Proton NMR studies of transforming and nontransforming H-ras p21 mutants

Abstract

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+.

Mechanism of Free Radical Nitric Oxide-mediated Ras Guanine Nucleotide Dissociation

“…[15][16][17][18][19][20][21][22][23] This interaction is important for high affinity Ras nucleotide binding interactions. 34,35 The distance between the Phe 28 side-chain C 4 atom and the center of Ras-bound GDP guanine base is w3 Å . [15][16][17][18][19][20] The close n-p interaction between the side-chain of Phe 28 and the Ras-bound GDP guanine base may induce delocalization of the Phe 28 side-chain unpaired electrons to lower the redox potential of Phe 28 side-chain cation radical (Phe 28%C ) relative to the thiyl radical (i.e., Ras-S 118% ), 26-28 thereby facilitating electron transfer from the Phe 28 side-chain to Ras-S 118% .…”

Characterization of a Fast Cycling ADP-ribosylation Factor 6 Mutant

“…The fast cycling Ras mutation, F28L, reduces the size of the side chain at this position, which removes contacts between this residue and the guanine ring and weakens the affinity of the protein for guanine nucleotides (19). This reduced affinity allows the GTPase to spontaneously release GDP and bind GTP, even in the absence of a GEF.…”

“…These mutants have reduced affinity for nucleotides and spontaneously release GDP and bind GTP, thereby increasing levels of active GTPase within the cell (17,19). Importantly this pool of active GTPase can still hydrolyze GTP and go through the entire normal GTPase cycle (17,20).…”

Sequence-specific 1H and 15N resonance assignments and secondary structure of GDP-bound human c-Ha-Ras protein in solution

“…The conformation of the Ras protein in aqueous solution has been studied mainly by NMR spectroscopy (Schlichting et al, 1988(Schlichting et al, , 1990bCampbell-Burk, 1989;Campbell-Burk et al, 1989;Hata-Tanaka et al, 1989;Yamasaki et al, 1989;Redfield and Papastavros, 1990;Fujita-Yoshigaki et al, Miller et al, 1992;Yamasaki et al, 1992). For human N-Ras protein, selective 15N-labeling techniques have been applied for Gly and Lys residues, and selective ~3C-~SN labeling techniques (Kainosho and Tsuji, 1982) have been used for partial resonance assignments (Campbell-Burk, 1989;Campbell-Burk et al, 1989;Redfield and Papastavros, 1990).…”