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. 1997 Mar;6(3):657–665. doi: 10.1002/pro.5560060315

Thermal denaturation of iso-1-cytochrome c variants: comparison with solvent denaturation.

L M Herrmann 1, B E Bowler 1
PMCID: PMC2143682  PMID: 9070448

Abstract

Thermal denaturation studies as a function of pH were carried out on wild-type iso-1-cytochrome c and three variants of this protein at the solvent-exposed position 73 of the sequence. By examining the enthalpy and Tm at various pH values, the heat capacity increment (delta Cp), which is dominated by the degree of change in nonpolar hydration upon protein unfolding, was found for the wild type where lysine 73 is normally present and for three variants. For the Trp 73 variant, the delta Cp value (1.15 +/- 0.17 kcal/mol K) decreased slightly relative to wild-type iso-1-cytochrome c (1.40 +/- 0.06 kcal/mol K), while for the Ile 73 (1.65 +/- 0.07 kcal/mol K) and the Val 73 (1.50 +/- 0.06 kcal/mol K) variants, delta Cp increased slightly. In previous studies, the Trp 73, Ile 73, and Val 73 variants have been shown to have decreased m-values in guanidine hydrochloride denaturations relative to the wild-type protein (Hermann L, Bowler BE, Dong A, Caughey WS. 1995. The effects of hydrophilic to hydrophobic surface mutations on the denatured state of iso-1-cytochrome c: Investigation of aliphatic residues. Biochemistry 34:3040-3047). Both the m-value and delta Cp are related to the change in solvent exposure upon unfolding and other investigators have shown a correlation exists between these two parameters. However, for this subset of variants of iso-1-cytochrome c, a lack of correlation exists which implies that there may be basic differences between the guanidine hydrochloride and thermal denaturations of this protein. Spectroscopic data are consistent with different denatured states for thermal and guanidine hydrochloride unfolding. The different response of m-values and delta Cp for these variants will be discussed in this context.

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Selected References

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  1. Aune K. C., Salahuddin A., Zarlengo M. H., Tanford C. Evidence for residual structure in acid- and heat-denatured proteins. J Biol Chem. 1967 Oct 10;242(19):4486–4489. [PubMed] [Google Scholar]
  2. Babul J., Stellwagen E. The existence of heme-protein coordinate-covalent bonds in denaturing solvents. Biopolymers. 1971 Nov;10(11):2359–2361. doi: 10.1002/bip.360101125. [DOI] [PubMed] [Google Scholar]
  3. Becktel W. J., Schellman J. A. Protein stability curves. Biopolymers. 1987 Nov;26(11):1859–1877. doi: 10.1002/bip.360261104. [DOI] [PubMed] [Google Scholar]
  4. Betz S. F., Pielak G. J. Introduction of a disulfide bond into cytochrome c stabilizes a compact denatured state. Biochemistry. 1992 Dec 15;31(49):12337–12344. doi: 10.1021/bi00164a007. [DOI] [PubMed] [Google Scholar]
  5. Bowler B. E., Dong A., Caughey W. S. Characterization of the guanidine hydrochloride-denatured state of iso-1-cytochrome c by infrared spectroscopy. Biochemistry. 1994 Mar 8;33(9):2402–2408. doi: 10.1021/bi00175a008. [DOI] [PubMed] [Google Scholar]
  6. Bowler B. E., May K., Zaragoza T., York P., Dong A., Caughey W. S. Destabilizing effects of replacing a surface lysine of cytochrome c with aromatic amino acids: implications for the denatured state. Biochemistry. 1993 Jan 12;32(1):183–190. doi: 10.1021/bi00052a024. [DOI] [PubMed] [Google Scholar]
  7. Brandts J. F., Hunt L. The thermodynamics of protein denaturation. 3. The denaturation of ribonuclease in water and in aqueous urea and aqueous ethanol mixtures. J Am Chem Soc. 1967 Sep 13;89(19):4826–4838. doi: 10.1021/ja00995a002. [DOI] [PubMed] [Google Scholar]
  8. Cohen D. S., Pielak G. J. Stability of yeast iso-1-ferricytochrome c as a function of pH and temperature. Protein Sci. 1994 Aug;3(8):1253–1260. doi: 10.1002/pro.5560030811. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Cohen J. S., Hayes M. B. Nuclear magnetic resonance titration curves of histidine ring protons. V. Comparative study of cytochrome c from three species and the assignment of individual proton resonances. J Biol Chem. 1974 Sep 10;249(17):5472–5477. [PubMed] [Google Scholar]
  10. Creamer T. P., Srinivasan R., Rose G. D. Modeling unfolded states of peptides and proteins. Biochemistry. 1995 Dec 19;34(50):16245–16250. doi: 10.1021/bi00050a003. [DOI] [PubMed] [Google Scholar]
  11. Dill K. A. Dominant forces in protein folding. Biochemistry. 1990 Aug 7;29(31):7133–7155. doi: 10.1021/bi00483a001. [DOI] [PubMed] [Google Scholar]
  12. Dill K. A., Shortle D. Denatured states of proteins. Annu Rev Biochem. 1991;60:795–825. doi: 10.1146/annurev.bi.60.070191.004051. [DOI] [PubMed] [Google Scholar]
  13. 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]
  14. 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]
  15. Gómez J., Hilser V. J., Xie D., Freire E. The heat capacity of proteins. Proteins. 1995 Aug;22(4):404–412. doi: 10.1002/prot.340220410. [DOI] [PubMed] [Google Scholar]
  16. Herrmann L., Bowler B. E., Dong A., Caughey W. S. The effects of hydrophilic to hydrophobic surface mutations on the denatured state of iso-1-cytochrome c: investigation of aliphatic residues. Biochemistry. 1995 Mar 7;34(9):3040–3047. doi: 10.1021/bi00009a035. [DOI] [PubMed] [Google Scholar]
  17. Kuroda Y., Kidokoro S., Wada A. Thermodynamic characterization of cytochrome c at low pH. Observation of the molten globule state and of the cold denaturation process. J Mol Biol. 1992 Feb 20;223(4):1139–1153. doi: 10.1016/0022-2836(92)90265-l. [DOI] [PubMed] [Google Scholar]
  18. 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]
  19. Matthews B. W. Structural and genetic analysis of protein stability. Annu Rev Biochem. 1993;62:139–160. doi: 10.1146/annurev.bi.62.070193.001035. [DOI] [PubMed] [Google Scholar]
  20. Miller W. G., Goebel C. V. Dimensions of protein random coils. Biochemistry. 1968 Nov;7(11):3925–3935. doi: 10.1021/bi00851a021. [DOI] [PubMed] [Google Scholar]
  21. Monera O. D., Kay C. M., Hodges R. S. Protein denaturation with guanidine hydrochloride or urea provides a different estimate of stability depending on the contributions of electrostatic interactions. Protein Sci. 1994 Nov;3(11):1984–1991. doi: 10.1002/pro.5560031110. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Muthukrishnan K., Nall B. T. Effective concentrations of amino acid side chains in an unfolded protein. Biochemistry. 1991 May 14;30(19):4706–4710. doi: 10.1021/bi00233a010. [DOI] [PubMed] [Google Scholar]
  23. Myers J. K., Pace C. N., Scholtz J. M. Denaturant m values and heat capacity changes: relation to changes in accessible surface areas of protein unfolding. Protein Sci. 1995 Oct;4(10):2138–2148. doi: 10.1002/pro.5560041020. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Oliveberg M., Vuilleumier S., Fersht A. R. Thermodynamic study of the acid denaturation of barnase and its dependence on ionic strength: evidence for residual electrostatic interactions in the acid/thermally denatured state. Biochemistry. 1994 Jul 26;33(29):8826–8832. doi: 10.1021/bi00195a026. [DOI] [PubMed] [Google Scholar]
  25. 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]
  26. Pakula A. A., Sauer R. T. Reverse hydrophobic effects relieved by amino-acid substitutions at a protein surface. Nature. 1990 Mar 22;344(6264):363–364. doi: 10.1038/344363a0. [DOI] [PubMed] [Google Scholar]
  27. Pfeil W., Privalov P. L. Thermodynamic investigations of proteins. III. Thermodynamic description of lysozyme. Biophys Chem. 1976 Jan;4(1):41–50. doi: 10.1016/0301-4622(76)80005-1. [DOI] [PubMed] [Google Scholar]
  28. Privalov P. L., Gill S. J. Stability of protein structure and hydrophobic interaction. Adv Protein Chem. 1988;39:191–234. doi: 10.1016/s0065-3233(08)60377-0. [DOI] [PubMed] [Google Scholar]
  29. 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]
  30. Privalov P. L., Makhatadze G. I. Contribution of hydration and non-covalent interactions to the heat capacity effect on protein unfolding. J Mol Biol. 1992 Apr 5;224(3):715–723. doi: 10.1016/0022-2836(92)90555-x. [DOI] [PubMed] [Google Scholar]
  31. 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]
  32. 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]
  33. Privalov P. L., Tiktopulo E. I., Venyaminov SYu, Griko YuV, Makhatadze G. I., Khechinashvili N. N. Heat capacity and conformation of proteins in the denatured state. J Mol Biol. 1989 Feb 20;205(4):737–750. doi: 10.1016/0022-2836(89)90318-5. [DOI] [PubMed] [Google Scholar]
  34. Rose G. D., Gierasch L. M., Smith J. A. Turns in peptides and proteins. Adv Protein Chem. 1985;37:1–109. doi: 10.1016/s0065-3233(08)60063-7. [DOI] [PubMed] [Google Scholar]
  35. Shortle D., Chan H. S., Dill K. A. Modeling the effects of mutations on the denatured states of proteins. Protein Sci. 1992 Feb;1(2):201–215. doi: 10.1002/pro.5560010202. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Shortle D., Meeker A. K., Freire E. Stability mutants of staphylococcal nuclease: large compensating enthalpy-entropy changes for the reversible denaturation reaction. Biochemistry. 1988 Jun 28;27(13):4761–4768. doi: 10.1021/bi00413a027. [DOI] [PubMed] [Google Scholar]
  37. 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]
  38. Tanford C., Aune K. C. Thermodynamics of the denaturation of lysozyme by guanidine hydrochloride. 3. Dependence on temperature. Biochemistry. 1970 Jan 20;9(2):206–211. doi: 10.1021/bi00804a003. [DOI] [PubMed] [Google Scholar]
  39. Tanford C. Protein denaturation. Adv Protein Chem. 1968;23:121–282. doi: 10.1016/s0065-3233(08)60401-5. [DOI] [PubMed] [Google Scholar]
  40. Tiffany M. L., Krimm S. Effect of temperature on the circular dichroism spectra of polypeptides in the extended state. Biopolymers. 1972;11(11):2309–2316. doi: 10.1002/bip.1972.360111109. [DOI] [PubMed] [Google Scholar]
  41. Tsong T. Y. An acid induced conformational transition of denatured cytochrome c in urea and guanidine hydrochloride solutions. Biochemistry. 1975 Apr 8;14(7):1542–1547. doi: 10.1021/bi00678a031. [DOI] [PubMed] [Google Scholar]
  42. Tsong T. Y. The Trp-59 fluorescence of ferricytochrome c as a sensitive measure of the over-all protein conformation. J Biol Chem. 1974 Mar 25;249(6):1988–1990. [PubMed] [Google Scholar]

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