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 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-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 A) Xaa H alpha-Pro H delta interproton distances are diagnostic for the trans configuration, whereas short (< 2.5 A) Xaa H alpha-Pro H alpha interproton distances are diagnostic for the cis configuration. Biosynthetic incorporation of [alpha-13C]Xaa and [delta-13C]proline facilitates detection of trans Xaa-Pro peptide bonds, whereas incorporation of [alpha-13C]Xaa and [alpha-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 1H epsilon 1 NMR signals.(ABSTRACT TRUNCATED AT 250 WORDS)
Hinck
A P
AP
Department of Biochemistry, College of Agricultural and Life Sciences, University of Wisconsin, Madison 53706.
Eberhardt
E S
ES
Markley
J L
JL
D000595:Amino Acid Sequence; D008239:Lysine; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008836:Micrococcal Nuclease / Q000737:chemistry*; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D011392:Proline; D011487:Protein Conformation; D011506:Proteins / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry
D011506:Proteins; D011994:Recombinant Proteins; D011392:Proline; D008836:Micrococcal Nuclease; D008239:Lysine