NMR Strategy for Determining Xaa-Pro Peptide Bond Configurations in Proteins: Mutants of Staphylococcal Nuclease with Altered Configuration at Proline-117

Andrew P. Hinck, Eric S. Eberhardt, John L. Markley

Research output: Contribution to journalArticlepeer-review

33 Scopus citations

Abstract

A general approach has been developed for configurational analysis (cis or trans) of Xaa-Pro peptide bonds in proteins. This approach, which entails selective 13C labeling of Xaa and Pro residues in the protein and isotope-edited NMR, has been applied to mutants of staphylococcal nuclease with suspected altered configurations of the Lys116-Pro'17 peptide bond. The technique for monitoring proline configurations is based on differences in interproton distances between the Hα of residue Xaa and the proline Hδ or Hα protons. Short (<2.5 Å) Xaa Hα-Pro Hδ interproton distances are diagnostic for the trans configuration, whereas short (<2.5 Å) Xaa Hα-Pro Hδ interproton distances are diagnostic for the cis configuration. Biosynthetic incorporation of [α−13C]Xaa and [δ−13C]proline facilitates detection of trans Xaa-Pro peptide bonds, whereas incorporation of [α−13C]Xaa and [α-13C]proline facilitates detection of cis Xaa-Pro peptide bonds. Provided that the Xaa-Pro peptide bond is unique within the protein sequence, symmetric off-diagonal NOE cross peaks in the isotope-edited NOE spectrum allow for simultaneous chemical shift assignment and determination of the prolyl peptide bond geometry. We have used this technique to determine the predominant configuration of the Lys116-Pro117 peptide bond in recombinant V8 staphylococcal nuclease A (H124L) and two of its single amino acid mutants (D77A+H124L and G79S+H124L). The results are consistent with conclusions reached on the basis of indirect arguments concerning changes in the chemical shifts of histidine 1Hϵ1 NMR signals. The Lys116-Pro117 peptide bond was found by this direct isotope-edited NOE method to be predominantly cis in H124L but predominantly trans in D77A+H124L and G79S+H124L. However, when a saturating amount of an inhibitor (pdTp plus Ca2+) was added to either D77A+H124L or G79S+H124L, the peptide bond became predominantly cis.

Original languageEnglish (US)
Pages (from-to)11810-11818
Number of pages9
JournalBiochemistry
Volume32
Issue number44
DOIs
StatePublished - 1993
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry

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