Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Molten globular characteristics of the native state of apomyoglobin

Nature Structural Biology volume 1, pages 447–452 (1994)Cite this article

Abstract

Apomyoglobin, myoglobin lacking the haem group, is a natural intermediate in biosynthesis of myoglobin, and has some structural features in common with the haem-containing native state. Unfolding or refolding studies of apomyoglobin have identified a molten globule intermediate at acid pH. We show here that both the native state of apomyoglobin and the molten globule intermediate have highly plastic structures. Substitution of single amino acids on the surface or in the interior of helices in the native protein produce dramatic changes in the helix content and tryptophan emission of apomyoglobin at neutral and acidic pH. The signals from the intermediate and native apomyoglobin correlate closely suggesting that apomyoglobin itself has a molten globule-like character, its structure representing a population of interconverting substates rather than a fixed conformation.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Similar content being viewed by others

References

  1. Kim, P.S. & Baldwin, R.L. Intermediates in the folding reactions of small proteins. Annu. Rev. Biochem. 59, 631–660 (1990).

    Article  CAS  Google Scholar 

  2. Matthews, C.R. Pathways of protein folding. Annu. Rev. Biochem. 62, 653–683 (1993).

    Article  CAS  Google Scholar 

  3. Matthews, B.W. Rev. Biochem. 62, 139–160 (1993).

    Article  CAS  Google Scholar 

  4. Karplus, M. & Shakhnovich, E. in Protein Folding. (ed. Creighton, T. E.) 127–195 (W. H. Freeman and Company, New York, 1992).

    Google Scholar 

  5. Levinthal, C. Are there pathways for protein folding? J. chim. Phys. 65, 44–45 (1968).

    Article  Google Scholar 

  6. Englander, S.W. & Mayne, L. Protein folding studied using hydrogen-exchange labeling and two-dimensional NMR. Annu. Rev. Biophys. biomolec. Struct. 21, 243–265 (1992).

    Article  CAS  Google Scholar 

  7. Kuwajima, K. The molten globule state as a clue for understanding the folding and cooperativity of globular-protein structure. Proteins 6, 87–103 (1989).

    Article  CAS  Google Scholar 

  8. Ptitsyn, O.B. Secondary structure formation and stability. Curr. Opin. struct. Biol. 2, 13–20 (1992).

    Article  CAS  Google Scholar 

  9. Uversky, V.N. Use of fast protein size-exclusion liquid chromatography to study the unfolding of proteins which denature through the molten globule. Biochemistry 32, 13288–13298 (1993).

    Article  CAS  Google Scholar 

  10. Kuwajima, K., Yamaya, H., Miwa, S., Sugai, S. & Nagamura, T. Rapid formation of secomdary structure framework in protein folding studied by stopped-flow circular dichroism. FEBS Lett. 221, 115–118 (1987).

    Article  CAS  Google Scholar 

  11. Goldberg, M.E., Semisotnov, G.V., Friguet, B., Kuwajima, K., Ptitsyn, O.B. & Sugai, S. An early immunoreactive folding intermediate of the tryptophan synthase β2 subunit is a ‘molten globule’. FEBS Lett. 263, 51–56 (1990).

    Article  CAS  Google Scholar 

  12. Griko, Y.V. & Privalov, P.L. Thermodynamic puzzle of apomyoglobin unfolding. J. molec. Biol. 235, 1318–1325 (1994).

    Article  CAS  Google Scholar 

  13. Semisotnov, G.V., Rodionova, N.A., Razgulyaev, O. I., Uversky, V. N., Gripas, A. F. & Gilmanshin, R. I. Study of the “molten globule” intermediate state in protein folding by a hydrophobic fluorescent probe. Biopolymers 31, 119–128 (1991).

    Article  CAS  Google Scholar 

  14. Feng, Y., Sligar, S.G. & Wand, J.A. Solution structure of apocytochrome b562 . Nature struct. Biol. 1, 30–35 (1994).

    Article  CAS  Google Scholar 

  15. Radford, S.E., Dobson, C.M. & Evans, P.A. The folding of hen lysozyme involves partially structured intermediates and multiple pathways. Nature 358, 302–307 (1992).

    Article  CAS  Google Scholar 

  16. Chyan, C., Wormald, C., Dobson, C., Evans, P.A. & Baum, J. Structure and stability of the molten globule state of guinea-pig α-lactalbumin: A hydrogen exchange study. Biochemistry 32, 5681–5691 (1993).

    Article  CAS  Google Scholar 

  17. Gittis, A.G., Stites, W.E. & Lattman, E.E. The phase transition between a compact denatured state and a random coil state in Staphylococcal nuclease is first-order. J. molec. Biol. 232, 718–724 (1993).

    Article  CAS  Google Scholar 

  18. Griko, Y.V., Privalov, P.L. & Venyaminov, S. Y. Thermodynamic study of the apomyoglobin structure. J. molec. Biol. 202, 127–138 (1988).

    Article  CAS  Google Scholar 

  19. Goto, Y. & Fink, A.L. Phase diagram for acidic conformational states of apomyoglobin. J. molec. Biol. 214, 803–805 (1990).

    Article  CAS  Google Scholar 

  20. Irace, G., Balestrieri, C., Parlato, G., Servillo, L. & Colonna, G. Tryptophanyl fluorescence heterogeneity of apomyoglobins. Correlation with the presence of two distinct structural domains. Biochemistry 20, 792–799 (1981).

    Article  CAS  Google Scholar 

  21. Hughson, F.M., Barrick, D. & Baldwin, R.L. Probing the stability of a partly folded apomyoglobin intermediate by site-directed Mutagenesis. Biochemistry 30, 4113–4118 (1991).

    Article  CAS  Google Scholar 

  22. Cocco, M.J. & Lecomte, J.T.J. The native state of apomyoglobin described by proton NMR spectroscopy: Interaction with the paramagnetic probe HyTEMPO and the fluorescent dye ANS. Prot. Sci. 3, 267–281 (1994).

    Article  CAS  Google Scholar 

  23. Goto, Y., Calciano, L. J. & Fink, A. Acid-induced folding of proteins. Proc. natn. Acad. Sci. U.S.A. 87, 573–577 (1990).

    Article  CAS  Google Scholar 

  24. Hughson, F.M., Wright, P.E. & Baldwin, R.L. Structural characterization of a partly folded apomyoglobin intermediate. Science 249, 1544–1548 (1990).

    Article  CAS  Google Scholar 

  25. Barrick, D. & Baldwin, R.L. The molten globule intermediate of apomyoglobin and the process of protein folding. Prot. Sci. 2, 869–876 (1993).

    Article  CAS  Google Scholar 

  26. Hughson, F.M. & Baldwin, R.L. Use of site-directed mutagenesis to destabilize native apomyoglobin relative to folding intermediates. Biochemistry 28, 4415–4422 (1989).

    Article  CAS  Google Scholar 

  27. Pinker, R.J., Lin, L., Rose, G.D. & Kallenbach, N.R. Effects of alanine substitutions in a-helices of sperm whale myoglobin on protein stability. Prot. Sci. 2, 1099–1106 (1993).

    Article  CAS  Google Scholar 

  28. Lin, L., Pinker, R.J. & Kallenbach, N.R. α-helix stability and the native state of myoglobin. Biochemistry 32, 12638–12643 (1993).

    Article  CAS  Google Scholar 

  29. Woody, R.W. Circular dichroism of peptides. (Academic Press, New York, 1988).

    Google Scholar 

  30. Puett, D. The equilibrium unfolding parameters of horse and sperm whale myoglobin. J. biol. Chem. 248, 4623–4634 (1973).

    CAS  PubMed  Google Scholar 

  31. Takano, T. Structure of myoglobin refined at 2.0 Å resolution I. Crystallographic refinemant of metmyoglobin from sperm whale. J. molec. Biol. 110, 537–568 (1977).

    Article  CAS  Google Scholar 

  32. Barrick, D. & Baldwin, R.L. Three-state analysis of sperm whale apomyoglobin folding. Biochemistry 32, 3790–3796 (1993).

    Article  CAS  Google Scholar 

  33. Young, L.R.D., Dill, K. & Fink, A. Aggregation and denaturation of apomyoglobin in aqueous urea solutions. Biochemistry 32, 3877–3886 (1993).

    Article  Google Scholar 

  34. Merutka, G., Lipton, W., Shalongo, W., Park, S. & Stellwagen, E. Effect of central-residue replacements on the helical stability of a monomeric peptide. Biochemistry 29, 7511–7515 (1990).

    Article  CAS  Google Scholar 

  35. Harding, M.M., Williams, D.H. & Woolfson, D.N. Characterization of a partially denatured state of a protein by two-dimensional NMR: reduction of the hydrophobic interactions in Ubiquitin. Biochemistry 30, 3120–3128 (1991).

    Article  CAS  Google Scholar 

  36. Chakrabartty, A., Kortemme, T., Padmanabhan, S. & Baldwin, R. L. Aromatic side-chain contribution to far-ultraviolet circular dichroism of helical peptides and its effect on measurement of helix propensities. Biochemistry 32, 5560–5565 (1993).

    Article  CAS  Google Scholar 

  37. Waltho, J.P., Feher, V.A., Merutka, G., Dyson, H.J. & Wright, P.E. Peptide models of protein folding initiation sites. 1. Secondary structure formation by peptides corresponding to the G- and H- helices of myoglobin. Biochemistry 32, 6337–6347 (1993).

    Article  CAS  Google Scholar 

  38. Jeng, M., Englander, W.S., Elove, G.A., Wand, J.A. & Roder, H. Structural description of acid-denatured cytochrome c by hydrogen exchange and 2D NMR. Biochemistry 29, 10433–10437 (1990).

    Article  CAS  Google Scholar 

  39. Riddihough, G. Snapshots of the ‘molten globule’. Nature 367, 98 (1994).

    Article  Google Scholar 

  40. Shortle, D. & Meeker, A.K. Residual structure in large fragments of Staphylococcal nuclease: effects of amino acid substitutions. Biochemistry 28, 936–944 (1989).

    Article  CAS  Google Scholar 

  41. Pfeil, W., Nolting, B.O. & Jung, C. Apocytochrome P450cam is a native protein with some intermediate-like properties. Biochemistry 32, 8856–8862 (1993).

    Article  CAS  Google Scholar 

  42. Koshland, D.L., Jr., Nemethy, G. & Filmer, D. Comparison of experimental binding data and theoretical models in proteins containing subunits. Biochemistry 5, 365–385 (1966).

    Article  CAS  Google Scholar 

  43. Springer, B.A. & Sligar, S.G. High level expressin of sperm whale myoglobin in E. coli. Proc. natn. Acad. Sci. U.S.A. 84, 8961–8965 (1987).

    Article  CAS  Google Scholar 

  44. Teale, F.W.J. Cleavage of the haem-protein link by acid methylethylketone. Biochim. biophys. Acta 35, 543 (1959).

    Article  CAS  Google Scholar 

  45. Edelhoch, H. Spectroscopic determination of tryptophan and tyrosine in proteins. Biochemistry 6, 1948–1954 (1967).

    Article  CAS  Google Scholar 

  46. Cantor, C.R. & Schimmel, P.R. in Biophysical Chemistry II. Techniques for the study of biological structure and function. (W. H. Freeman and Company, New York, 1980).

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Chemistry, New York University, New York, 10003, USA

    Laura Lin, Rachel J. Pinker, Kirk Forde & Neville R. Kallenbach

  2. Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, MO, 63110, U.S.A.

    George D. Rose

Authors
  1. Laura Lin
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Rachel J. Pinker
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Kirk Forde
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. George D. Rose
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Neville R. Kallenbach
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Lin, L., Pinker, R., Forde, K. et al. Molten globular characteristics of the native state of apomyoglobin. Nat Struct Mol Biol 1, 447–452 (1994). https://doi.org/10.1038/nsb0794-447

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nsb0794-447

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing