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. 2001 Jul;81(1):345–351. doi: 10.1016/S0006-3495(01)75704-9

Folding properties of functional domains of tropomodulin.

A S Kostyukova 1, E I Tiktopulo 1, Y Maéda 1
PMCID: PMC1301516  PMID: 11423419

Abstract

Tropomodulin (Tmod) stabilizes the actin-tropomyosin filament by capping the slow-growing end (P-end). The N- and C-terminal halves play distinct roles; the N-terminal half interacts with the N-terminal region of tropomyosin, whereas the C-terminal half interacts with actin. Our previous study (A. Kostyukova, K. Maeda, E. Yamauchi, I. Krieger, and Y. Maéda Y., 2000, Eur. J. Biochem. 267:6470-6475) suggested that the two halves are also structurally distinct from each other. We have now studied the folding properties of the two halves, by circular dichroism spectroscopy and by differential scanning calorimetry of the expressed chicken E-type tropomodulin and its large fragments. The results showed that the C-terminal half represents a single, independently folded unit that melts cooperatively through a two-state transition. In contrast, the N-terminal half lacks a definite tertiary structure in solution. The binding of N11, a fragment that corresponds to the first 91 amino acids of the tropomodulin, to tropomyosin substantially stabilized the tropomyosin. This may indicate that the flexible structure of the N-terminal half of tropomodulin in solution is required for binding to tropomyosin and that the N-terminal half acquires its tertiary structure upon binding to tropomyosin.

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Selected References

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  1. Almenar-Queralt A., Lee A., Conley C. A., Ribas de Pouplana L., Fowler V. M. Identification of a novel tropomodulin isoform, skeletal tropomodulin, that caps actin filament pointed ends in fast skeletal muscle. J Biol Chem. 1999 Oct 1;274(40):28466–28475. doi: 10.1074/jbc.274.40.28466. [DOI] [PubMed] [Google Scholar]
  2. Appel R. D., Bairoch A., Hochstrasser D. F. A new generation of information retrieval tools for biologists: the example of the ExPASy WWW server. Trends Biochem Sci. 1994 Jun;19(6):258–260. doi: 10.1016/0968-0004(94)90153-8. [DOI] [PubMed] [Google Scholar]
  3. Cox P. R., Zoghbi H. Y. Sequencing, expression analysis, and mapping of three unique human tropomodulin genes and their mouse orthologs. Genomics. 2000 Jan 1;63(1):97–107. doi: 10.1006/geno.1999.6061. [DOI] [PubMed] [Google Scholar]
  4. Dye C. A., Lee J. K., Atkinson R. C., Brewster R., Han P. L., Bellen H. J. The Drosophila sanpodo gene controls sibling cell fate and encodes a tropomodulin homolog, an actin/tropomyosin-associated protein. Development. 1998 May;125(10):1845–1856. doi: 10.1242/dev.125.10.1845. [DOI] [PubMed] [Google Scholar]
  5. Fischer R. S., Lee A., Fowler V. M. Tropomodulin and tropomyosin mediate lens cell actin cytoskeleton reorganization in vitro. Invest Ophthalmol Vis Sci. 2000 Jan;41(1):166–174. [PubMed] [Google Scholar]
  6. Fowler V. M. Identification and purification of a novel Mr 43,000 tropomyosin-binding protein from human erythrocyte membranes. J Biol Chem. 1987 Sep 15;262(26):12792–12800. [PubMed] [Google Scholar]
  7. Fowler V. M. Tropomodulin: a cytoskeletal protein that binds to the end of erythrocyte tropomyosin and inhibits tropomyosin binding to actin. J Cell Biol. 1990 Aug;111(2):471–481. doi: 10.1083/jcb.111.2.471. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Gregorio C. C., Weber A., Bondad M., Pennise C. R., Fowler V. M. Requirement of pointed-end capping by tropomodulin to maintain actin filament length in embryonic chick cardiac myocytes. Nature. 1995 Sep 7;377(6544):83–86. doi: 10.1038/377083a0. [DOI] [PubMed] [Google Scholar]
  9. Gudkov A. T., Khechinashvili N. N., Bushuev V. N. Studies on the structure of protein L7/L12 from Escherichia coli ribosomes. Eur J Biochem. 1978 Oct;90(2):313–318. doi: 10.1111/j.1432-1033.1978.tb12606.x. [DOI] [PubMed] [Google Scholar]
  10. KAUZMANN W. Some factors in the interpretation of protein denaturation. Adv Protein Chem. 1959;14:1–63. doi: 10.1016/s0065-3233(08)60608-7. [DOI] [PubMed] [Google Scholar]
  11. Kostyukova A. S., Pyatibratov M. G., Filimonov V. V., Fedorov O. V. Flagellin parts acquiring a regular structure during polymerization are disposed on the molecule ends. FEBS Lett. 1988 Dec 5;241(1-2):141–144. doi: 10.1016/0014-5793(88)81047-0. [DOI] [PubMed] [Google Scholar]
  12. Kostyukova A., Maeda K., Yamauchi E., Krieger I., Maéda Y. Domain structure of tropomodulin: distinct properties of the N-terminal and C-terminal halves. Eur J Biochem. 2000 Nov;267(21):6470–6475. doi: 10.1046/j.1432-1327.2000.01738.x. [DOI] [PubMed] [Google Scholar]
  13. Krieger I., Kostyukova A. S., Maéda Y. Crystallization and preliminary characterization of crystals of the C-terminal half fragment of tropomodulin. Acta Crystallogr D Biol Crystallogr. 2001 Apr 24;57(Pt 5):743–744. doi: 10.1107/s0907444901003924. [DOI] [PubMed] [Google Scholar]
  14. 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]
  15. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  16. Littlefield R., Fowler V. M. Defining actin filament length in striated muscle: rulers and caps or dynamic stability? Annu Rev Cell Dev Biol. 1998;14:487–525. doi: 10.1146/annurev.cellbio.14.1.487. [DOI] [PubMed] [Google Scholar]
  17. McElhinny A. S., Kolmerer B., Fowler V. M., Labeit S., Gregorio C. C. The N-terminal end of nebulin interacts with tropomodulin at the pointed ends of the thin filaments. J Biol Chem. 2001 Jan 5;276(1):583–592. doi: 10.1074/jbc.M005693200. [DOI] [PubMed] [Google Scholar]
  18. Potekhin S. A., Privalov P. L. Co-operative blocks in tropomyosin. J Mol Biol. 1982 Aug 15;159(3):519–535. doi: 10.1016/0022-2836(82)90299-6. [DOI] [PubMed] [Google Scholar]
  19. Privalov P. L., Khechinashvili N. N. A thermodynamic approach to the problem of stabilization of globular protein structure: a calorimetric study. J Mol Biol. 1974 Jul 5;86(3):665–684. doi: 10.1016/0022-2836(74)90188-0. [DOI] [PubMed] [Google Scholar]
  20. Privalov P. L., Potekhin S. A. Scanning microcalorimetry in studying temperature-induced changes in proteins. Methods Enzymol. 1986;131:4–51. doi: 10.1016/0076-6879(86)31033-4. [DOI] [PubMed] [Google Scholar]
  21. Privalov P. L. Stability of proteins: small globular proteins. Adv Protein Chem. 1979;33:167–241. doi: 10.1016/s0065-3233(08)60460-x. [DOI] [PubMed] [Google Scholar]
  22. Sturtevant J. M. Heat capacity and entropy changes in processes involving proteins. Proc Natl Acad Sci U S A. 1977 Jun;74(6):2236–2240. doi: 10.1073/pnas.74.6.2236. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Sung L. A., Fowler V. M., Lambert K., Sussman M. A., Karr D., Chien S. Molecular cloning and characterization of human fetal liver tropomodulin. A tropomyosin-binding protein. J Biol Chem. 1992 Feb 5;267(4):2616–2621. [PubMed] [Google Scholar]
  24. Sung L. A., Lin J. J. Erythrocyte tropomodulin binds to the N-terminus of hTM5, a tropomyosin isoform encoded by the gamma-tropomyosin gene. Biochem Biophys Res Commun. 1994 Jun 15;201(2):627–634. doi: 10.1006/bbrc.1994.1747. [DOI] [PubMed] [Google Scholar]
  25. Tiktopulo E. I., Privalov P. L., Odintsova T. I., Ermokhina T. M., Krasheninnikov I. A., Aviles F. X., Cary P. D., Crane-Robinson C. The central tryptic fragment of histones H1 and H5 is a fully compacted domain and is the only folded region in the polypeptide chain. A thermodynamic study. Eur J Biochem. 1982 Feb;122(2):327–331. doi: 10.1111/j.1432-1033.1982.tb05884.x. [DOI] [PubMed] [Google Scholar]
  26. Vera C., Sood A., Gao K. M., Yee L. J., Lin J. J., Sung L. A. Tropomodulin-binding site mapped to residues 7-14 at the N-terminal heptad repeats of tropomyosin isoform 5. Arch Biochem Biophys. 2000 Jun 1;378(1):16–24. doi: 10.1006/abbi.2000.1802. [DOI] [PubMed] [Google Scholar]
  27. Watakabe A., Kobayashi R., Helfman D. M. N-tropomodulin: a novel isoform of tropomodulin identified as the major binding protein to brain tropomyosin. J Cell Sci. 1996 Sep;109(Pt 9):2299–2310. doi: 10.1242/jcs.109.9.2299. [DOI] [PubMed] [Google Scholar]
  28. Weber A., Pennise C. R., Babcock G. G., Fowler V. M. Tropomodulin caps the pointed ends of actin filaments. J Cell Biol. 1994 Dec;127(6 Pt 1):1627–1635. doi: 10.1083/jcb.127.6.1627. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Wolf E., Kim P. S., Berger B. MultiCoil: a program for predicting two- and three-stranded coiled coils. Protein Sci. 1997 Jun;6(6):1179–1189. doi: 10.1002/pro.5560060606. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Xu P., Mitchelhill K. I., Kobe B., Kemp B. E., Zot H. G. The myosin-I-binding protein Acan125 binds the SH3 domain and belongs to the superfamily of leucine-rich repeat proteins. Proc Natl Acad Sci U S A. 1997 Apr 15;94(8):3685–3690. doi: 10.1073/pnas.94.8.3685. [DOI] [PMC free article] [PubMed] [Google Scholar]

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