Abstract
The guanine nucleotide binding protein Ras plays a central role as molecular switch in cellular signal transduction. Ras cycles between a GDP-bound '' off '' state and a GTP-bound '' on '' state. Specific oncogenic mutations in the Ras protein are found in up to 30% of all human tumors. Previous P-31 NMR studies had demonstrated that in liquid solution different conformational states in the ...
Abstract
The guanine nucleotide binding protein Ras plays a central role as molecular switch in cellular signal transduction. Ras cycles between a GDP-bound '' off '' state and a GTP-bound '' on '' state. Specific oncogenic mutations in the Ras protein are found in up to 30% of all human tumors. Previous P-31 NMR studies had demonstrated that in liquid solution different conformational states in the GDP-bound as well as in the GTP-bound form coexist. High-field EPR spectroscopy of the GDP complexes in solution displayed differences in the ligand sphere of the wild-type complex as compared to its oncogenic mutant Ras(G12V). Only three water ligands were found in the former with respect to four in the G12V mutant [Rohrer, M. et al. (2001) Biochemistry 40, 1884-1889]. These differences were not detected in previous X-ray structures in the crystalline state. In this paper, we employ high-frequency electron nuclear double resonance (ENDOR) spectroscopy to probe the ligand sphere of the metal ion in the GDP-bound state. This technique in combination with selective isotope labeling has enabled us to detect the resonances of nuclei in the first ligand sphere of the ion with high spectral resolution. We have observed the 170 ENDOR spectra of the water ligands, and we have accurately determined the 170 hyperfine coupling with a(iso) = -0.276 mT, supporting the results of previous line shape analysis in solution. Further, the distinct resonances of the alpha-, beta-, and gamma-phosphorus of the bound nucleotides are illustrated in the P-31 ENDOR spectra, and their hyperfine tensors lead to distances in agreement with the X-ray structures. Finally, C-13 ENDOR spectra of uniformly C-13-labeled Ras(wt)(circle)GDP and Ras(G12V)(circle)GDP complexes as well as of the Ras(wt)(circle)GppNHp and the selectively 1,4-C-13-Asp labeled Ras(wt)(circle)GDP complexes have revealed that in frozen solution only one amino acid is ligated to the ion in the GDP state, whereas two are bound in the GppNHp complex. Our results suggest that a second conformational state of the protein, if correlated with a different ligand sphere of the Mn2+ ion, is not populated in the GDP form of Ras at low temperatures in frozen solution.
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