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. 1994 Nov 22;91(24):11507–11511. doi: 10.1073/pnas.91.24.11507

Folding of apominimyoglobin.

G De Sanctis 1, F Ascoli 1, M Brunori 1
PMCID: PMC45260  PMID: 7972092

Abstract

The acid unfolding pathway of apominimyoglobin (apo-mini-Mb), a 108-aa fragment (aa 32-139) of horse heart apomyoglobin has been studied by means of circular dichroism, in comparison with the native apoprotein. Similar to sperm whale apomyoglobin [Hughson, F. M., Wright, P. E. & Baldwin, R. L. (1990) Science 249, 1544-1548], a partly folded intermediate (alpha-helical content approximately 35%) is populated at pH 4.2 for horse heart apomyoglobin. For this intermediate, Hughson et al. proposed a structural model with a compact subdomain involving tertiary interactions between the folded A, G, and H helices, with the remainder of the protein essentially unfolded. As described in this paper, a folding intermediate with an alpha-helical content of approximately 33% is populated at pH 4.3-5.0 also in apo-mini-Mb. The acid unfolding pathway is similarly affected in both the native and the mini apoprotein by 15% trifluoroethanol, a helix-stabilizing compound. Thus, the folding of the apo-mini-Mb intermediate is similar to that observed for the native apoprotein, in spite of the absence in the miniprotein of the A helix and of a large part of the H helix, which are crucial for the stability of apo-Mb intermediate. Our results suggest that acquisition of a folded state in apo-mini-Mb occurs through an alternative pathway, which may or may not be shared also by apo-Mb.

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Selected References

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

  1. Baldwin R. L. How does protein folding get started? Trends Biochem Sci. 1989 Jul;14(7):291–294. doi: 10.1016/0968-0004(89)90067-4. [DOI] [PubMed] [Google Scholar]
  2. Barrick D., Baldwin R. L. Stein and Moore Award address. The molten globule intermediate of apomyoglobin and the process of protein folding. Protein Sci. 1993 Jun;2(6):869–876. doi: 10.1002/pro.5560020601. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Barrow C. J., Yasuda A., Kenny P. T., Zagorski M. G. Solution conformations and aggregational properties of synthetic amyloid beta-peptides of Alzheimer's disease. Analysis of circular dichroism spectra. J Mol Biol. 1992 Jun 20;225(4):1075–1093. doi: 10.1016/0022-2836(92)90106-t. [DOI] [PubMed] [Google Scholar]
  4. Blanchetot A., Price M., Jeffreys A. J. The mouse myoglobin gene. Characterisation and sequence comparison with other mammalian myoglobin genes. Eur J Biochem. 1986 Sep 15;159(3):469–474. doi: 10.1111/j.1432-1033.1986.tb09909.x. [DOI] [PubMed] [Google Scholar]
  5. Blanchetot A., Wilson V., Wood D., Jeffreys A. J. The seal myoglobin gene: an unusually long globin gene. Nature. 1983 Feb 24;301(5902):732–734. doi: 10.1038/301732a0. [DOI] [PubMed] [Google Scholar]
  6. Brooks C. L., 3rd Characterization of "native" apomyoglobin by molecular dynamics simulation. J Mol Biol. 1992 Sep 20;227(2):375–380. doi: 10.1016/0022-2836(92)90893-o. [DOI] [PubMed] [Google Scholar]
  7. Chen Y. H., Yang J. T., Martinez H. M. Determination of the secondary structures of proteins by circular dichroism and optical rotatory dispersion. Biochemistry. 1972 Oct 24;11(22):4120–4131. doi: 10.1021/bi00772a015. [DOI] [PubMed] [Google Scholar]
  8. Christensen H., Pain R. H. Molten globule intermediates and protein folding. Eur Biophys J. 1991;19(5):221–229. doi: 10.1007/BF00183530. [DOI] [PubMed] [Google Scholar]
  9. Cocco M. J., Kao Y. H., Phillips A. T., Lecomte J. T. Structural comparison of apomyoglobin and metaquomyoglobin: pH titration of histidines by NMR spectroscopy. Biochemistry. 1992 Jul 21;31(28):6481–6491. doi: 10.1021/bi00143a018. [DOI] [PubMed] [Google Scholar]
  10. Cocco M. J., Lecomte J. T. Characterization of hydrophobic cores in apomyoglobin: a proton NMR spectroscopy study. Biochemistry. 1990 Dec 18;29(50):11067–11072. doi: 10.1021/bi00502a008. [DOI] [PubMed] [Google Scholar]
  11. De Sanctis G., Falcioni G., Giardina B., Ascoli F., Brunori M. Mini-myoglobin. The structural significance of haem-ligand interactions. J Mol Biol. 1988 Apr 20;200(4):725–733. doi: 10.1016/0022-2836(88)90483-4. [DOI] [PubMed] [Google Scholar]
  12. De Sanctis G., Falcioni G., Giardina B., Ascoli F., Brunori M. Mini-myoglobin: preparation and reaction with oxygen and carbon monoxide. J Mol Biol. 1986 Mar 5;188(1):73–76. doi: 10.1016/0022-2836(86)90481-x. [DOI] [PubMed] [Google Scholar]
  13. De Sanctis G., Falcioni G., Grelloni F., Desideri A., Polizio F., Giardina B., Ascoli F., Brunori M. Mini-myoglobin. Electron paramagnetic resonance and reversible oxygenation of the cobalt derivative. J Mol Biol. 1991 Dec 5;222(3):637–643. doi: 10.1016/0022-2836(91)90501-v. [DOI] [PubMed] [Google Scholar]
  14. De Sanctis G., Falcioni G., Polizio F., Desideri A., Giardina B., Ascoli F., Brunori M. Mini-myoglobin: native-like folding of the NO-derivative. Biochim Biophys Acta. 1994 Jan 11;1204(1):28–32. doi: 10.1016/0167-4838(94)90028-0. [DOI] [PubMed] [Google Scholar]
  15. Di Iorio E. E., Yu W., Calonder C., Winterhalter K. H., De Sanctis G., Falcioni G., Ascoli F., Giardina B., Brunori M. Protein dynamics in minimyoglobin: is the central core of myoglobin the conformational domain? Proc Natl Acad Sci U S A. 1993 Mar 1;90(5):2025–2029. doi: 10.1073/pnas.90.5.2025. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Dill K. A., Fiebig K. M., Chan H. S. Cooperativity in protein-folding kinetics. Proc Natl Acad Sci U S A. 1993 Mar 1;90(5):1942–1946. doi: 10.1073/pnas.90.5.1942. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Dyson H. J., Rance M., Houghten R. A., Wright P. E., Lerner R. A. Folding of immunogenic peptide fragments of proteins in water solution. II. The nascent helix. J Mol Biol. 1988 May 5;201(1):201–217. doi: 10.1016/0022-2836(88)90447-0. [DOI] [PubMed] [Google Scholar]
  18. Edelhoch H. Spectroscopic determination of tryptophan and tyrosine in proteins. Biochemistry. 1967 Jul;6(7):1948–1954. doi: 10.1021/bi00859a010. [DOI] [PubMed] [Google Scholar]
  19. Evans S. V., Brayer G. D. High-resolution study of the three-dimensional structure of horse heart metmyoglobin. J Mol Biol. 1990 Jun 20;213(4):885–897. doi: 10.1016/S0022-2836(05)80270-0. [DOI] [PubMed] [Google Scholar]
  20. Fersht A. R., Matouschek A., Serrano L. The folding of an enzyme. I. Theory of protein engineering analysis of stability and pathway of protein folding. J Mol Biol. 1992 Apr 5;224(3):771–782. doi: 10.1016/0022-2836(92)90561-w. [DOI] [PubMed] [Google Scholar]
  21. 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]
  22. 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]
  23. 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]
  24. 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]
  25. Kim P. S., Baldwin R. L. Intermediates in the folding reactions of small proteins. Annu Rev Biochem. 1990;59:631–660. doi: 10.1146/annurev.bi.59.070190.003215. [DOI] [PubMed] [Google Scholar]
  26. 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]
  27. Miranker A., Radford S. E., Karplus M., Dobson C. M. Demonstration by NMR of folding domains in lysozyme. Nature. 1991 Feb 14;349(6310):633–636. doi: 10.1038/349633a0. [DOI] [PubMed] [Google Scholar]
  28. Pain R. H. Molecular biology. Brevity is the soul of wit. Nature. 1986 Mar 20;320(6059):216–217. doi: 10.1038/320216a0. [DOI] [PubMed] [Google Scholar]
  29. Ptitsyn O. B., Pain R. H., Semisotnov G. V., Zerovnik E., Razgulyaev O. I. Evidence for a molten globule state as a general intermediate in protein folding. FEBS Lett. 1990 Mar 12;262(1):20–24. doi: 10.1016/0014-5793(90)80143-7. [DOI] [PubMed] [Google Scholar]
  30. Schacterle G. R., Pollack R. L. A simplified method for the quantitative assay of small amounts of protein in biologic material. Anal Biochem. 1973 Feb;51(2):654–655. doi: 10.1016/0003-2697(73)90523-x. [DOI] [PubMed] [Google Scholar]
  31. Stryer L. The interaction of a naphthalene dye with apomyoglobin and apohemoglobin. A fluorescent probe of non-polar binding sites. J Mol Biol. 1965 Sep;13(2):482–495. doi: 10.1016/s0022-2836(65)80111-5. [DOI] [PubMed] [Google Scholar]
  32. Tirado-Rives J., Jorgensen W. L. Molecular dynamics simulations of the unfolding of apomyoglobin in water. Biochemistry. 1993 Apr 27;32(16):4175–4184. doi: 10.1021/bi00067a004. [DOI] [PubMed] [Google Scholar]
  33. WEBER G., YOUNG L. B. FRAGMENTATION OF BOVINE SERUM ALBUMIN BY PEPSIN. I. THE ORIGIN OF THE ACID EXPANSION OF THE ALBUMIN MOLECULE. J Biol Chem. 1964 May;239:1415–1423. [PubMed] [Google Scholar]
  34. Weller P., Jeffreys A. J., Wilson V., Blanchetot A. Organization of the human myoglobin gene. EMBO J. 1984 Feb;3(2):439–446. doi: 10.1002/j.1460-2075.1984.tb01825.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Woodward C. Is the slow exchange core the protein folding core? Trends Biochem Sci. 1993 Oct;18(10):359–360. doi: 10.1016/0968-0004(93)90086-3. [DOI] [PubMed] [Google Scholar]

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