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 (ci s or trans) of Xaa-Pro peptide bonds in proteins. This approach, which entails selective C-13 labeling of Xaa and Pro residues in the protei n and isotope-edited NMR, has been applied to mutants of staphylococca l nuclease with suspected altered configurations of the Lys116-Pro117 peptide bond. The technique for monitoring proline configurations is b ased on differences in interproton distances between the H(alpha) of r esidue Xaa and the proline H(delta) or H(alpha) protons. Short (<2.5 a ngstrom) Xaa H(alpha)-Pro H(delta) interproton distances are diagnosti c for the trans configuration, whereas short (<2.5 angstrom) Xaa H(alp ha)-Pro H(alpha) interproton distances are diagnostic for the cis conf iguration. Biosynthetic incorporation of [alpha-C-13]Xaa and [delta-C- 13]proline facilitates detection of trans Xaa-Pro peptide bonds, where as incorporation of [alpha-C-13]Xaa and [alpha-C-13] proline facilitat es detection of cis Xaa-Pro peptide bonds. Provided that the Xaa-Pro p eptide bond is unique within the protein sequence, symmetric off-diago nal NOE cross peaks in the isotope-edited NOE spectrum allow for simul taneous chemical shift assignment and determination of the prolyl pept ide bond geometry. We have used this technique to determine the predom inant configuration of the Lys116-Pro117 peptide bond in recombinant V 8 staphylococcal nuclease A (H124L) and two of its single amino acid m utants (D77A+H124L and G79S+H124L). The results are consistent with co nclusions reached on the basis of indirect arguments concerning change s in the chemical shifts of histidine H-1epsilon1 NMR signals. The Lys 116-Pro117 peptide bond was found by this direct isotope-edited NOE me thod to be predominantly cis in H124L but predominantly trans in D77AH124L and G79S+H124L. However, when a saturating amount of an inhibito r (pdTp plus Ca2+) was added to either D77A+H124L or G79S+H124L, the p eptide bond became predominantly cis.