Identification of resonances from an oncogenic activating locus of human N-RAS-encoded p21 protein using isotope-edited NMR.
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.
Burk
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Department of Biochemistry, Brandeis University, Waltham, MA 02254.
Papastavros
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McCormick
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Redfield
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D002247:Carbon Isotopes; D002471:Cell Transformation, Neoplastic; D004926:Escherichia coli / Q000235:genetics; D011905:Genes, ras; D005998:Glycine; D006801:Humans; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008565:Membrane Proteins / Q000502:physiology*; D009587:Nitrogen Isotopes; D010957:Plasmids; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000502:physiology*; D016283:Proto-Oncogene Proteins p21(ras); D014633:Valine
D002247:Carbon Isotopes; D008565:Membrane Proteins; D009587:Nitrogen Isotopes; D011518:Proto-Oncogene Proteins; C501009:HRAS protein, human; D016283:Proto-Oncogene Proteins p21(ras); D014633:Valine; D005998:Glycine