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. 1992 Oct;1(10):1319–1325. doi: 10.1002/pro.5560011011

Unfolding domains of recombinant fusion alpha alpha-tropomyosin.

Y Ishii 1, S Hitchcock-DeGregori 1, K Mabuchi 1, S S Lehrer 1
PMCID: PMC2142099  PMID: 1303750

Abstract

The thermal unfolding of the coiled-coil alpha-helix of recombinant alpha alpha-tropomyosin from rat striated muscle containing an additional 80-residue peptide of influenza virus NS1 protein at the N-terminus (fusion-tropomyosin) was studied with circular dichroism and fluorescence techniques. Fusion-tropomyosin unfolded in four cooperative transitions: (1) a pretransition starting at 35 degrees C involving the middle of the molecule; (2) a major transition at 46 degrees C involving no more than 36% of the helix from the C-terminus; (3) a major transition at 56 degrees C involving about 46% of the helix from the N-terminus; and (4) a transition from the nonhelical fusion domain at about 70 degrees C. Rabbit skeletal muscle tropomyosin, which lacks the fusion peptide but has the same tropomyosin sequence, does not exhibit the 56 degrees C or 70 degrees C transition. The very stable fusion unfolding domain of fusion-tropomyosin, which appears in electron micrographs as a globular structural domain at one end of the tropomyosin rod, acts as a cross-link to stabilize the adjacent N-terminal domain. The least stable middle of the molecule, when unfolded, acts as a boundary to allow the independent unfolding of the C-terminal domain at 46 degrees C from the stabilized N-terminal unfolding domain at 56 degrees C. Thus, strong localized interchain interactions in coiled-coil molecules can increase the stability of neighboring domains.

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Selected References

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  1. Betcher-Lange S. L., Lehrer S. S. Pyrene excimer fluorescence in rabbit skeletal alphaalphatropomyosin labeled with N-(1-pyrene)maleimide. A probe of sulfhydryl proximity and local chain separation. J Biol Chem. 1978 Jun 10;253(11):3757–3760. [PubMed] [Google Scholar]
  2. Betteridge D. R., Lehrer S. S. Two conformational states of didansylcystine-labeled rabbit cardiac tropomyosin. J Mol Biol. 1983 Jun 25;167(2):481–496. doi: 10.1016/s0022-2836(83)80346-5. [DOI] [PubMed] [Google Scholar]
  3. Brandts J. F., Hu C. Q., Lin L. N., Mos M. T. A simple model for proteins with interacting domains. Applications to scanning calorimetry data. Biochemistry. 1989 Oct 17;28(21):8588–8596. doi: 10.1021/bi00447a048. [DOI] [PubMed] [Google Scholar]
  4. Chao Y. Y., Holtzer A. Spin-label studies of tropomyosin. Biochemistry. 1975 May 20;14(10):2164–2170. doi: 10.1021/bi00681a019. [DOI] [PubMed] [Google Scholar]
  5. Cho Y. J., Hitchcock-DeGregori S. E. Relationship between alternatively spliced exons and functional domains in tropomyosin. Proc Natl Acad Sci U S A. 1991 Nov 15;88(22):10153–10157. doi: 10.1073/pnas.88.22.10153. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Gilbert W. Genes-in-pieces revisited. Science. 1985 May 17;228(4701):823–824. doi: 10.1126/science.4001923. [DOI] [PubMed] [Google Scholar]
  7. Graceffa P., Lehrer S. S. Dynamic equilibrium between the two conformational states of spin-labeled tropomyosin. Biochemistry. 1984 Jun 5;23(12):2606–2612. doi: 10.1021/bi00307a011. [DOI] [PubMed] [Google Scholar]
  8. Graceffa P., Lehrer S. S. The excimer fluorescence of pyrene-labeled tropomyosin. A probe of conformational dynamics. J Biol Chem. 1980 Dec 10;255(23):11296–11300. [PubMed] [Google Scholar]
  9. Heald R. W., Hitchcock-DeGregori S. E. The structure of the amino terminus of tropomyosin is critical for binding to actin in the absence and presence of troponin. J Biol Chem. 1988 Apr 15;263(11):5254–5259. [PubMed] [Google Scholar]
  10. 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]
  11. Holtzer M. E., Bracken W. C., Holtzer A. Alpha-helix to random coil transitions of two-chain coiled coils: experiments on the thermal denaturation of beta beta tropomyosin cross-linked selectively at C36. Biopolymers. 1990 May-Jun;29(6-7):1045–1056. doi: 10.1002/bip.360290615. [DOI] [PubMed] [Google Scholar]
  12. 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]
  13. Leavis P. C., Gergely J. Thin filament proteins and thin filament-linked regulation of vertebrate muscle contraction. CRC Crit Rev Biochem. 1984;16(3):235–305. doi: 10.3109/10409238409108717. [DOI] [PubMed] [Google Scholar]
  14. Lehrer S. S. Effects of an interchain disulfide bond on tropomyosin structure: intrinsic fluorescence and circular dichroism studies. J Mol Biol. 1978 Jan 15;118(2):209–226. doi: 10.1016/0022-2836(78)90413-8. [DOI] [PubMed] [Google Scholar]
  15. Lehrer S. S. Intramolecular crosslinking of tropomyosin via disulfide bond formation: evidence for chain register. Proc Natl Acad Sci U S A. 1975 Sep;72(9):3377–3381. doi: 10.1073/pnas.72.9.3377. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Mabuchi K. Melting of myosin and tropomyosin: electron microscopic observations. J Struct Biol. 1990 May;103(3):249–256. doi: 10.1016/1047-8477(90)90043-c. [DOI] [PubMed] [Google Scholar]
  17. Morrongiello M. P., Dales S. Characterization of cytoplasmic inclusions formed during influenza/WSN virus infection of chick embryo fibroblast cells. Intervirology. 1977;8(5):281–293. doi: 10.1159/000148903. [DOI] [PubMed] [Google Scholar]
  18. O'Neil K. T., Hoess R. H., DeGrado W. F. Design of DNA-binding peptides based on the leucine zipper motif. Science. 1990 Aug 17;249(4970):774–778. doi: 10.1126/science.2389143. [DOI] [PubMed] [Google Scholar]
  19. O'Shea E. K., Rutkowski R., Kim P. S. Evidence that the leucine zipper is a coiled coil. Science. 1989 Jan 27;243(4890):538–542. doi: 10.1126/science.2911757. [DOI] [PubMed] [Google Scholar]
  20. 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]
  21. Pont M. J., Woods E. F. Denaturation of tropomyosin by guanidine hydrochloride. Int J Protein Res. 1971;3(4):177–183. doi: 10.1111/j.1399-3011.1971.tb01710.x. [DOI] [PubMed] [Google Scholar]
  22. 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]
  23. Ramsay G., Freire E. Linked thermal and solute perturbation analysis of cooperative domain interactions in proteins. Structural stability of diphtheria toxin. Biochemistry. 1990 Sep 18;29(37):8677–8683. doi: 10.1021/bi00489a024. [DOI] [PubMed] [Google Scholar]
  24. Regan L., DeGrado W. F. Characterization of a helical protein designed from first principles. Science. 1988 Aug 19;241(4868):976–978. doi: 10.1126/science.3043666. [DOI] [PubMed] [Google Scholar]
  25. Ruiz-Opazo N., Nadal-Ginard B. Alpha-tropomyosin gene organization. Alternative splicing of duplicated isotype-specific exons accounts for the production of smooth and striated muscle isoforms. J Biol Chem. 1987 Apr 5;262(10):4755–4765. [PubMed] [Google Scholar]
  26. Ruiz-Opazo N., Weinberger J., Nadal-Ginard B. Comparison of alpha-tropomyosin sequences from smooth and striated muscle. Nature. 1985 May 2;315(6014):67–70. doi: 10.1038/315067a0. [DOI] [PubMed] [Google Scholar]
  27. Shaw M. W., Compans R. W. Isolation and characterization of cytoplasmic inclusions from influenza A virus-infected cells. J Virol. 1978 Feb;25(2):608–615. doi: 10.1128/jvi.25.2.608-615.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Shuman J. D., Vinson C. R., McKnight S. L. Evidence of changes in protease sensitivity and subunit exchange rate on DNA binding by C/EBP. Science. 1990 Aug 17;249(4970):771–774. doi: 10.1126/science.2202050. [DOI] [PubMed] [Google Scholar]
  29. Skolnick J., Holtzer A. Alpha-helix-to-random-coil transitions of two-chain, coiled coils: a theoretical model for the "pretransition" in cysteine-190-cross-linked tropomyosin. Biochemistry. 1986 Oct 7;25(20):6192–6202. doi: 10.1021/bi00368a054. [DOI] [PubMed] [Google Scholar]
  30. Stone D., Smillie L. B. The amino acid sequence of rabbit skeletal alpha-tropomyosin. The NH2-terminal half and complete sequence. J Biol Chem. 1978 Feb 25;253(4):1137–1148. [PubMed] [Google Scholar]
  31. Talanian R. V., McKnight C. J., Kim P. S. Sequence-specific DNA binding by a short peptide dimer. Science. 1990 Aug 17;249(4970):769–771. doi: 10.1126/science.2389142. [DOI] [PubMed] [Google Scholar]
  32. Ueno H. Local structural changes in tropomyosin detected by a trypsin-probe method. Biochemistry. 1984 Sep 25;23(20):4791–4798. doi: 10.1021/bi00315a040. [DOI] [PubMed] [Google Scholar]
  33. Weiss M. A. Thermal unfolding studies of a leucine zipper domain and its specific DNA complex: implications for scissor's grip recognition. Biochemistry. 1990 Sep 4;29(35):8020–8024. doi: 10.1021/bi00487a004. [DOI] [PubMed] [Google Scholar]
  34. Woods E. F. Studies on the denaturation of tropomyosin and light meromyosin. Int J Protein Res. 1969;1(1):29–43. doi: 10.1111/j.1399-3011.1969.tb01624.x. [DOI] [PubMed] [Google Scholar]
  35. Woods E. F. The conformational stabilities of tropomyosins. Aust J Biol Sci. 1976 Dec;29(5-6):405–418. doi: 10.1071/bi9760405. [DOI] [PubMed] [Google Scholar]
  36. Zot A. S., Potter J. D. Structural aspects of troponin-tropomyosin regulation of skeletal muscle contraction. Annu Rev Biophys Biophys Chem. 1987;16:535–559. doi: 10.1146/annurev.bb.16.060187.002535. [DOI] [PubMed] [Google Scholar]

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