Skip to main content
PMC home page
Biophysical Journal logoLink to Biophysical Journal
. 2002 Oct;83(4):2259–2269. doi: 10.1016/S0006-3495(02)73986-6

Pretransitional structural changes in the thermal denaturation of ribonuclease S and S protein.

Simona D Stelea 1, Timothy A Keiderling 1
PMCID: PMC1302314  PMID: 12324443

Abstract

Two mechanisms have been proposed for the thermal unfolding of ribonuclease S (RNase S). The first is a sequential partial unfolding of the S peptide/S protein complex followed by dissociation, whereas the second is a concerted denaturation/dissociation. The thermal denaturation of ribonuclease S and its fragment, the S protein, were followed with circular dichroism and infrared spectra. These spectra were analyzed by the principal component method of factor analysis. The use of multiple spectral techniques and of factor analysis monitored different aspects of the denaturation simultaneously. The unfolding pathway was compared with that of the parent enzyme ribonuclease A (RNase A), and a model was devised to assess the importance of the dissociation in the unfolding. The unfolding patterns obtained from the melting curves of each protein imply the existence of multiple intermediate states and/or processes. Our data provide evidence that the pretransition in the unfolding of ribonuclease S is due to partial unfolding of the S protein/S peptide complex and that the dissociation occurs at higher temperature. Our observations are consistent with a sequential denaturation mechanism in which at least one partial unfolding step comes before the main conformational transition, which is instead a concerted, final unfolding/dissociation step.

Full Text

The Full Text of this article is available as a PDF (191.7 KB).

Selected References

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

  1. Baello B. I., Pancoska P., Keiderling T. A. Enhanced prediction accuracy of protein secondary structure using hydrogen exchange Fourier transform infrared spectroscopy. Anal Biochem. 2000 Apr 10;280(1):46–57. doi: 10.1006/abio.2000.4483. [DOI] [PubMed] [Google Scholar]
  2. Baumruk V., Pancoska P., Keiderling T. A. Predictions of secondary structure using statistical analyses of electronic and vibrational circular dichroism and Fourier transform infrared spectra of proteins in H2O. J Mol Biol. 1996 Jun 21;259(4):774–791. doi: 10.1006/jmbi.1996.0357. [DOI] [PubMed] [Google Scholar]
  3. Catanzano F., Giancola C., Graziano G., Barone G. Temperature-induced denaturation of ribonuclease S: a thermodynamic study. Biochemistry. 1996 Oct 15;35(41):13378–13385. doi: 10.1021/bi960855h. [DOI] [PubMed] [Google Scholar]
  4. Chakshusmathi G., Ratnaparkhi G. S., Madhu P. K., Varadarajan R. Native-state hydrogen-exchange studies of a fragment complex can provide structural information about the isolated fragments. Proc Natl Acad Sci U S A. 1999 Jul 6;96(14):7899–7904. doi: 10.1073/pnas.96.14.7899. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Goldberg J. M., Baldwin R. L. Kinetic mechanism of a partial folding reaction. 2. Nature of the transition state. Biochemistry. 1998 Feb 24;37(8):2556–2563. doi: 10.1021/bi972403q. [DOI] [PubMed] [Google Scholar]
  6. Goldberg M. S., Zhang J., Sondek S., Matthews C. R., Fox R. O., Horwich A. L. Native-like structure of a protein-folding intermediate bound to the chaperonin GroEL. Proc Natl Acad Sci U S A. 1997 Feb 18;94(4):1080–1085. doi: 10.1073/pnas.94.4.1080. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Graziano G., Catanzano F., Giancola C., Barone G. DSC study of the thermal stability of S-protein and S-peptide/S-protein. Biochemistry. 1996 Oct 15;35(41):13386–13392. doi: 10.1021/bi960856+. [DOI] [PubMed] [Google Scholar]
  8. Haris P. I., Lee D. C., Chapman D. A Fourier transform infrared investigation of the structural differences between ribonuclease A and ribonuclease S. Biochim Biophys Acta. 1986 Dec 12;874(3):255–265. doi: 10.1016/0167-4838(86)90024-5. [DOI] [PubMed] [Google Scholar]
  9. Hearn R. P., Richards F. M., Sturtevant J. M., Watt G. D. Thermodynamics of the binding of S-peptide to S-protein to form ribonuclease S.. Biochemistry. 1971 Mar 2;10(5):806–817. doi: 10.1021/bi00781a013. [DOI] [PubMed] [Google Scholar]
  10. Horwitz J., Strickland E. H. Absorption and circular dichroism spectra of ribonuclease-S at 77 degrees K. J Biol Chem. 1971 Jun 10;246(11):3749–3752. doi: 10.2172/4043504. [DOI] [PubMed] [Google Scholar]
  11. Kim E. E., Varadarajan R., Wyckoff H. W., Richards F. M. Refinement of the crystal structure of ribonuclease S. Comparison with and between the various ribonuclease A structures. Biochemistry. 1992 Dec 15;31(49):12304–12314. doi: 10.1021/bi00164a004. [DOI] [PubMed] [Google Scholar]
  12. Klee W. A. Studies on the conformation of ribonuclease S-peptide. Biochemistry. 1968 Aug;7(8):2731–2736. doi: 10.1021/bi00848a006. [DOI] [PubMed] [Google Scholar]
  13. Kurapkat G., Krüger P., Wollmer A., Fleischhauer J., Kramer B., Zobel E., Koslowski A., Botterweck H., Woody R. W. Calculations of the CD spectrum of bovine pancreatic ribonuclease. Biopolymers. 1997 Mar;41(3):267–287. doi: 10.1002/(SICI)1097-0282(199703)41:3<267::AID-BIP3>3.0.CO;2-Q. [DOI] [PubMed] [Google Scholar]
  14. Labhardt A. M., Baldwin R. L. Recombination of S-peptide with S-protein during folding of ribonuclease S. I. Folding pathways of the slow-folding and fast-folding classes of unfolded S-protein. J Mol Biol. 1979 Nov 25;135(1):231–244. doi: 10.1016/0022-2836(79)90349-8. [DOI] [PubMed] [Google Scholar]
  15. 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]
  16. Lumry R., Biltonen R. Validity of the "two-state" hypothesis for conformational transitions of proteins. Biopolymers. 1966 Sep;4(8):917–944. doi: 10.1002/bip.1966.360040808. [DOI] [PubMed] [Google Scholar]
  17. Makhatadze G. I., Clore G. M., Gronenborn A. M. Solvent isotope effect and protein stability. Nat Struct Biol. 1995 Oct;2(10):852–855. doi: 10.1038/nsb1095-852. [DOI] [PubMed] [Google Scholar]
  18. Pancoska P., Bitto E., Janota V., Keiderling T. A. Quantitative analysis of vibrational circular dichroism spectra of proteins. Problems and perspectives. Faraday Discuss. 1994;(99):287–310. doi: 10.1039/fd9949900287. [DOI] [PubMed] [Google Scholar]
  19. Pancoska P., Bitto E., Janota V., Urbanova M., Gupta V. P., Keiderling T. A. Comparison of and limits of accuracy for statistical analyses of vibrational and electronic circular dichroism spectra in terms of correlations to and predictions of protein secondary structure. Protein Sci. 1995 Jul;4(7):1384–1401. doi: 10.1002/pro.5560040713. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Pancoska P., Yasui S. C., Keiderling T. A. Statistical analyses of the vibrational circular dichroism of selected proteins and relationship to secondary structures. Biochemistry. 1991 May 21;30(20):5089–5103. doi: 10.1021/bi00234a036. [DOI] [PubMed] [Google Scholar]
  21. RICHARDS F. M., VITHAYATHIL P. J. The preparation of subtilisn-modified ribonuclease and the separation of the peptide and protein components. J Biol Chem. 1959 Jun;234(6):1459–1465. [PubMed] [Google Scholar]
  22. Rico M., Bruix M., Santoro J., Gonzalez C., Neira J. L., Nieto J. L., Herranz J. Sequential 1H-NMR assignment and solution structure of bovine pancreatic ribonuclease A. Eur J Biochem. 1989 Aug 15;183(3):623–638. doi: 10.1111/j.1432-1033.1989.tb21092.x. [DOI] [PubMed] [Google Scholar]
  23. SELA M., ANFINSEN C. B. Some spectrophotometric and polarimetric experiments with ribonuclease. Biochim Biophys Acta. 1957 May;24(2):229–235. doi: 10.1016/0006-3002(57)90186-5. [DOI] [PubMed] [Google Scholar]
  24. Schreier A. A., Baldwin R. L. Concentration-dependent hydrogen exchange kinetics of 3H-labeled S-peptide in ribonuclease S. J Mol Biol. 1976 Aug 15;105(3):409–426. doi: 10.1016/0022-2836(76)90101-7. [DOI] [PubMed] [Google Scholar]
  25. Schreier A. A., Baldwin R. L. Mechanism of dissociation of S-peptide from ribonuclease S. Biochemistry. 1977 Sep 20;16(19):4203–4209. doi: 10.1021/bi00638a012. [DOI] [PubMed] [Google Scholar]
  26. Seshadri S., Oberg K. A., Fink A. L. Thermally denatured ribonuclease A retains secondary structure as shown by FTIR. Biochemistry. 1994 Feb 15;33(6):1351–1355. doi: 10.1021/bi00172a010. [DOI] [PubMed] [Google Scholar]
  27. Sherwood L. M., Potts J. T., Jr Conformational studies of pancreatic ribonuclease and its subtilisin-produced derivatives. J Biol Chem. 1965 Oct;240(10):3799–3805. [PubMed] [Google Scholar]
  28. Shindo H., Matsuura S., Cohen J. S. Conformation of ribonuclease S-protein. Experientia. 1979 Oct 15;35(10):1284–1285. doi: 10.1007/BF01963958. [DOI] [PubMed] [Google Scholar]
  29. Simons E. R., Schneider E. G., Blout E. R. Thermal effects on the circular dichroism spectra of ribonuclease A and of ribonuclease S-protein. J Biol Chem. 1969 Aug 10;244(15):4023–4026. [PubMed] [Google Scholar]
  30. 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]
  31. Stelea S. D., Pancoska P., Benight A. S., Keiderling T. A. Thermal unfolding of ribonuclease A in phosphate at neutral pH: deviations from the two-state model. Protein Sci. 2001 May;10(5):970–978. doi: 10.1110/ps.47101. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Strickland E. H. Interactions contributing to the tyrosyl circular dichroism bands of ribonuclease S and A. Biochemistry. 1972 Aug 29;11(18):3465–3474. doi: 10.1021/bi00768a022. [DOI] [PubMed] [Google Scholar]
  33. Varadarajan R., Richards F. M. Crystallographic structures of ribonuclease S variants with nonpolar substitution at position 13: packing and cavities. Biochemistry. 1992 Dec 15;31(49):12315–12327. doi: 10.1021/bi00164a005. [DOI] [PubMed] [Google Scholar]
  34. Wlodawer A., Bott R., Sjölin L. The refined crystal structure of ribonuclease A at 2.0 A resolution. J Biol Chem. 1982 Feb 10;257(3):1325–1332. [PubMed] [Google Scholar]
  35. Wyckoff H. W., Tsernoglou D., Hanson A. W., Knox J. R., Lee B., Richards F. M. The three-dimensional structure of ribonuclease-S. Interpretation of an electron density map at a nominal resolution of 2 A. J Biol Chem. 1970 Jan 25;245(2):305–328. [PubMed] [Google Scholar]

Articles from Biophysical Journal are provided here courtesy of The Biophysical Society

RESOURCES