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
. 1999 Apr;8(4):832–840. doi: 10.1110/ps.8.4.832

Thermodynamic stability of ribonuclease A in alkylurea solutions and preferential solvation changes accompanying its thermal denaturation: a calorimetric and spectroscopic study.

N Poklar 1, N Petrovcic 1, M Oblak 1, G Vesnaver 1
PMCID: PMC2144304  PMID: 10211829

Abstract

The effect of methylurea, N,N'-dimethylurea, ethylurea, and butylurea as well as guanidine hydrochloride (GuHCl), urea and pH on the thermal stability, structural properties, and preferential solvation changes accompanying the thermal unfolding of ribonuclease A (RNase A) has been investigated by differential scanning calorimetry (DSC), UV, and circular dichroism (CD) spectroscopy. The results show that the thermal stability of RNase A decreases with increasing concentration of denaturants and the size of the hydrophobic group substituted on the urea molecule. From CD measurements in the near- and far-UV range, it has been observed that the tertiary structure of RNase A melts at about 3 degrees C lower temperature than its secondary structure, which means that the hierarchy in structural building blocks exists for RNase A even at conditions at which according to DSC and UV measurements the RNase A unfolding can be interpreted in terms of a two-state approximation. The far-UV CD spectra also show that the final denatured states of RNase A at high temperatures in the presence of different denaturants including 4.5 M GuHCl are similar to each other but different from the one obtained in 4.5 M GuHCl at 25 degrees C. The concentration dependence of the preferential solvation change delta r23, expressed as the number of cosolvent molecules entering or leaving the solvation shell of the protein upon denaturation and calculated from DSC data, shows the same relative denaturation efficiency of alkylureas as other methods.

Full Text

The Full Text of this article is available as a PDF (222.2 KB).

Selected References

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

  1. Bierzynski A., Baldwin R. L. Local secondary structure in ribonuclease A denatured by guanidine . HCl near 1 degree C. J Mol Biol. 1982 Nov 25;162(1):173–186. doi: 10.1016/0022-2836(82)90167-x. [DOI] [PubMed] [Google Scholar]
  2. Bychkova V. E., Dujsekina A. E., Klenin S. I., Tiktopulo E. I., Uversky V. N., Ptitsyn O. B. Molten globule-like state of cytochrome c under conditions simulating those near the membrane surface. Biochemistry. 1996 May 14;35(19):6058–6063. doi: 10.1021/bi9522460. [DOI] [PubMed] [Google Scholar]
  3. Dunbar J., Yennawar H. P., Banerjee S., Luo J., Farber G. K. The effect of denaturants on protein structure. Protein Sci. 1997 Aug;6(8):1727–1733. doi: 10.1002/pro.5560060813. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Fabian H., Mantsch H. H. Ribonuclease A revisited: infrared spectroscopic evidence for lack of native-like secondary structures in the thermally denatured state. Biochemistry. 1995 Oct 17;34(41):13651–13655. doi: 10.1021/bi00041a046. [DOI] [PubMed] [Google Scholar]
  5. Fink A. L., Calciano L. J., Goto Y., Kurotsu T., Palleros D. R. Classification of acid denaturation of proteins: intermediates and unfolded states. Biochemistry. 1994 Oct 18;33(41):12504–12511. doi: 10.1021/bi00207a018. [DOI] [PubMed] [Google Scholar]
  6. Freer S. T., Kraut J., Robertus J. D., Wright H. T., Xuong N. H. Chymotrypsinogen: 2.5-angstrom crystal structure, comparison with alpha-chymotrypsin, and implications for zymogen activation. Biochemistry. 1970 Apr 28;9(9):1997–2009. doi: 10.1021/bi00811a022. [DOI] [PubMed] [Google Scholar]
  7. Fujita Y., Noda Y. Effect of reductive alkylation on thermal stability of ribonuclease A and chymotrypsinogen A. Int J Pept Protein Res. 1991 Nov;38(5):445–452. doi: 10.1111/j.1399-3011.1991.tb01525.x. [DOI] [PubMed] [Google Scholar]
  8. GORDON J. A., JENCKS W. P. The relationship of structure to the effectiveness of denaturing agents for proteins. Biochemistry. 1963 Jan-Feb;2:47–57. doi: 10.1021/bi00901a011. [DOI] [PubMed] [Google Scholar]
  9. Greene R. F., Jr, Pace C. N. Urea and guanidine hydrochloride denaturation of ribonuclease, lysozyme, alpha-chymotrypsin, and beta-lactoglobulin. J Biol Chem. 1974 Sep 10;249(17):5388–5393. [PubMed] [Google Scholar]
  10. Haynie D. T., Freire E. Estimation of the folding/unfolding energetics of marginally stable proteins using differential scanning calorimetry. Anal Biochem. 1994 Jan;216(1):33–41. doi: 10.1006/abio.1994.1004. [DOI] [PubMed] [Google Scholar]
  11. Hirota N., Mizuno K., Goto Y. Cooperative alpha-helix formation of beta-lactoglobulin and melittin induced by hexafluoroisopropanol. Protein Sci. 1997 Feb;6(2):416–421. doi: 10.1002/pro.5560060218. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Houry W. A., Rothwarf D. M., Scheraga H. A. Circular dichroism evidence for the presence of burst-phase intermediates on the conformational folding pathway of ribonuclease A. Biochemistry. 1996 Aug 6;35(31):10125–10133. doi: 10.1021/bi960617m. [DOI] [PubMed] [Google Scholar]
  13. Jayaraman G., Kumar T. K., Arunkumar A. I., Yu C. 2,2,2-Trifluoroethanol induces helical conformation in an all beta-sheet protein. Biochem Biophys Res Commun. 1996 May 6;222(1):33–37. doi: 10.1006/bbrc.1996.0693. [DOI] [PubMed] [Google Scholar]
  14. Kamatari Y. O., Konno T., Kataoka M., Akasaka K. The methanol-induced globular and expanded denatured states of cytochrome c: a study by CD fluorescence, NMR and small-angle X-ray scattering. J Mol Biol. 1996 Jun 14;259(3):512–523. doi: 10.1006/jmbi.1996.0336. [DOI] [PubMed] [Google Scholar]
  15. Kovrigin E. L., Potekhin S. A. Preferential solvation changes upon lysozyme heat denaturation in mixed solvents. Biochemistry. 1997 Jul 29;36(30):9195–9199. doi: 10.1021/bi9630164. [DOI] [PubMed] [Google Scholar]
  16. Kugimiya M., Bigelow C. C. The denatured states of lysozyme. Can J Biochem. 1973 May;51(5):581–585. doi: 10.1139/o73-072. [DOI] [PubMed] [Google Scholar]
  17. Labhardt A. M. Secondary structure in ribonuclease. I. Equilibrium folding transitions seen by amide circular dichroism. J Mol Biol. 1982 May 15;157(2):331–355. doi: 10.1016/0022-2836(82)90238-8. [DOI] [PubMed] [Google Scholar]
  18. Lapanje S., Poklar N. Calorimetric and circular dichroic studies of the thermal denaturation of beta-lactoglobulin. Biophys Chem. 1989 Oct;34(2):155–162. doi: 10.1016/0301-4622(89)80053-5. [DOI] [PubMed] [Google Scholar]
  19. Liu Y., Sturtevant J. M. The observed change in heat capacity accompanying the thermal unfolding of proteins depends on the composition of the solution and on the method employed to change the temperature of unfolding. Biochemistry. 1996 Mar 5;35(9):3059–3062. doi: 10.1021/bi952198j. [DOI] [PubMed] [Google Scholar]
  20. 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]
  21. Marky L. A., Breslauer K. J. Calculating thermodynamic data for transitions of any molecularity from equilibrium melting curves. Biopolymers. 1987 Sep;26(9):1601–1620. doi: 10.1002/bip.360260911. [DOI] [PubMed] [Google Scholar]
  22. Pace C. N. Contribution of the hydrophobic effect to globular protein stability. J Mol Biol. 1992 Jul 5;226(1):29–35. doi: 10.1016/0022-2836(92)90121-y. [DOI] [PubMed] [Google Scholar]
  23. Plaza del Pino I. M., Sanchez-Ruiz J. M. An osmolyte effect on the heat capacity change for protein folding. Biochemistry. 1995 Jul 11;34(27):8621–8630. doi: 10.1021/bi00027a011. [DOI] [PubMed] [Google Scholar]
  24. Poklar N., Lah J., Salobir M., Macek P., Vesnaver G. pH and temperature-induced molten globule-like denatured states of equinatoxin II: a study by UV-melting, DSC, far- and near-UV CD spectroscopy, and ANS fluorescence. Biochemistry. 1997 Nov 25;36(47):14345–14352. doi: 10.1021/bi971719v. [DOI] [PubMed] [Google Scholar]
  25. Poklar N., Lapanje S. Solvation of beta-lactoglobulin in alkylurea solutions. Biophys Chem. 1992 Apr;42(3):283–290. doi: 10.1016/0301-4622(92)80020-6. [DOI] [PubMed] [Google Scholar]
  26. Poklar N., Vesnaver G., Lapanje S. Interactions of alpha-chymotrypsinogen A with alkylureas. Biophys Chem. 1996 Jan;57(2-3):279–289. doi: 10.1016/0301-4622(95)00073-1. [DOI] [PubMed] [Google Scholar]
  27. Poklar N., Vesnaver G., Lapanje S. Thermodynamics of denaturation of alpha-chymotrypsinogen A in aqueous urea and alkylurea solutions. J Protein Chem. 1995 Nov;14(8):709–719. doi: 10.1007/BF01886910. [DOI] [PubMed] [Google Scholar]
  28. 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]
  29. Privalov P. L., Makhatadze G. I. Heat capacity of proteins. II. Partial molar heat capacity of the unfolded polypeptide chain of proteins: protein unfolding effects. J Mol Biol. 1990 May 20;213(2):385–391. doi: 10.1016/S0022-2836(05)80198-6. [DOI] [PubMed] [Google Scholar]
  30. Reinstädler D., Fabian H., Backmann J., Naumann D. Refolding of thermally and urea-denatured ribonuclease A monitored by time-resolved FTIR spectroscopy. Biochemistry. 1996 Dec 10;35(49):15822–15830. doi: 10.1021/bi961810j. [DOI] [PubMed] [Google Scholar]
  31. Schellman J. A. A simple model for solvation in mixed solvents. Applications to the stabilization and destabilization of macromolecular structures. Biophys Chem. 1990 Aug 31;37(1-3):121–140. doi: 10.1016/0301-4622(90)88013-i. [DOI] [PubMed] [Google Scholar]
  32. Schellman J. A. The thermodynamics of solvent exchange. Biopolymers. 1994 Aug;34(8):1015–1026. doi: 10.1002/bip.360340805. [DOI] [PubMed] [Google Scholar]
  33. Scheraga H. A. Structural studies of pancreatic ribonuclease. Fed Proc. 1967 Sep;26(5):1380–1387. [PubMed] [Google Scholar]
  34. Schrier E. E., Mackey L. D. The effect of salts of organic acids and bases on the thermal transition of ribonuclease. J Phys Chem. 1968 Feb;72(2):733–736. doi: 10.1021/j100848a057. [DOI] [PubMed] [Google Scholar]
  35. Shirley B. A., Stanssens P., Hahn U., Pace C. N. Contribution of hydrogen bonding to the conformational stability of ribonuclease T1. Biochemistry. 1992 Jan 28;31(3):725–732. doi: 10.1021/bi00118a013. [DOI] [PubMed] [Google Scholar]
  36. Sosnick T. R., Trewhella J. Denatured states of ribonuclease A have compact dimensions and residual secondary structure. Biochemistry. 1992 Sep 8;31(35):8329–8335. doi: 10.1021/bi00150a029. [DOI] [PubMed] [Google Scholar]
  37. Tsong T. Y., Hearn R. P., Wrathall D. P., Sturtevant J. M. A calorimetric study of thermally induced conformational transitions of ribonuclease A and certain of its derivatives. Biochemistry. 1970 Jun 23;9(13):2666–2677. doi: 10.1021/bi00815a015. [DOI] [PubMed] [Google Scholar]
  38. Von Hippel P. H., Wong K. Y. On the conformational stability of globular proteins. The effects of various electrolytes and nonelectrolytes on the thermal ribonuclease transition. J Biol Chem. 1965 Oct;240(10):3909–3923. [PubMed] [Google Scholar]
  39. Xie G., Timasheff S. N. Mechanism of the stabilization of ribonuclease A by sorbitol: preferential hydration is greater for the denatured then for the native protein. Protein Sci. 1997 Jan;6(1):211–221. doi: 10.1002/pro.5560060123. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Xie G., Timasheff S. N. Temperature dependence of the preferential interactions of ribonuclease A in aqueous co-solvent systems: thermodynamic analysis. Protein Sci. 1997 Jan;6(1):222–232. doi: 10.1002/pro.5560060124. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES