Subject(s)
Mitogen-Activated Protein Kinases , Nitric Oxide/metabolism , Proto-Oncogene Proteins p21(ras)/metabolism , Signal Transduction , Binding Sites , Calcium-Calmodulin-Dependent Protein Kinases/metabolism , Cell Line , Cyanogen Bromide , Cysteine/chemistry , Cysteine/metabolism , Enzyme Activation , Mitogen-Activated Protein Kinase 1 , Mitogen-Activated Protein Kinase 3 , Oxidation-Reduction , Proto-Oncogene Proteins p21(ras)/chemistryABSTRACT
A sample of Escherichia coli-expressed human N-RAS-encoded p21, a 21-kDa protein, was selectively labeled with 15N at each of the 14 glycine amide positions. Two-dimensional proton-observe 15N correlation spectra showed one peak for each glycine residue. Five glycine resonances were identified with residues near the nucleotide binding site and provide useful reporters of several oncogene-activating positions. Three of these resonances were assigned to residues 10, 15, and 115 from the spectrum of a sample that was also labeled with [13C]valine. These resonances showed extra splitting or broadening due to the 13C label, which could be eliminated by 13C decoupling. Two other peaks were unambiguously identified as Gly-12 and Gly-13 using a one-dimensional edited nuclear Overhauser experiment and by spectral comparison with an Asp-12 mutant. These assignments have provided several site-specific probes of critical domains in p21.
