The crystal structure of the cis-proline to glycine variant (P114G) of ribonuclease A

David A. Schultz, Alan M. Friedman, Mark White, Robert O. Fox

Research output: Contribution to journalArticle

17 Citations (Scopus)

Abstract

Replacement of a cis-proline by glycine at position 114 in ribonuclease A leads to a large decrease in thermal stability and simplifies the refolding kinetics. A crystallographic approach was used to determine whether the decrease in thermal stability results from the presence of a cis glycine peptide bond, or from a localized structural rearrangement caused by the isomerization of the mutated cis 114 peptide bond. The structure was solved at 2.0 Å resolution and refined to an R-factor of 19.5% and an Rfree of 21.9%. The overall conformation of the protein was similar to that of wild-type ribonuclease A; however, there was a large localized rearrangement of the mutated loop (residues 110-117 - a 9.3 Å shift of the Cα atom of residue 114). The peptide bond before Gly114 is in the trans configuration. Interestingly, a large anomalous difference density was found near residue 114, and was attributed to a bound cesium ion present in the crystallization experiment. The trans isomeric configuration of the peptide bond in the folded state of this mutant is consistent with the refolding kinetics previously reported, and the associated protein conformational change provides an explanation for the decreased thermal stability.

Original languageEnglish (US)
Pages (from-to)2862-2870
Number of pages9
JournalProtein Science
Volume14
Issue number11
DOIs
StatePublished - Nov 2005

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Pancreatic Ribonuclease
Proline
Glycine
Crystal structure
Peptides
Thermodynamic stability
Hot Temperature
R388
Cesium
Protein Conformation
Kinetics
Isomerization
Crystallization
Conformations
Proteins
Ions
Atoms
Experiments

Keywords

  • Cesium binding site
  • cis
  • Conformational changes
  • P114G
  • Proline
  • Protein structure/folding
  • Ribonuclease A
  • RNase A
  • Stability and mutagenesis
  • Structure
  • trans
  • X-ray crystallography

ASJC Scopus subject areas

  • Biochemistry

Cite this

The crystal structure of the cis-proline to glycine variant (P114G) of ribonuclease A. / Schultz, David A.; Friedman, Alan M.; White, Mark; Fox, Robert O.

In: Protein Science, Vol. 14, No. 11, 11.2005, p. 2862-2870.

Research output: Contribution to journalArticle

Schultz, David A. ; Friedman, Alan M. ; White, Mark ; Fox, Robert O. / The crystal structure of the cis-proline to glycine variant (P114G) of ribonuclease A. In: Protein Science. 2005 ; Vol. 14, No. 11. pp. 2862-2870.
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abstract = "Replacement of a cis-proline by glycine at position 114 in ribonuclease A leads to a large decrease in thermal stability and simplifies the refolding kinetics. A crystallographic approach was used to determine whether the decrease in thermal stability results from the presence of a cis glycine peptide bond, or from a localized structural rearrangement caused by the isomerization of the mutated cis 114 peptide bond. The structure was solved at 2.0 {\AA} resolution and refined to an R-factor of 19.5{\%} and an Rfree of 21.9{\%}. The overall conformation of the protein was similar to that of wild-type ribonuclease A; however, there was a large localized rearrangement of the mutated loop (residues 110-117 - a 9.3 {\AA} shift of the Cα atom of residue 114). The peptide bond before Gly114 is in the trans configuration. Interestingly, a large anomalous difference density was found near residue 114, and was attributed to a bound cesium ion present in the crystallization experiment. The trans isomeric configuration of the peptide bond in the folded state of this mutant is consistent with the refolding kinetics previously reported, and the associated protein conformational change provides an explanation for the decreased thermal stability.",
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