Proton NMR studies of transforming and nontransforming H-ras p21 mutants.
One- and two-dimensional nuclear magnetic resonance spectroscopy (1D and 2D NMR) and site-directed mutagenesis were used to study the influence of mutations on the conformation of the H-ras oncogene product p21. No severe structural differences between the different mutants, whether they were transforming or nontransforming, could be detected. Initially, selective incorporation of 3,5-deuterated tyrosyl residues into p21 and 2D NMR were used to identify the resonances representing the spin systems of the imidazole rings of the three histidyl residues in the protein, of six of the nine tyrosyl rings, and of four of the five phenylalanyl rings. The spin systems of the phenyl rings of Phe28, Phe78, and Phe82 could be assigned by using mutant proteins, since no severe structure-induced spectral changes in the aromatic part of the spectra of the mutant proteins were detected. Sequence-specific assignments of the histidine imidazole resonances could be obtained by comparison of the distance information obtained by nuclear Overhauser enhancement spectroscopy (NOESY) experiments with the crystal structure. The change in the chemical shift values of the Hl' proton and the alpha-phosphate of the bound GDP in the NMR spectra of the p21(F28L) mutant and the 28-fold increase in the GDP dissociation rate constants of this mutant suggest a strong interaction between Phe28 and the p21-bound nucleotide. In solution, the p21-bound GDP.Mg2+ has an anti conformation, and the phenyl ring of Phe28 is close to the ribose of the bound GDP.Mg2+.
Schlichting
I
I
Department of Biophysics, Max-Planck-Institut for Medical Research, Heidelberg, West Germany.
John
J
J
Frech
M
M
Chardin
P
P
Wittinghofer
A
A
Zimmermann
H
H
Rösch
P
P
D001483:Base Sequence; D002471:Cell Transformation, Neoplastic / Q000235:genetics*; D004247:DNA / Q000235:genetics; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D008274:Magnesium / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D009154:Mutation; D015689:Oncogene Protein p21(ras) / Q000235:genetics* / Q000378:metabolism; D010649:Phenylalanine; D011487:Protein Conformation; D014443:Tyrosine
D006153:Guanosine Diphosphate; D014443:Tyrosine; D010649:Phenylalanine; D006160:Guanosine Triphosphate; D004247:DNA; D015689:Oncogene Protein p21(ras); D008274:Magnesium