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

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From unfolding/refolding kinetic studies of the disulfide‐intact wild‐type protein and of a variant in which Pro93 had been replaced by Ala, it had been suggested that the Tyr92‐Ala93 peptide group also exists in the cis conformation in the native state.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.

[1]  R. L. Baldwin,et al.  Both the fast and slow refolding reactions of ribonuclease A yield native enzyme. , 1973, Proceedings of the National Academy of Sciences of the United States of America.

[2]  H. Halvorson,et al.  Consideration of the Possibility that the slow step in protein denaturation reactions is due to cis-trans isomerism of proline residues. , 1975, Biochemistry.

[3]  H. Scheraga,et al.  Stability of cis, trans, and nonplanar peptide groups. , 1976, Macromolecules.

[4]  B. Edwards,et al.  The refined crystal structure of a fully active semisynthetic ribonuclease at 1.8-A resolution. , 1990, The Journal of biological chemistry.

[5]  A. Wlodawer,et al.  Structure of phosphate-free ribonuclease A refined at 1.26 A. , 1988, Biochemistry.

[6]  J. Sack,et al.  CHAIN — A crystallographic modeling program , 1988 .

[7]  A. Brunger.  Free R value: a novel statistical quantity for assessing the accuracy of crystal structures. , 1992 .

[8]  H. Scheraga,et al.  A very fast phase in the refolding of disulfide-intact ribonuclease A: implications for the refolding and unfolding pathways. , 1994, Biochemistry.

[9]  Harold A. Scheraga,et al.  Mechanism of reductive protein unfolding , 1995, Nature Structural Biology.

[10]  H. Scheraga,et al.  Folding and unfolding kinetics of the proline-to-alanine mutants of bovine pancreatic ribonuclease A. , 1996, Biochemistry.

[11]  H. Scheraga,et al.  Nature of the unfolded state of ribonuclease A: effect of cis-trans X-Pro peptide bond isomerization. , 1996, Biochemistry.

[12]  P. Karplus.  Experimentally observed conformation‐dependent geometry and hidden strain in proteins , 1996, Protein science : a publication of the Protein Society.

[13]  P. Andrew Karplus,et al.  Improved R-factors for diffraction data analysis in macromolecular crystallography , 1997, Nature Structural Biology.

[14]  H. Scheraga,et al.  Tyrosyl interactions in the folding and unfolding of bovine pancreatic ribonuclease A: a study of tyrosine-to-phenylalanine mutants. , 1997, Biochemistry.

[15]  Z. Otwinowski,et al.  [20] Processing of X-ray diffraction data collected in oscillation mode. , 1997, Methods in enzymology.

[16]  H. Scheraga,et al.  Structural characterization of an analog of the major rate-determining disulfide folding intermediate of bovine pancreatic ribonuclease A. , 1997, Biochemistry.

[17]  H. Scheraga,et al.  Regeneration of bovine pancreatic ribonuclease A: identification of two nativelike three-disulfide intermediates involved in separate pathways. , 1998, Biochemistry.