Advertisement

Molecular Biology

, Volume 52, Issue 1, pp 52–61 | Cite as

Effect of Substitutions in Surface Amino Acid on Energy Profile of Apomyoglobin

  • M. A. Majorina
  • K. A. Glukhova
  • V. V. Marchenkov
  • B. S. Melnik
Structural Functional Analysis of Biopolymers and Their Complexes
  • 21 Downloads

Abstract

Studies on the process of spontaneous protein folding into a unique native state are an important issue of molecular biology. Apomyoglobin from the sperm whale is a convenient model for these studies in vitro. Here, we present the results of equilibrium and kinetic experiments carried out in a study on the folding and unfolding of eight mutant apomyoglobin forms of with hydrophobic amino acid substitutions on the protein surface. Calculated values of apparent constants of folding/unfolding rates, as well as the data on equilibrium conformational transitions in the urea concentration range of 0–6 M at 11°C are given. Based on the obtained information on the kinetic properties of the studied proteins, a Φ-value analysis of the transition state has been performed and values of urea concentrations corresponding to the midpoint of the transition from the native to intermediate state have been determined for the given forms of mutant apomyoglobin. It has been found that a significant increase in the stability of the native state can be achieved by a small number of amino acid substitutions on the protein surface. It has been shown that the substitution of only one amino acid residue exclusively affects the height of the energy barrier that separates different states of apomyoglobin.

Keywords

apomyoglobin protein folding tryptophan fluorescence circular dichroism stopped-flow experiments chevron plot 

Abbreviations

CD

circular dichroism

a.r.

amino-acid residue

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. 1.
    Matouschek A., Kellis J.T. Jr., Serrano L., Fersht A.R. 1989. Mapping the transition state and pathway of protein folding by protein engineering. Nature. 340, 122–126.CrossRefPubMedGoogle Scholar
  2. 2.
    Matouschek A., Kellis J.T. Jr., Serrano L., Fersht A.R. 1990. Transient folding intermediates characterized by protein engineering. Nature. 346, 440–445.CrossRefPubMedGoogle Scholar
  3. 3.
    Fersht A.R., Matouschek A., Serrano L. 1992. The folding of an enzyme: 1. Theory of protein engineering analysis and pathway of protein folding}. J. Mol. Biol. 224, 771–782.CrossRefPubMedGoogle Scholar
  4. 4.
    Matouschek A., Kellis Jr., Serrano L., Fersht A.R. 1998. Mapping the transition state and pathway of protein folding by protein engineering. Nature. 340, 122–126.CrossRefGoogle Scholar
  5. 5.
    Kawamura-Konishi Y., Kihara H., Suzuki H. 1988. Reconstitution of myoglobin from apoprotein and heme, monitored by stopped-flow absorption, fluorescence and circular dichroism. Eur. J. Biochem. 170, 589–595CrossRefPubMedGoogle Scholar
  6. 6.
    Baldwin R.L. 1996. On-pathway versus off-pathway folding intermediates. Fold. Des. 1, R1–R8.CrossRefPubMedGoogle Scholar
  7. 7.
    Barrick D., Baldwin R.L. 1993. The molten globuzle intermediate of apomyoglobin and the process of protein folding. Protein Sci. 2, 869–876.CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Barrick D., Baldwin R.L. 1993. Three-state analysis of sperm whale apomyoglobin folding. Biochemistry. 32, 3790–3796.CrossRefPubMedGoogle Scholar
  9. 9.
    Baryshnikova E.N., Sharapov M.G., Kashparov I.A., et al. 2005. Investigation of apomyoglobin stability depending on urea and temperature at two different pH values. Mol. Biol. 39, 292–297.CrossRefGoogle Scholar
  10. 10.
    Cavagnero S., Dyson H.J., Wright P.E. 1999. Effect of H helix destabilizing mutations on the kinetic and equilibrium folding of apomyoglobin. J. Mol. Biol. 285, 269–282.CrossRefPubMedGoogle Scholar
  11. 11.
    Schwarzinger S., Mohana-Borges R., Kroon G. J.A., et al. 2007. Structural characterization of partially folded intermediates of apomyoglobin H64F. Protein Sci. 17, 313–321.CrossRefGoogle Scholar
  12. 12.
    Xu M., Beresneva O., Rosario R., Roder H. 2012. Microsecond folding dynamics of apomyoglobin at acidic pH. J. Phys. Chem. B. 116, 7014–7025.CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Griko Y.V., Privalov P.L., Venyaminov S.Y., et al. 1988. Thermodynamic study of the apomyoglobin structure. J. Mol. Biol. 202, 127–138.CrossRefPubMedGoogle Scholar
  14. 14.
    Hughon F.M., Wright P.E., Baldwin R.L. 1990. Structural characterization of a partly folded apomyoglobin intermediate. Science. 249, 1544–1548.CrossRefGoogle Scholar
  15. 15.
    Jamin M., Antalic M., Loh S.N., et al. 2000. The unfolding enthalpy of the pH 4 molten globule of apomyoglobin measured by isothermal titration calorimetry. Protein Sci. 9, 1340–1346.CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Dolgikh D.A., Gilmanshin R.I., Brazhnikov E.V., et al. 1981. α-Lactalbumin: Compact state with fluctuating tertiary structure? FEBS Lett. 136, 311–315.CrossRefPubMedGoogle Scholar
  17. 17.
    Ptitsyn O.B. 1995. Molten globule and protein folding. Adv. Protein Chem. 47, 83–229.CrossRefPubMedGoogle Scholar
  18. 18.
    Eliezer D., Wright P.E. 1996. Is apomyoglobin a molten globule? Structural characterization by NMR. J. Mol. Biol. 263, 531–538.CrossRefPubMedGoogle Scholar
  19. 19.
    Jennings P.A., Wright P.E. 1993. Formation of a molten globule intermediate early in the kinetic folding pathway of apomyoglobin. Science. 262, 892–896.CrossRefPubMedGoogle Scholar
  20. 20.
    Baryshnikova E.N., Melnik B.S., Finkelstein A.V., et al. 2005. Three-state protein folding: Experimental determination of free-energy profile. Protein Sci. 14, 2658–2667.CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Samatova E.N., Katina N.S., Balobanov V.A., et al. 2009. How strong are side chain interactions in the folding intermediate? Protein Sci. 18, 2152–2159.CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Melnik T.N., Majorina M.A., Larina D.S., et al. 2014. Independent of their localization in protein the hydrophobic amino acid residues have no effect on the molten globule state of apomyoglobin and the disulfide bond on the surface of apomyoglobin stabilizes this intermediate state. PLOS ONE. 9, e98645.CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Samatova E.N., Melnik B.S., Balobanov V.A., et al. 2010. Folding intermediate and folding nucleus for I→N and U→I→N transitions in apomyoglobin: Contributions by conserved and nonconserved residues. Biophys. J. }}98}}, 1694–1702.Google Scholar
  24. 24.
    Guzman-Luna V., Quezada A.G., Diaz-Salazar A.J., et al. 2017. The effect of specific proline residues on the kinetic stability of the triosephosphate isomerases of two trypanosomes. Proteins. 85 (4), 571–579. doi doi 10.1002/prot.25231CrossRefPubMedGoogle Scholar
  25. 25.
    Frare E., Polverino de Laureto P., Scaramella E., et al. 2005. Chemical synthesis of the RGD-protein decorsin: ProAla replacement reduces protein thermostability. Protein Eng. Des. Sel. 18, 487–495.CrossRefPubMedGoogle Scholar
  26. 26.
    Schweiker K.L., Zarrine-Afsar A., Davidson A.R., et al. 2007. Computational design of the Fyn SH3 domain with increased stability through optimization of surface charge-charge interactions. Protein Sci. 16, 2694–2702.CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Strub C., Alies C., Lougarre A., et al. 2004. Mutation of exposed hydrophobic amino acids to arginine to increase protein stability. Biochemistry. 5, 1471–2091.Google Scholar
  28. 28.
    Ermolenko D.N., Richardson J.M., Makhatadze G.I. 2003. Noncharged amino acid residues at the solvent exposed positions in the middle and at the C terminus of the α-helix have the same helical propensity. Protein Sci. 12, 1169–1176.CrossRefPubMedPubMedCentralGoogle Scholar
  29. 29.
    Schweiker K.L., Makhatadze G.I. 2009. A computational approach for the rational design of stable proteins and enzymes: Optimization of surface charge-charge interactions. Methods Enzymol. 454, 175–211.CrossRefPubMedGoogle Scholar
  30. 30.
    Permyakov S.E., Makhatadze G.I., Owenius R., et al. 2005. How to improve nature: Study of the electrostatic properties of the surface of a-lactalbumin. Protein Eng. Des. Sel. 18, 425–433.CrossRefPubMedGoogle Scholar
  31. 31.
    Oda M., Kanaori K., Akasaka K. 2007. Increased thermodynamic stability by hydrophilic amino acid substitutions on the surface of Streptomyces subtilisin inhibitor. J. Biol. Macromol. 7, 3–8.CrossRefGoogle Scholar
  32. 32.
    Smulders R., Merck K.B., Aendekerk J., et al. 1995. The mutation Asp69-Ser affects the chaperone-like activity of αA-crystallin. Eur. J. Biochem. 232, 834–838.CrossRefPubMedGoogle Scholar
  33. 33.
    Jennings P.A., Stone M.J., Wright P.E. 1995. Overexpression of myoglobin and assignment of its amide, Ca and Cb resonances. J. Biomol. NMR. 6, 271–276.CrossRefPubMedGoogle Scholar
  34. 34.
    Aoto P.C., Nishimura C., Dyson H.J., et al. 2014. Probing the non-native H helix translocation in apomyoglobin folding intermediates. Biochemistry. 53, 3767–3780.CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Pleiades Publishing, Inc. 2018

Authors and Affiliations

  • M. A. Majorina
    • 1
  • K. A. Glukhova
    • 1
  • V. V. Marchenkov
    • 1
  • B. S. Melnik
    • 1
  1. 1.Institute of Protein ResearchRussian Academy of SciencesPushchino, Moscow oblastRussia

Personalised recommendations