Case study of protein structure, stability, and function: NMR investigations of the proline residues in staphylococcal nuclease

John L. Markley, Kenneth Prehoda, Dagmar Truckses, Jinfeng Wang, Andrew P. Hinck, Stewart N. Loh, William F. Walkenhorst

Research output: Contribution to journalArticle

4 Scopus citations

Abstract

We have used NMR spectroscopy to determine peptide bond configurations and to measure the rates and equilibria of interconversion at individual Xaa-Pro peptide bond linkages in staphylococcal nuclease and several variants produced by site-directed mutagenesis. In general, tertiary interactions, rather than short-range interactions, have been found to be critical for stabilizing the cis linkage at Lys116- Pro117which predominates in the wild-type enzyme. A correlation has been found between the position of the cis/trans equilibrium at the Lys11-Pro11peptide bond and thermal stability of the variant. Enthalpic interactions that stabilize the folded protein appear to be present when the peptide bond is cis but not when it is trans. Hydrogen exchange protection factors correlate with the mole fraction of the protein that is in the cis configuration. Nuclease variants in which the peptide bond is predominantly cis are more stable against denaturation (by heat, pressure, or guanidinium chloride) than those that are predominantly trans. Disulfide bridges have been engineered and introduced by mutagenesis that stabilize certain conformational states; one of these shows coupling between the oxidation state of the engineered cysteine pair and the cis/trans configuration about the Lys116-Pro117peptide bond. A rough correlation is seen between the catalytic activities of mutants and the cis/trans ratio; the effect is primarily on kcat rather than on Km.

Original languageEnglish (US)
Pages (from-to)65-69
Number of pages5
JournalPure and Applied Chemistry
Volume66
Issue number1
DOIs
StatePublished - Jan 1 1994
Externally publishedYes

ASJC Scopus subject areas

  • Chemistry(all)
  • Chemical Engineering(all)

Fingerprint Dive into the research topics of 'Case study of protein structure, stability, and function: NMR investigations of the proline residues in staphylococcal nuclease'. Together they form a unique fingerprint.

  • Cite this