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
. 1993 Dec;2(12):2037–2049. doi: 10.1002/pro.5560021205

Thermodynamics of unfolding for turkey ovomucoid third domain: thermal and chemical denaturation.

L Swint 1, A D Robertson 1
PMCID: PMC2142319  PMID: 8298454

Abstract

We have used thermal and chemical denaturation to characterize the thermodynamics of unfolding for turkey ovomucoid third domain (OMTKY3). Thermal denaturation was monitored spectroscopically at a number of wave-lengths and data were subjected to van't Hoff analysis; at pH 2.0, the midpoint of denaturation (Tm) occurs at 58.6 +/- 0.4 degrees C and the enthalpy of unfolding at this temperature (delta Hm) is 40.8 +/- 0.3 kcal/mol. When Tm was perturbed by varying pH and denaturant concentration, the resulting plots of delta Hm versus Tm yield a mean value of 590 +/- 120 cal/(mol.K) for the change in heat capacity upon unfolding (delta Cp). A global fit of the same data to an equation that includes the temperature dependence for the enthalpy of unfolding yielded a value of 640 +/- 110 cal/(mol.K). We also performed a variation of the linear extrapolation method described by Pace and Laurents, which is an independent method for determining delta Cp (Pace, C.N. & Laurents, D., 1989, Biochemistry 28, 2520-2525). First, OMTKY3 was thermally denatured in the presence of a variety of denaturant concentrations. Linear extrapolations were then made from isothermal slices through the transition region of the denaturation curves. When extrapolated free energies of unfolding (delta Gu) were plotted versus temperature, the resulting curve appeared linear; therefore, delta Cp could not be determined. However, the data for delta Gu versus denaturant concentration are linear over an extraordinarily wide range of concentrations. Moreover, extrapolated values of delta Gu in urea are identical to values measured directly.

Full Text

The Full Text of this article is available as a PDF (1.3 MB).

Selected References

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

  1. Adler A. J., Greenfield N. J., Fasman G. D. Circular dichroism and optical rotatory dispersion of proteins and polypeptides. Methods Enzymol. 1973;27:675–735. doi: 10.1016/s0076-6879(73)27030-1. [DOI] [PubMed] [Google Scholar]
  2. Alexander P., Fahnestock S., Lee T., Orban J., Bryan P. Thermodynamic analysis of the folding of the streptococcal protein G IgG-binding domains B1 and B2: why small proteins tend to have high denaturation temperatures. Biochemistry. 1992 Apr 14;31(14):3597–3603. doi: 10.1021/bi00129a007. [DOI] [PubMed] [Google Scholar]
  3. Alonso D. O., Dill K. A. Solvent denaturation and stabilization of globular proteins. Biochemistry. 1991 Jun 18;30(24):5974–5985. doi: 10.1021/bi00238a023. [DOI] [PubMed] [Google Scholar]
  4. Appel P., Yang J. T. Helix-coil transition of polyl-glutamic acid and polyl-lysine in D2O. Biochemistry. 1965 Jul;4(7):1244–1249. doi: 10.1021/bi00883a005. [DOI] [PubMed] [Google Scholar]
  5. Ardelt W., Laskowski M., Jr Effect of single amino acid replacements on the thermodynamics of the reactive site peptide bond hydrolysis in ovomucoid third domain. J Mol Biol. 1991 Aug 20;220(4):1041–1053. doi: 10.1016/0022-2836(91)90370-l. [DOI] [PubMed] [Google Scholar]
  6. Brumbaugh E. E., Huang C. Parameter estimation in binary mixtures of phospholipids. Methods Enzymol. 1992;210:521–539. doi: 10.1016/0076-6879(92)10027-b. [DOI] [PubMed] [Google Scholar]
  7. Das B. K., Agarwal S. K., Khan M. Y. Ovomucoid domains: preparation and physico-chemical characterization. Biochem Int. 1990 Dec;22(6):993–1004. [PubMed] [Google Scholar]
  8. Doig A. J., Williams D. H. Is the hydrophobic effect stabilizing or destabilizing in proteins? The contribution of disulphide bonds to protein stability. J Mol Biol. 1991 Jan 20;217(2):389–398. doi: 10.1016/0022-2836(91)90551-g. [DOI] [PubMed] [Google Scholar]
  9. Griko Y. V., Privalov P. L. Calorimetric study of the heat and cold denaturation of beta-lactoglobulin. Biochemistry. 1992 Sep 22;31(37):8810–8815. doi: 10.1021/bi00152a017. [DOI] [PubMed] [Google Scholar]
  10. HERMANS J., Jr, SCHERAGA H. A. The thermally induced configurational change of ribonuclease in water and deuterium. Biochim Biophys Acta. 1959 Dec;36:534–535. doi: 10.1016/0006-3002(59)90197-0. [DOI] [PubMed] [Google Scholar]
  11. Hildebrandt P. G., Copeland R. A., Spiro T. G., Otlewski J., Laskowski M., Jr, Prendergast F. G. Tyrosine hydrogen-bonding and environmental effects in proteins probed by ultraviolet resonance Raman spectroscopy. Biochemistry. 1988 Jul 26;27(15):5426–5433. doi: 10.1021/bi00415a007. [DOI] [PubMed] [Google Scholar]
  12. Johnson M. L., Faunt L. M. Parameter estimation by least-squares methods. Methods Enzymol. 1992;210:1–37. doi: 10.1016/0076-6879(92)10003-v. [DOI] [PubMed] [Google Scholar]
  13. Kato I., Schrode J., Kohr W. J., Laskowski M., Jr Chicken ovomucoid: determination of its amino acid sequence, determination of the trypsin reactive site, and preparation of all three of its domains. Biochemistry. 1987 Jan 13;26(1):193–201. doi: 10.1021/bi00375a027. [DOI] [PubMed] [Google Scholar]
  14. Kim K. S., Tao F., Fuchs J., Danishefsky A. T., Housset D., Wlodawer A., Woodward C. Crevice-forming mutants of bovine pancreatic trypsin inhibitor: stability changes and new hydrophobic surface. Protein Sci. 1993 Apr;2(4):588–596. doi: 10.1002/pro.5560020410. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Kim P. S., Baldwin R. L. Specific intermediates in the folding reactions of small proteins and the mechanism of protein folding. Annu Rev Biochem. 1982;51:459–489. doi: 10.1146/annurev.bi.51.070182.002331. [DOI] [PubMed] [Google Scholar]
  16. Laskowski M., Jr, Apostol I., Ardelt W., Cook J., Giletto A., Kelly C. A., Lu W. Y., Park S. J., Qasim M. A., Whatley H. E. Amino acid sequences of ovomucoid third domain from 25 additional species of birds. J Protein Chem. 1990 Dec;9(6):715–725. doi: 10.1007/BF01024766. [DOI] [PubMed] [Google Scholar]
  17. Laskowski M., Jr, Kato I., Ardelt W., Cook J., Denton A., Empie M. W., Kohr W. J., Park S. J., Parks K., Schatzley B. L. Ovomucoid third domains from 100 avian species: isolation, sequences, and hypervariability of enzyme-inhibitor contact residues. Biochemistry. 1987 Jan 13;26(1):202–221. doi: 10.1021/bi00375a028. [DOI] [PubMed] [Google Scholar]
  18. Lemm U., Wenzel M. Stabilisierung von Enzymen und Antiseren durch Schweres Wasser. Eur J Biochem. 1981 Jun 1;116(3):441–445. doi: 10.1111/j.1432-1033.1981.tb05355.x. [DOI] [PubMed] [Google Scholar]
  19. Makhatadze G. I., Privalov P. L. Protein interactions with urea and guanidinium chloride. A calorimetric study. J Mol Biol. 1992 Jul 20;226(2):491–505. doi: 10.1016/0022-2836(92)90963-k. [DOI] [PubMed] [Google Scholar]
  20. Murphy K. P., Gill S. J. Solid model compounds and the thermodynamics of protein unfolding. J Mol Biol. 1991 Dec 5;222(3):699–709. doi: 10.1016/0022-2836(91)90506-2. [DOI] [PubMed] [Google Scholar]
  21. Murphy K. P., Privalov P. L., Gill S. J. Common features of protein unfolding and dissolution of hydrophobic compounds. Science. 1990 Feb 2;247(4942):559–561. doi: 10.1126/science.2300815. [DOI] [PubMed] [Google Scholar]
  22. Pace C. N. Determination and analysis of urea and guanidine hydrochloride denaturation curves. Methods Enzymol. 1986;131:266–280. doi: 10.1016/0076-6879(86)31045-0. [DOI] [PubMed] [Google Scholar]
  23. Pace C. N., Laurents D. V. A new method for determining the heat capacity change for protein folding. Biochemistry. 1989 Mar 21;28(6):2520–2525. doi: 10.1021/bi00432a026. [DOI] [PubMed] [Google Scholar]
  24. Pace C. N., Laurents D. V., Thomson J. A. pH dependence of the urea and guanidine hydrochloride denaturation of ribonuclease A and ribonuclease T1. Biochemistry. 1990 Mar 13;29(10):2564–2572. doi: 10.1021/bi00462a019. [DOI] [PubMed] [Google Scholar]
  25. Pace C. N., Vanderburg K. E. Determining globular protein stability: guanidine hydrochloride denaturation of myoglobin. Biochemistry. 1979 Jan 23;18(2):288–292. doi: 10.1021/bi00569a008. [DOI] [PubMed] [Google Scholar]
  26. Pfeil W., Privalov P. L. Thermodynamic investigations of proteins. II. Calorimetric study of lysozyme denaturation by guanidine hydrochloride. Biophys Chem. 1976 Jan;4(1):33–40. doi: 10.1016/0301-4622(76)80004-x. [DOI] [PubMed] [Google Scholar]
  27. Privalov P. L. Cold denaturation of proteins. Crit Rev Biochem Mol Biol. 1990;25(4):281–305. doi: 10.3109/10409239009090612. [DOI] [PubMed] [Google Scholar]
  28. 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]
  29. Privalov P. L., Khechinashvili N. N. A thermodynamic approach to the problem of stabilization of globular protein structure: a calorimetric study. J Mol Biol. 1974 Jul 5;86(3):665–684. doi: 10.1016/0022-2836(74)90188-0. [DOI] [PubMed] [Google Scholar]
  30. Privalov P. L. Stability of proteins: small globular proteins. Adv Protein Chem. 1979;33:167–241. doi: 10.1016/s0065-3233(08)60460-x. [DOI] [PubMed] [Google Scholar]
  31. Read R. J., Fujinaga M., Sielecki A. R., James M. N. Structure of the complex of Streptomyces griseus protease B and the third domain of the turkey ovomucoid inhibitor at 1.8-A resolution. Biochemistry. 1983 Sep 13;22(19):4420–4433. doi: 10.1021/bi00288a012. [DOI] [PubMed] [Google Scholar]
  32. Renner M., Hinz H. J., Scharf M., Engels J. W. Thermodynamics of unfolding of the alpha-amylase inhibitor tendamistat. Correlations between accessible surface area and heat capacity. J Mol Biol. 1992 Feb 5;223(3):769–779. doi: 10.1016/0022-2836(92)90988-v. [DOI] [PubMed] [Google Scholar]
  33. Robertson A. D., Westler W. M., Markley J. L. Two-dimensional NMR studies of Kazal proteinase inhibitors. 1. Sequence-specific assignments and secondary structure of turkey ovomucoid third domain. Biochemistry. 1988 Apr 5;27(7):2519–2529. doi: 10.1021/bi00407a039. [DOI] [PubMed] [Google Scholar]
  34. Santoro M. M., Bolen D. W. A test of the linear extrapolation of unfolding free energy changes over an extended denaturant concentration range. Biochemistry. 1992 May 26;31(20):4901–4907. doi: 10.1021/bi00135a022. [DOI] [PubMed] [Google Scholar]
  35. Scopes R. K. Measurement of protein by spectrophotometry at 205 nm. Anal Biochem. 1974 May;59(1):277–282. doi: 10.1016/0003-2697(74)90034-7. [DOI] [PubMed] [Google Scholar]
  36. Spolar R. S., Livingstone J. R., Record M. T., Jr Use of liquid hydrocarbon and amide transfer data to estimate contributions to thermodynamic functions of protein folding from the removal of nonpolar and polar surface from water. Biochemistry. 1992 Apr 28;31(16):3947–3955. doi: 10.1021/bi00131a009. [DOI] [PubMed] [Google Scholar]
  37. Tanford C. Protein denaturation. C. Theoretical models for the mechanism of denaturation. Adv Protein Chem. 1970;24:1–95. [PubMed] [Google Scholar]
  38. Timasheff S. N. Water as ligand: preferential binding and exclusion of denaturants in protein unfolding. Biochemistry. 1992 Oct 20;31(41):9857–9864. doi: 10.1021/bi00156a001. [DOI] [PubMed] [Google Scholar]
  39. WYMAN J., Jr LINKED FUNCTIONS AND RECIPROCAL EFFECTS IN HEMOGLOBIN: A SECOND LOOK. Adv Protein Chem. 1964;19:223–286. doi: 10.1016/s0065-3233(08)60190-4. [DOI] [PubMed] [Google Scholar]
  40. Waheed A., Qasim M. A., Salahuddin A. Characterization of stable conformational states in urea-induced transition in ovomucoid. Eur J Biochem. 1977 Jun 15;76(2):383–390. doi: 10.1111/j.1432-1033.1977.tb11606.x. [DOI] [PubMed] [Google Scholar]
  41. Watanabe K., Matsuda T., Sato Y. The secondary structure of ovomucoid and its domains as studied by circular dichroism. Biochim Biophys Acta. 1981 Feb 27;667(2):242–250. doi: 10.1016/0005-2795(81)90189-6. [DOI] [PubMed] [Google Scholar]

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

RESOURCES