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

Authors

  • David A. Schultz,

    1. Department of Physics, University of California, San Diego, La Jolla, California 92093, USA
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  • Alan M. Friedman,

    1. Department of Biological Sciences, Purdue University, West Lafayette, Indiana 47907, USA
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  • Mark A. White,

    1. Department of Human Biological Chemistry and Genetics, and The Sealy Center for Structural Biology, University of Texas Medical Branch at Galveston, Galveston, Texas 77555, USA
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  • Robert O. Fox

    Corresponding author
    1. Department of Human Biological Chemistry and Genetics, and The Sealy Center for Structural Biology, University of Texas Medical Branch at Galveston, Galveston, Texas 77555, USA
    • Department of Human Biological Chemistry and Genetics, University of Texas Medical Branch, 301 University Boulevard, Mail Route 0647, Galveston, TX 77555, USA; fax: (409) 747-4745.

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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 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.

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