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. 1994 Apr;3(4):638–649. doi: 10.1002/pro.5560030412

Slow-folding kinetics of ribonuclease-A by volume change and circular dichroism: evidence for two independent reactions.

J A Ybe 1, P C Kahn 1
PMCID: PMC2142864  PMID: 8003982

Abstract

The slow refolding of guanidine-HCl-denatured ribonuclease-A was studied by volume change and by kinetic CD at 222 and 276 nm. Dilatometric measurements revealed that on refolding there is a fast volume change of +232 mL/mol of protein. This is followed by a very slow nonexponential change that takes about 25 min to reach equilibrium. By adding varying amounts of (NH4)2SO4, the slow volume change curve was resolved into 2 concurrent reactions. The faster of the 2 slow events entails a negative volume change of -64 mL/mol of protein and appears to arise from proline isomerization. The slower process, attended by a positive change of +53 mL/mol of protein, has properties consistent with the "XY" reaction of Lin and Brands (1983, Biochemistry 22:563-573). This reaction is so named because the conformational nature of neither its initial (Y) nor its final state (X) is known; the transition is characterized solely by its absorbance and fluorescence kinetics. These are the first direct physical measures attributable to the "XY" process. The early formation of a compact structure in the event responsible for the rapid +232-mL/mol volume change, however, is consistent with the sequential model of folding (Cook KH, Schmid FX, Baldwin RL, 1979, Proc Natl Acad Sci USA 76:6157-6161; Kim PS, Baldwin RL, 1980, Biochemistry 19:6124-6129). The usefulness of volume change measurements as a method of detecting structural rearrangements was confirmed by finding agreement between time constants obtained from parallel volume change and kinetic CD experiments. The measured volume changes arise from both changes in hydration and changes in the packing of atoms in the interior of the protein.

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Selected References

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  1. Adler M., Scheraga H. A. Nonnative isomers of proline-93 and -114 predominate in heat-unfolded ribonuclease A. Biochemistry. 1990 Sep 11;29(36):8211–8216. doi: 10.1021/bi00488a003. [DOI] [PubMed] [Google Scholar]
  2. Brandts J. F., Halvorson H. R., Brennan M. Consideration of the Possibility that the slow step in protein denaturation reactions is due to cis-trans isomerism of proline residues. Biochemistry. 1975 Nov 4;14(22):4953–4963. doi: 10.1021/bi00693a026. [DOI] [PubMed] [Google Scholar]
  3. Bycroft M., Matouschek A., Kellis J. T., Jr, Serrano L., Fersht A. R. Detection and characterization of a folding intermediate in barnase by NMR. Nature. 1990 Aug 2;346(6283):488–490. doi: 10.1038/346488a0. [DOI] [PubMed] [Google Scholar]
  4. Cooke R., Kuntz I. D. The properties of water in biological systems. Annu Rev Biophys Bioeng. 1974;3(0):95–126. doi: 10.1146/annurev.bb.03.060174.000523. [DOI] [PubMed] [Google Scholar]
  5. Evans P. A., Dobson C. M., Kautz R. A., Hatfull G., Fox R. O. Proline isomerism in staphylococcal nuclease characterized by NMR and site-directed mutagenesis. Nature. 1987 Sep 17;329(6136):266–268. doi: 10.1038/329266a0. [DOI] [PubMed] [Google Scholar]
  6. Fischer G., Bang H., Mech C. Nachweis einer Enzymkatalyse für die cis-trans-Isomerisierung der Peptidbindung in prolinhaltigen Peptiden. Biomed Biochim Acta. 1984;43(10):1101–1111. [PubMed] [Google Scholar]
  7. Fischer G., Bang H. The refolding of urea-denatured ribonuclease A is catalyzed by peptidyl-prolyl cis-trans isomerase. Biochim Biophys Acta. 1985 Mar 22;828(1):39–42. doi: 10.1016/0167-4838(85)90006-8. [DOI] [PubMed] [Google Scholar]
  8. Garel J. R., Baldwin R. L. Both the fast and slow refolding reactions of ribonuclease A yield native enzyme. Proc Natl Acad Sci U S A. 1973 Dec;70(12):3347–3351. doi: 10.1073/pnas.70.12.3347. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Garel J. R., Nall B. T., Baldwin R. L. Guanidine-unfolded state of ribonuclease A contains both fast- and slow-refolding species. Proc Natl Acad Sci U S A. 1976 Jun;73(6):1853–1857. doi: 10.1073/pnas.73.6.1853. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Gittelman M. S., Matthews C. R. Folding and stability of trp aporepressor from Escherichia coli. Biochemistry. 1990 Jul 31;29(30):7011–7020. doi: 10.1021/bi00482a009. [DOI] [PubMed] [Google Scholar]
  11. Goux W. J., Hooker T. M., Jr Letter: Contribution of tyrosine residues to the optical activity of ribonuclease S. J Am Chem Soc. 1975 Mar 19;97(6):1605–1606. doi: 10.1021/ja00839a071. [DOI] [PubMed] [Google Scholar]
  12. Harrison S. C., Durbin R. Is there a single pathway for the folding of a polypeptide chain? Proc Natl Acad Sci U S A. 1985 Jun;82(12):4028–4030. doi: 10.1073/pnas.82.12.4028. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Horwitz J., Strickland E. H., Billups C. Analysis of the vibrational structure in the near-ultraviolet circular dichroism and absorption spectra of tyrosine derivatives and ribonuclease-A at 77 degrees K. J Am Chem Soc. 1970 Apr 8;92(7):2119–2129. doi: 10.1021/ja00710a054. [DOI] [PubMed] [Google Scholar]
  14. KAUZMANN W. Some factors in the interpretation of protein denaturation. Adv Protein Chem. 1959;14:1–63. doi: 10.1016/s0065-3233(08)60608-7. [DOI] [PubMed] [Google Scholar]
  15. 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]
  16. 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]
  17. Kim P. S., Baldwin R. L. Structural intermediates trapped during the folding of ribonuclease A by amide proton exchange. Biochemistry. 1980 Dec 23;19(26):6124–6129. doi: 10.1021/bi00567a027. [DOI] [PubMed] [Google Scholar]
  18. Kuntz I. D., Jr, Kauzmann W. Hydration of proteins and polypeptides. Adv Protein Chem. 1974;28:239–345. doi: 10.1016/s0065-3233(08)60232-6. [DOI] [PubMed] [Google Scholar]
  19. Kuwajima K., Garvey E. P., Finn B. E., Matthews C. R., Sugai S. Transient intermediates in the folding of dihydrofolate reductase as detected by far-ultraviolet circular dichroism spectroscopy. Biochemistry. 1991 Aug 6;30(31):7693–7703. doi: 10.1021/bi00245a005. [DOI] [PubMed] [Google Scholar]
  20. Kuwajima K., Hiraoka Y., Ikeguchi M., Sugai S. Comparison of the transient folding intermediates in lysozyme and alpha-lactalbumin. Biochemistry. 1985 Feb 12;24(4):874–881. doi: 10.1021/bi00325a010. [DOI] [PubMed] [Google Scholar]
  21. 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]
  22. Kuwajima K. The molten globule state as a clue for understanding the folding and cooperativity of globular-protein structure. Proteins. 1989;6(2):87–103. doi: 10.1002/prot.340060202. [DOI] [PubMed] [Google Scholar]
  23. Kuwajima K., Yamaya H., Miwa S., Sugai S., Nagamura T. Rapid formation of secondary structure framework in protein folding studied by stopped-flow circular dichroism. FEBS Lett. 1987 Aug 31;221(1):115–118. doi: 10.1016/0014-5793(87)80363-0. [DOI] [PubMed] [Google Scholar]
  24. Lin L. N., Brandts J. F. Evidence showing that a proline-specific endopeptidase has an absolute requirement for a trans peptide bond immediately preceding the active bond. Biochemistry. 1983 Sep 13;22(19):4480–4485. doi: 10.1021/bi00288a020. [DOI] [PubMed] [Google Scholar]
  25. Lin L. N., Brandts J. F. Isomerization of proline-93 during the unfolding and refolding of ribonuclease A. Biochemistry. 1983 Feb 1;22(3):559–563. doi: 10.1021/bi00272a006. [DOI] [PubMed] [Google Scholar]
  26. Lin L. N., Brandts J. F. Mechanism for the unfolding and refolding of ribonuclease A. Kinetic studies utilizing spectroscopic methods. Biochemistry. 1983 Feb 1;22(3):564–573. doi: 10.1021/bi00272a007. [DOI] [PubMed] [Google Scholar]
  27. Lin L. N., Brandts J. F. Mechanism for the unfolding and refolding of ribonuclease A. Simulations using a simple model with no structural intermediates. Biochemistry. 1983 Feb 1;22(3):573–580. doi: 10.1021/bi00272a008. [DOI] [PubMed] [Google Scholar]
  28. Lin L. N., Brandts J. F. Separation of the nativelike intermediate from unfolded forms during refolding of ribonuclease A. Biochemistry. 1988 Dec 13;27(25):9037–9042. doi: 10.1021/bi00425a023. [DOI] [PubMed] [Google Scholar]
  29. Pflumm M., Luchins J., Beychok S. Stopped-flow circular dichroism. Methods Enzymol. 1986;130:519–534. doi: 10.1016/0076-6879(86)30024-7. [DOI] [PubMed] [Google Scholar]
  30. Ptitsyn O. B., Pain R. H., Semisotnov G. V., Zerovnik E., Razgulyaev O. I. Evidence for a molten globule state as a general intermediate in protein folding. FEBS Lett. 1990 Mar 12;262(1):20–24. doi: 10.1016/0014-5793(90)80143-7. [DOI] [PubMed] [Google Scholar]
  31. Radford S. E., Dobson C. M., Evans P. A. The folding of hen lysozyme involves partially structured intermediates and multiple pathways. Nature. 1992 Jul 23;358(6384):302–307. doi: 10.1038/358302a0. [DOI] [PubMed] [Google Scholar]
  32. Roder H., Elöve G. A., Englander S. W. Structural characterization of folding intermediates in cytochrome c by H-exchange labelling and proton NMR. Nature. 1988 Oct 20;335(6192):700–704. doi: 10.1038/335700a0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Schmid F. X. A native-like intermediate on the ribonuclease A folding pathway. 1. Detection by tyrosine fluorescence changes. Eur J Biochem. 1981;114(1):105–109. doi: 10.1111/j.1432-1033.1981.tb06179.x. [DOI] [PubMed] [Google Scholar]
  34. Schmid F. X., Baldwin R. L. Acid catalysis of the formation of the slow-folding species of RNase A: evidence that the reaction is proline isomerization. Proc Natl Acad Sci U S A. 1978 Oct;75(10):4764–4768. doi: 10.1073/pnas.75.10.4764. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Schmid F. X., Blaschek H. A native-like intermediate on the ribonuclease A folding pathway. 2. Comparison of its properties to native ribonuclease A. Eur J Biochem. 1981;114(1):111–117. doi: 10.1111/j.1432-1033.1981.tb06180.x. [DOI] [PubMed] [Google Scholar]
  36. Schmid F. X. Proline isomerization during refolding of ribonuclease A is accelerated by the presence of folding intermediates. FEBS Lett. 1986 Mar 31;198(2):217–220. doi: 10.1016/0014-5793(86)80408-2. [DOI] [PubMed] [Google Scholar]
  37. Schultz D. A., Baldwin R. L. Cis proline mutants of ribonuclease A. I. Thermal stability. Protein Sci. 1992 Jul;1(7):910–916. doi: 10.1002/pro.5560010709. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Strickland E. H. Aromatic contributions to circular dichroism spectra of proteins. CRC Crit Rev Biochem. 1974 Jan;2(1):113–175. doi: 10.3109/10409237409105445. [DOI] [PubMed] [Google Scholar]
  39. Sugawara T., Kuwajima K., Sugai S. Folding of staphylococcal nuclease A studied by equilibrium and kinetic circular dichroism spectra. Biochemistry. 1991 Mar 12;30(10):2698–2706. doi: 10.1021/bi00224a018. [DOI] [PubMed] [Google Scholar]
  40. Texter F. L., Spencer D. B., Rosenstein R., Matthews C. R. Intramolecular catalysis of a proline isomerization reaction in the folding of dihydrofolate reductase. Biochemistry. 1992 Jun 30;31(25):5687–5691. doi: 10.1021/bi00140a001. [DOI] [PubMed] [Google Scholar]
  41. Udgaonkar J. B., Baldwin R. L. NMR evidence for an early framework intermediate on the folding pathway of ribonuclease A. Nature. 1988 Oct 20;335(6192):694–699. doi: 10.1038/335694a0. [DOI] [PubMed] [Google Scholar]
  42. Wilkins J. A., Cone J., Randhawa Z. I., Wood D., Warren M. K., Witkowska H. E. A study of intermediates involved in the folding pathway for recombinant human macrophage colony-stimulating factor (M-CSF): evidence for two distinct folding pathways. Protein Sci. 1993 Feb;2(2):244–254. doi: 10.1002/pro.5560020213. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Yang J. T., Wu C. S., Martinez H. M. Calculation of protein conformation from circular dichroism. Methods Enzymol. 1986;130:208–269. doi: 10.1016/0076-6879(86)30013-2. [DOI] [PubMed] [Google Scholar]

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