge interactions between nebulin and actin, we propose a model for the structure of the F-actin-nebulin complex in vivo. According to that, two nebulin molecules occupy symmetrical positions along the central cleft of the actin filament bridging the two strands of the actin two-start helix. The consistency of this model with experimental data is discussed.

Full text

PDF
1784

Images in this article

1783Image
on p.1783
1785Image
on p.1785
1787Image
on p.1787

Selected References

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

  1. Brahms S., Brahms J. Determination of protein secondary structure in solution by vacuum ultraviolet circular dichroism. J Mol Biol. 1980 Apr;138(2):149–178. doi: 10.1016/0022-2836(80)90282-x. [DOI] [PubMed] [Google Scholar]
  2. Braun W., Go N. Calculation of protein conformations by proton-proton distance constraints. A new efficient algorithm. J Mol Biol. 1985 Dec 5;186(3):611–626. doi: 10.1016/0022-2836(85)90134-2. [DOI] [PubMed] [Google Scholar]
  3. Chen M. J., Shih C. L., Wang K. Nebulin as an actin zipper. A two-module nebulin fragment promotes actin nucleation and stabilizes actin filaments. J Biol Chem. 1993 Sep 25;268(27):20327–20334. [PubMed] [Google Scholar]
  4. Diamond R. On the multiple simultaneous superposition of molecular structures by rigid body transformations. Protein Sci. 1992 Oct;1(10):1279–1287. doi: 10.1002/pro.5560011006. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Dyson H. J., Merutka G., Waltho J. P., Lerner R. A., Wright P. E. Folding of peptide fragments comprising the complete sequence of proteins. Models for initiation of protein folding. I. Myohemerythrin. J Mol Biol. 1992 Aug 5;226(3):795–817. doi: 10.1016/0022-2836(92)90633-u. [DOI] [PubMed] [Google Scholar]
  6. Dyson H. J., Rance M., Houghten R. A., Lerner R. A., Wright P. E. Folding of immunogenic peptide fragments of proteins in water solution. I. Sequence requirements for the formation of a reverse turn. J Mol Biol. 1988 May 5;201(1):161–200. doi: 10.1016/0022-2836(88)90446-9. [DOI] [PubMed] [Google Scholar]
  7. Ebashi S., Endo M., Otsuki I. Control of muscle contraction. Q Rev Biophys. 1969 Nov;2(4):351–384. doi: 10.1017/s0033583500001190. [DOI] [PubMed] [Google Scholar]
  8. Güntert P., Braun W., Wüthrich K. Efficient computation of three-dimensional protein structures in solution from nuclear magnetic resonance data using the program DIANA and the supporting programs CALIBA, HABAS and GLOMSA. J Mol Biol. 1991 Feb 5;217(3):517–530. doi: 10.1016/0022-2836(91)90754-t. [DOI] [PubMed] [Google Scholar]
  9. Holmes K. C., Popp D., Gebhard W., Kabsch W. Atomic model of the actin filament. Nature. 1990 Sep 6;347(6288):44–49. doi: 10.1038/347044a0. [DOI] [PubMed] [Google Scholar]
  10. Jin J. P., Wang K. Nebulin as a giant actin-binding template protein in skeletal muscle sarcomere. Interaction of actin and cloned human nebulin fragments. FEBS Lett. 1991 Apr 9;281(1-2):93–96. doi: 10.1016/0014-5793(91)80366-b. [DOI] [PubMed] [Google Scholar]
  11. Kabsch W., Mannherz H. G., Suck D., Pai E. F., Holmes K. C. Atomic structure of the actin:DNase I complex. Nature. 1990 Sep 6;347(6288):37–44. doi: 10.1038/347037a0. [DOI] [PubMed] [Google Scholar]
  12. Kruger M., Wright J., Wang K. Nebulin as a length regulator of thin filaments of vertebrate skeletal muscles: correlation of thin filament length, nebulin size, and epitope profile. J Cell Biol. 1991 Oct;115(1):97–107. doi: 10.1083/jcb.115.1.97. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Labeit S., Gibson T., Lakey A., Leonard K., Zeviani M., Knight P., Wardale J., Trinick J. Evidence that nebulin is a protein-ruler in muscle thin filaments. FEBS Lett. 1991 May 6;282(2):313–316. doi: 10.1016/0014-5793(91)80503-u. [DOI] [PubMed] [Google Scholar]
  14. 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]
  15. Marion D., Wüthrich K. Application of phase sensitive two-dimensional correlated spectroscopy (COSY) for measurements of 1H-1H spin-spin coupling constants in proteins. Biochem Biophys Res Commun. 1983 Jun 29;113(3):967–974. doi: 10.1016/0006-291x(83)91093-8. [DOI] [PubMed] [Google Scholar]
  16. Matsudaira P. T., Burgess D. R. SDS microslab linear gradient polyacrylamide gel electrophoresis. Anal Biochem. 1978 Jul 1;87(2):386–396. doi: 10.1016/0003-2697(78)90688-7. [DOI] [PubMed] [Google Scholar]
  17. Motta A., Pastore A., Goud N. A., Castiglione Morelli M. A. Solution conformation of salmon calcitonin in sodium dodecyl sulfate micelles as determined by two-dimensional NMR and distance geometry calculations. Biochemistry. 1991 Oct 29;30(43):10444–10450. doi: 10.1021/bi00107a012. [DOI] [PubMed] [Google Scholar]
  18. Nicholls A., Sharp K. A., Honig B. Protein folding and association: insights from the interfacial and thermodynamic properties of hydrocarbons. Proteins. 1991;11(4):281–296. doi: 10.1002/prot.340110407. [DOI] [PubMed] [Google Scholar]
  19. Pardee J. D., Spudich J. A. Purification of muscle actin. Methods Enzymol. 1982;85(Pt B):164–181. doi: 10.1016/0076-6879(82)85020-9. [DOI] [PubMed] [Google Scholar]
  20. Reynaud J. A., Grivet J. P., Sy D., Trudelle Y. Interactions of basic amphiphilic peptides with dimyristoylphosphatidylcholine small unilamellar vesicles: optical, NMR, and electron microscopy studies and conformational calculations. Biochemistry. 1993 May 18;32(19):4997–5008. doi: 10.1021/bi00070a005. [DOI] [PubMed] [Google Scholar]
  21. Schägger H., von Jagow G. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal Biochem. 1987 Nov 1;166(2):368–379. doi: 10.1016/0003-2697(87)90587-2. [DOI] [PubMed] [Google Scholar]
  22. Takahashi H., Matuoka S., Kato S., Ohki K., Hatta I. Electrostatic interaction of poly(L-lysine) with dipalmitoylphosphatidic acid studied by X-ray diffraction. Biochim Biophys Acta. 1991 Nov 4;1069(2):229–234. doi: 10.1016/0005-2736(91)90129-v. [DOI] [PubMed] [Google Scholar]
  23. Wang K. Purification of titin and nebulin. Methods Enzymol. 1982;85(Pt B):264–274. doi: 10.1016/0076-6879(82)85025-8. [DOI] [PubMed] [Google Scholar]
  24. Wang K., Williamson C. L. Identification of an N2 line protein of striated muscle. Proc Natl Acad Sci U S A. 1980 Jun;77(6):3254–3258. doi: 10.1073/pnas.77.6.3254. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Wang K., Wright J. Architecture of the sarcomere matrix of skeletal muscle: immunoelectron microscopic evidence that suggests a set of parallel inextensible nebulin filaments anchored at the Z line. J Cell Biol. 1988 Dec;107(6 Pt 1):2199–2212. doi: 10.1083/jcb.107.6.2199. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Way M., Pope B., Cross R. A., Kendrick-Jones J., Weeds A. G. Expression of the N-terminal domain of dystrophin in E. coli and demonstration of binding to F-actin. FEBS Lett. 1992 Apr 27;301(3):243–245. doi: 10.1016/0014-5793(92)80249-g. [DOI] [PubMed] [Google Scholar]
  27. Wegner A. Equilibrium of the actin-tropomyosin interaction. J Mol Biol. 1979 Jul 15;131(4):839–853. doi: 10.1016/0022-2836(79)90204-3. [DOI] [PubMed] [Google Scholar]
  28. Wieland T. Modification of actins by phallotoxins. Naturwissenschaften. 1977 Jun;64(6):303–309. doi: 10.1007/BF00446784. [DOI] [PubMed] [Google Scholar]
  29. Winder S. J., Walsh M. P. Smooth muscle calponin. Inhibition of actomyosin MgATPase and regulation by phosphorylation. J Biol Chem. 1990 Jun 15;265(17):10148–10155. [PubMed] [Google Scholar]
  30. Wright J., Huang Q. Q., Wang K. Nebulin is a full-length template of actin filaments in the skeletal muscle sarcomere: an immunoelectron microscopic study of its orientation and span with site-specific monoclonal antibodies. J Muscle Res Cell Motil. 1993 Oct;14(5):476–483. doi: 10.1007/BF00297210. [DOI] [PubMed] [Google Scholar]

Articles from The EMBO Journal are provided here courtesy of Nature Publishing Group

RESOURCES