NMR STRATEGY FOR DETERMINING XAA-PRO PEPTIDE-BOND CONFIGURATIONS IN PROTEINS - MUTANTS OF STAPHYLOCOCCAL NUCLEASE WITH ALTERED CONFIGURATION AT PROLINE-117

A general approach has been developed for configurational analysis (cis or trans) of Xaa-Pro peptide bonds in proteins. This approach, which entails selective C-13 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-Pro117 peptide bond. The technique for monitoring proline configurations is based on differences in interproton distances between the H(alpha) of residue Xaa and the proline H(delta) or H(alpha) protons. Short (<2.5 angstrom) Xaa H(alpha)-Pro H(delta) interproton distances are diagnostic for the trans configuration, whereas short (<2.5 angstrom) Xaa H(alpha)-Pro H(alpha) interproton distances are diagnostic for the cis configuration. Biosynthetic incorporation of [alpha-C-13]Xaa and [delta-C-13]proline facilitates detection of trans Xaa-Pro peptide bonds, whereas incorporation of [alpha-C-13]Xaa and [alpha-C-13] 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 H-1epsilon1 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.