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
. 1998 May;7(5):1255–1258. doi: 10.1002/pro.5560070522

Crystal structures of two mutants that have implications for the folding of bovine pancreatic ribonuclease A.

M A Pearson 1, P A Karplus 1, R W Dodge 1, J H Laity 1, H A Scheraga 1
PMCID: PMC2143996  PMID: 9605332

Abstract

The Tyr92-Pro93 peptide group of bovine pancreatic ribonuclease A (RNase A) exists in the cis conformation in the native state. From unfolding/refolding kinetic studies of the disulfide-intact wild-type protein and of a variant in which Pro93 had been replaced by Ala, it had been suggested that the Tyr92-Ala93 peptide group also exists in the cis conformation in the native state. Here, we report the crystal structure of the P93A variant. Although there is disorder in the region of residues 92 and 93, the best structural model contains a cis peptide at this position, lending support to the results of the kinetics experiments. We also report the crystal structure of the C[40, 95]A variant, which is an analog of the major rate-determining three-disulfide intermediate in the oxidative folding of RNase A, missing the 40-95 disulfide bond. As had been detected by NMR spectroscopy, the crystal structure of this analog shows disorder in the region surrounding the missing disulfide. However, the global chain fold of the remainder of the protein, including the disulfide bond between Cys65 and Cys72, appears to be unaffected by the mutation.

Full Text

The Full Text of this article is available as a PDF (361.5 KB).

Selected References

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

  1. Dodge R. W., Scheraga H. A. Folding and unfolding kinetics of the proline-to-alanine mutants of bovine pancreatic ribonuclease A. Biochemistry. 1996 Feb 6;35(5):1548–1559. doi: 10.1021/bi952348q. [DOI] [PubMed] [Google Scholar]
  2. Houry W. A., Rothwarf D. M., Scheraga H. A. A very fast phase in the refolding of disulfide-intact ribonuclease A: implications for the refolding and unfolding pathways. Biochemistry. 1994 Mar 8;33(9):2516–2530. doi: 10.1021/bi00175a022. [DOI] [PubMed] [Google Scholar]
  3. Houry W. A., Scheraga H. A. Nature of the unfolded state of ribonuclease A: effect of cis-trans X-Pro peptide bond isomerization. Biochemistry. 1996 Sep 10;35(36):11719–11733. doi: 10.1021/bi960745a. [DOI] [PubMed] [Google Scholar]
  4. Juminaga D., Wedemeyer W. J., Garduño-Júarez R., McDonald M. A., Scheraga H. A. Tyrosyl interactions in the folding and unfolding of bovine pancreatic ribonuclease A: a study of tyrosine-to-phenylalanine mutants. Biochemistry. 1997 Aug 19;36(33):10131–10145. doi: 10.1021/bi970711i. [DOI] [PubMed] [Google Scholar]
  5. Karplus P. A. Experimentally observed conformation-dependent geometry and hidden strain in proteins. Protein Sci. 1996 Jul;5(7):1406–1420. doi: 10.1002/pro.5560050719. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Laity J. H., Lester C. C., Shimotakahara S., Zimmerman D. E., Montelione G. T., Scheraga H. A. Structural characterization of an analog of the major rate-determining disulfide folding intermediate of bovine pancreatic ribonuclease A. Biochemistry. 1997 Oct 21;36(42):12683–12699. doi: 10.1021/bi970878b. [DOI] [PubMed] [Google Scholar]
  7. Li Y. J., Rothwarf D. M., Scheraga H. A. Mechanism of reductive protein unfolding. Nat Struct Biol. 1995 Jun;2(6):489–494. doi: 10.1038/nsb0695-489. [DOI] [PubMed] [Google Scholar]
  8. Martin P. D., Doscher M. S., Edwards B. F. The refined crystal structure of a fully active semisynthetic ribonuclease at 1.8-A resolution. J Biol Chem. 1987 Nov 25;262(33):15930–15938. doi: 10.2210/pdb1srn/pdb. [DOI] [PubMed] [Google Scholar]
  9. Rothwarf D. M., Li Y. J., Scheraga H. A. Regeneration of bovine pancreatic ribonuclease A: identification of two nativelike three-disulfide intermediates involved in separate pathways. Biochemistry. 1998 Mar 17;37(11):3760–3766. doi: 10.1021/bi972822n. [DOI] [PubMed] [Google Scholar]
  10. Shimotakahara S., Rios C. B., Laity J. H., Zimmerman D. E., Scheraga H. A., Montelione G. T. NMR structural analysis of an analog of an intermediate formed in the rate-determining step of one pathway in the oxidative folding of bovine pancreatic ribonuclease A: automated analysis of 1H, 13C, and 15N resonance assignments for wild-type and [C65S, C72S] mutant forms. Biochemistry. 1997 Jun 10;36(23):6915–6929. doi: 10.1021/bi963024k. [DOI] [PubMed] [Google Scholar]
  11. Wlodawer A., Svensson L. A., Sjölin L., Gilliland G. L. Structure of phosphate-free ribonuclease A refined at 1.26 A. Biochemistry. 1988 Apr 19;27(8):2705–2717. doi: 10.1021/bi00408a010. [DOI] [PubMed] [Google Scholar]
  12. Zimmerman S. S., Scheraga H. A. Stability of cis, trans, and nonplanar peptide groups. Macromolecules. 1976 May-Jun;9(3):408–416. doi: 10.1021/ma60051a005. [DOI] [PubMed] [Google Scholar]

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

RESOURCES