Skip to main content
PMC home page
Protein Science : A Publication of the Protein Society logoLink to Protein Science : A Publication of the Protein Society
. 1997 Oct;6(10):2134–2142. doi: 10.1002/pro.5560061008

Structural heterogeneity of the various forms of apomyoglobin: implications for protein folding.

R Gilmanshin 1, R B Dyer 1, R H Callender 1
PMCID: PMC2143565  PMID: 9336836

Abstract

Temperature-induced denaturation transitions of different structural forms of apomyoglobin were studied monitoring intrinsic tryptophan fluorescence. It was found that the tryptophans are effectively screened from solvent both in native and acid forms throughout most of the temperature range tested. Thus, the tryptophans' surrounding do not show a considerable change in structure where major protein conformational transitions have been found in apomyoglobin using other techniques. At high temperatures and under strong destabilizing conditions, the tryptophans' fluorescence parameters show sigmoidal thermal denaturation. These results, combined with previous studies, show that the structure of this protein is heterogeneous, including native-like (tightly packed) and molten globule-like substructures that exhibit conformation (denaturation) transitions under different conditions of pH and temperature (and denaturants). The results suggest that the folding of this protein proceeds via two "nucleation" events whereby native-like contacts are formed. One of these events, which involves AGH "core" formation, appears to occur very early in the folding process, even before significant hydrophobic collapse in the rest of the protein molecule. From the current studies and other results, a rather detailed picture of the folding of myoglobin is presented, on the level of specific structures and their thermodynamical properties as well as formation kinetics.

Full Text

The Full Text of this article is available as a PDF (977.6 KB).

Selected References

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

  1. Bai Y., Englander S. W. Future directions in folding: the multi-state nature of protein structure. Proteins. 1996 Feb;24(2):145–151. doi: 10.1002/(SICI)1097-0134(199602)24:2<145::AID-PROT1>3.0.CO;2-I. [DOI] [PubMed] [Google Scholar]
  2. CRUMPTON M. J., POLSON A. A COMPARISON OF THE CONFORMATION OF SPERM WHALE METMYOGLOBIN WITH THAT OF APOMYOGLOBIN. J Mol Biol. 1965 Apr;11:722–729. doi: 10.1016/s0022-2836(65)80030-4. [DOI] [PubMed] [Google Scholar]
  3. De Sanctis G., Ascoli F., Brunori M. Folding of apominimyoglobin. Proc Natl Acad Sci U S A. 1994 Nov 22;91(24):11507–11511. doi: 10.1073/pnas.91.24.11507. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Eftink M. R. The use of fluorescence methods to monitor unfolding transitions in proteins. Biophys J. 1994 Feb;66(2 Pt 1):482–501. doi: 10.1016/s0006-3495(94)80799-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Eliezer D., Jennings P. A., Wright P. E., Doniach S., Hodgson K. O., Tsuruta H. The radius of gyration of an apomyoglobin folding intermediate. Science. 1995 Oct 20;270(5235):487–488. doi: 10.1126/science.270.5235.487. [DOI] [PubMed] [Google Scholar]
  6. Eliezer D., Wright P. E. Is apomyoglobin a molten globule? Structural characterization by NMR. J Mol Biol. 1996 Nov 8;263(4):531–538. doi: 10.1006/jmbi.1996.0596. [DOI] [PubMed] [Google Scholar]
  7. Evans S. V., Brayer G. D. Horse heart metmyoglobin. A 2.8-A resolution three-dimensional structure determination. J Biol Chem. 1988 Mar 25;263(9):4263–4268. [PubMed] [Google Scholar]
  8. Fontana A., Zambonin M., Polverino de Laureto P., De Filippis V., Clementi A., Scaramella E. Probing the conformational state of apomyoglobin by limited proteolysis. J Mol Biol. 1997 Feb 21;266(2):223–230. doi: 10.1006/jmbi.1996.0787. [DOI] [PubMed] [Google Scholar]
  9. Gast K., Damaschun H., Misselwitz R., Müller-Frohne M., Zirwer D., Damaschun G. Compactness of protein molten globules: temperature-induced structural changes of the apomyoglobin folding intermediate. Eur Biophys J. 1994;23(4):297–305. doi: 10.1007/BF00213579. [DOI] [PubMed] [Google Scholar]
  10. Gilmanshin R., Williams S., Callender R. H., Woodruff W. H., Dyer R. B. Fast events in protein folding: relaxation dynamics of secondary and tertiary structure in native apomyoglobin. Proc Natl Acad Sci U S A. 1997 Apr 15;94(8):3709–3713. doi: 10.1073/pnas.94.8.3709. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Griko Y. V., Privalov P. L. Thermodynamic puzzle of apomyoglobin unfolding. J Mol Biol. 1994 Jan 28;235(4):1318–1325. doi: 10.1006/jmbi.1994.1085. [DOI] [PubMed] [Google Scholar]
  12. Griko Y. V., Privalov P. L., Venyaminov S. Y., Kutyshenko V. P. Thermodynamic study of the apomyoglobin structure. J Mol Biol. 1988 Jul 5;202(1):127–138. doi: 10.1016/0022-2836(88)90525-6. [DOI] [PubMed] [Google Scholar]
  13. Hughson F. M., Barrick D., Baldwin R. L. Probing the stability of a partly folded apomyoglobin intermediate by site-directed mutagenesis. Biochemistry. 1991 Apr 30;30(17):4113–4118. doi: 10.1021/bi00231a001. [DOI] [PubMed] [Google Scholar]
  14. Hughson F. M., Wright P. E., Baldwin R. L. Structural characterization of a partly folded apomyoglobin intermediate. Science. 1990 Sep 28;249(4976):1544–1548. doi: 10.1126/science.2218495. [DOI] [PubMed] [Google Scholar]
  15. Jamin M., Baldwin R. L. Refolding and unfolding kinetics of the equilibrium folding intermediate of apomyoglobin. Nat Struct Biol. 1996 Jul;3(7):613–618. doi: 10.1038/nsb0796-613. [DOI] [PubMed] [Google Scholar]
  16. Jennings P. A., Wright P. E. Formation of a molten globule intermediate early in the kinetic folding pathway of apomyoglobin. Science. 1993 Nov 5;262(5135):892–896. doi: 10.1126/science.8235610. [DOI] [PubMed] [Google Scholar]
  17. Johnson R. S., Walsh K. A. Mass spectrometric measurement of protein amide hydrogen exchange rates of apo- and holo-myoglobin. Protein Sci. 1994 Dec;3(12):2411–2418. doi: 10.1002/pro.5560031224. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Karplus M., Weaver D. L. Protein folding dynamics: the diffusion-collision model and experimental data. Protein Sci. 1994 Apr;3(4):650–668. doi: 10.1002/pro.5560030413. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Kataoka M., Nishii I., Fujisawa T., Ueki T., Tokunaga F., Goto Y. Structural characterization of the molten globule and native states of apomyoglobin by solution X-ray scattering. J Mol Biol. 1995 May 26;249(1):215–228. doi: 10.1006/jmbi.1995.0290. [DOI] [PubMed] [Google Scholar]
  20. Kay M. S., Baldwin R. L. Packing interactions in the apomyglobin folding intermediate. Nat Struct Biol. 1996 May;3(5):439–445. doi: 10.1038/nsb0596-439. [DOI] [PubMed] [Google Scholar]
  21. Lecomte J. T., Kao Y. H., Cocco M. J. The native state of apomyoglobin described by proton NMR spectroscopy: the A-B-G-H interface of wild-type sperm whale apomyoglobin. Proteins. 1996 Jul;25(3):267–285. doi: 10.1002/(SICI)1097-0134(199607)25:3<267::AID-PROT1>3.0.CO;2-D. [DOI] [PubMed] [Google Scholar]
  22. Loh S. N., Kay M. S., Baldwin R. L. Structure and stability of a second molten globule intermediate in the apomyoglobin folding pathway. Proc Natl Acad Sci U S A. 1995 Jun 6;92(12):5446–5450. doi: 10.1073/pnas.92.12.5446. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Makhatadze G. I., Clore G. M., Gronenborn A. M. Solvent isotope effect and protein stability. Nat Struct Biol. 1995 Oct;2(10):852–855. doi: 10.1038/nsb1095-852. [DOI] [PubMed] [Google Scholar]
  24. Nishii I., Kataoka M., Tokunaga F., Goto Y. Cold denaturation of the molten globule states of apomyoglobin and a profile for protein folding. Biochemistry. 1994 Apr 26;33(16):4903–4909. doi: 10.1021/bi00182a019. [DOI] [PubMed] [Google Scholar]
  25. Postnikova G. B., Komarov Y. E., Yumakova E. M. Fluorescence study of the conformational properties of myoglobin structure. 1. pH-dependent changes of tryptophanyl fluorescence in intact and chemically modified sperm whale apomyoglobins. Eur J Biochem. 1991 May 23;198(1):223–232. doi: 10.1111/j.1432-1033.1991.tb16005.x. [DOI] [PubMed] [Google Scholar]
  26. Privalov P. L., Griko YuV, Venyaminov SYu, Kutyshenko V. P. Cold denaturation of myoglobin. J Mol Biol. 1986 Aug 5;190(3):487–498. doi: 10.1016/0022-2836(86)90017-3. [DOI] [PubMed] [Google Scholar]
  27. Ptitsyn O. B. Structures of folding intermediates. Curr Opin Struct Biol. 1995 Feb;5(1):74–78. doi: 10.1016/0959-440x(95)80011-o. [DOI] [PubMed] [Google Scholar]
  28. Rischel C., Thyberg P., Rigler F., Poulsen F. M. Time-resolved fluorescence studies of the molten globule state of apomyoglobin. J Mol Biol. 1996 Apr 12;257(4):877–885. doi: 10.1006/jmbi.1996.0208. [DOI] [PubMed] [Google Scholar]
  29. Rothgeb T. M., Gurd F. R. Physical methods for the study of myoglobin. Methods Enzymol. 1978;52:473–486. doi: 10.1016/s0076-6879(78)52052-1. [DOI] [PubMed] [Google Scholar]
  30. Shakhnovich E. I., Finkelstein A. V. Theory of cooperative transitions in protein molecules. I. Why denaturation of globular protein is a first-order phase transition. Biopolymers. 1989 Oct;28(10):1667–1680. doi: 10.1002/bip.360281003. [DOI] [PubMed] [Google Scholar]
  31. Shin H. C., Merutka G., Waltho J. P., Tennant L. L., Dyson H. J., Wright P. E. Peptide models of protein folding initiation sites. 3. The G-H helical hairpin of myoglobin. Biochemistry. 1993 Jun 29;32(25):6356–6364. doi: 10.1021/bi00076a008. [DOI] [PubMed] [Google Scholar]
  32. Sirangelo I., Bismuto E., Irace G. Solvent and thermal denaturation of the acidic compact state of apomyoglobin. FEBS Lett. 1994 Jan 24;338(1):11–15. doi: 10.1016/0014-5793(94)80107-x. [DOI] [PubMed] [Google Scholar]
  33. TEALE F. W. Cleavage of the haem-protein link by acid methylethylketone. Biochim Biophys Acta. 1959 Oct;35:543–543. doi: 10.1016/0006-3002(59)90407-x. [DOI] [PubMed] [Google Scholar]
  34. 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. 1993 Jun 29;32(25):6337–6347. doi: 10.1021/bi00076a006. [DOI] [PubMed] [Google Scholar]
  35. Williams S., Causgrove T. P., Gilmanshin R., Fang K. S., Callender R. H., Woodruff W. H., Dyer R. B. Fast events in protein folding: helix melting and formation in a small peptide. Biochemistry. 1996 Jan 23;35(3):691–697. doi: 10.1021/bi952217p. [DOI] [PubMed] [Google Scholar]

Articles from Protein Science : A Publication of the Protein Society are provided here courtesy of The Protein Society

RESOURCES