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. 1998 Jul;75(1):484–492. doi: 10.1016/S0006-3495(98)77537-X

Chemical denaturation: potential impact of undetected intermediates in the free energy of unfolding and m-values obtained from a two-state assumption.

J L Soulages 1
PMCID: PMC1299722  PMID: 9649410

Abstract

The chemical unfolding transition of a protein was simulated, including the presence of an intermediate (I) in equilibrium with the native (N) and unfolded (U) states. The calculations included free energies of unfolding, DeltaGuw, in the range of 1.4 kcal/mol to 10 kcal/mol and three different global m-values. The simulations included a broad range of equilibrium constants for the N left arrow over right arrow I process. The dependence of the N <--> I equilibrium on the concentration of denaturant was also included in the simulations. Apparent DeltaGuw and m-values were obtained from the simulated unfolding transitions by fitting the data to a two-state unfolding process. The potential errors were calculated for two typical experimental situations: 1) the unfolding is monitored by a physical property that does not distinguish between native and intermediate states (case I), and 2) the physical property does not distinguish between intermediate and unfolded states (case II). The results obtained indicated that in the presence of an intermediate, and in both experimental situations, the free energy of unfolding and the m-values could be largely underestimated. The errors in DeltaGuw and m-values do not depend on the m-values that characterize the global N <--> U transition. They are dependent on the equilibrium constant for the N <--> I transition and its characteristic m1-value. The extent of the underestimation increases for higher energies of unfolding. Including no random error in the simulations, it was estimated that the underestimation in DeltaGuw could range between 25% and 35% for unfolding transitions of 3-10 kcal/mol (case I). In case II, the underestimation in DeltaGuw could be even larger than in case I. In the same energy range, a 50% error in the m-value could also take place. The fact that most of the mutant proteins are characterized by both a lower m-value and a lower stability than the wild-type protein suggests that in some cases the results could have been underestimated due to the application of the two-state assumption.

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Selected References

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  1. Barrick D., Baldwin R. L. Three-state analysis of sperm whale apomyoglobin folding. Biochemistry. 1993 Apr 13;32(14):3790–3796. doi: 10.1021/bi00065a035. [DOI] [PubMed] [Google Scholar]
  2. Carra J. H., Anderson E. A., Privalov P. L. Three-state thermodynamic analysis of the denaturation of staphylococcal nuclease mutants. Biochemistry. 1994 Sep 6;33(35):10842–10850. doi: 10.1021/bi00201a035. [DOI] [PubMed] [Google Scholar]
  3. Carra J. H., Privalov P. L. Energetics of denaturation and m values of staphylococcal nuclease mutants. Biochemistry. 1995 Feb 14;34(6):2034–2041. doi: 10.1021/bi00006a025. [DOI] [PubMed] [Google Scholar]
  4. Fink A. L. Molten globules. Methods Mol Biol. 1995;40:343–360. doi: 10.1385/0-89603-301-5:343. [DOI] [PubMed] [Google Scholar]
  5. 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]
  6. 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]
  7. Kuwajima K., Nitta K., Yoneyama M., Sugai S. Three-state denaturation of alpha-lactalbumin by guanidine hydrochloride. J Mol Biol. 1976 Sep 15;106(2):359–373. doi: 10.1016/0022-2836(76)90091-7. [DOI] [PubMed] [Google Scholar]
  8. Matthews B. W. Studies on protein stability with T4 lysozyme. Adv Protein Chem. 1995;46:249–278. doi: 10.1016/s0065-3233(08)60337-x. [DOI] [PubMed] [Google Scholar]
  9. 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]
  10. 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]
  11. Ptitsyn O. B. Molten globule and protein folding. Adv Protein Chem. 1995;47:83–229. doi: 10.1016/s0065-3233(08)60546-x. [DOI] [PubMed] [Google Scholar]
  12. 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]
  13. Santoro M. M., Bolen D. W. Unfolding free energy changes determined by the linear extrapolation method. 1. Unfolding of phenylmethanesulfonyl alpha-chymotrypsin using different denaturants. Biochemistry. 1988 Oct 18;27(21):8063–8068. doi: 10.1021/bi00421a014. [DOI] [PubMed] [Google Scholar]
  14. Shortle D. Probing the determinants of protein folding and stability with amino acid substitutions. J Biol Chem. 1989 Apr 5;264(10):5315–5318. [PubMed] [Google Scholar]
  15. Shortle D. Staphylococcal nuclease: a showcase of m-value effects. Adv Protein Chem. 1995;46:217–247. doi: 10.1016/s0065-3233(08)60336-8. [DOI] [PubMed] [Google Scholar]
  16. Yao M., Bolen D. W. How valid are denaturant-induced unfolding free energy measurements? Level of conformance to common assumptions over an extended range of ribonuclease A stability. Biochemistry. 1995 Mar 21;34(11):3771–3781. doi: 10.1021/bi00011a035. [DOI] [PubMed] [Google Scholar]

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