Stereospecific nuclear magnetic resonance assignments of the methyl groups of valine and leucine in the DNA-binding domain of the 434 repressor by biosynthetically directed fractional 13C labeling.
Stereospecific 1H and 13C NMR assignments were made for the two diastereotopic methyl groups of the 14 valyl and leucyl residues in the DNA-binding domain 1-69 of the 434 repressor. These results were obtained with a novel method, biosynthetically directed fractional 13C labeling, which should be quite widely applicable for peptides and proteins. The method is based on the use of a mixture of fully 13C-labeled and unlabeled glucose as the sole carbon source for the biosynthetic production of the protein studied, knowledge of the independently established stereoselectivity of the pathways for valine and leucine biosynthesis, and analysis of the distribution of 13C labels in the valyl and leucyl residues of the product by two-dimensional heteronuclear NMR correlation experiments. Experience gained with the present project and a previous application of the same principles with the cyclic polypeptide cyclosporin A provides a basis for the selection of the optimal NMR experiments to be used in conjunction with biosynthetic fractional 13C labeling of proteins and peptides.
Neri
D
D
Institut für Molekularbiologie und Biophysik, ETH-Hönggerberg, Zürich, Switzerland.
Szyperski
T
T
Otting
G
G
Senn
H
H
Wüthrich
K
K
D002247:Carbon Isotopes; D004268:DNA-Binding Proteins / Q000032:analysis*; D004926:Escherichia coli / Q000235:genetics; D007930:Leucine / Q000032:analysis*; D009682:Magnetic Resonance Spectroscopy; D008697:Methane / Q000032:analysis*; D015394:Molecular Structure; D010449:Peptide Mapping; D012097:Repressor Proteins / Q000032:analysis*; D014157:Transcription Factors / Q000032:analysis*; D014633:Valine / Q000032:analysis*
D002247:Carbon Isotopes; D004268:DNA-Binding Proteins; D012097:Repressor Proteins; D014157:Transcription Factors; D007930:Leucine; D014633:Valine; D008697:Methane