Within this review, we describe a home-built pulsed electron paramagnetic resonance (EPR) spectrometer operating at 180 GHz as well as the incorporation of two double resonance techniques, electron nuclear double resonance (ENDOR) and pulsed electron double resonance (PELDOR), along with first applications. Hahn-echo decays on a TEMPO/polystyrene sample are presented, demonstrating that the observation of anisotropic librational motions is possible in a very precise manner at high magnetic fields. Bisdiphenylene-phenyl-allyl is used as a model system to illustrate the performance of the setup for 1H-ENDOR using the Mims as well as the Davies sequence. Furthermore, first 1H-Mims and Davies ENDOR spectra on a biological sample, the wild-type Ras*Mn2+*GDP protein, are reported. The capability of the 180-GHz PELDOR setup is demonstrated using the three-pulse ELDOR sequence on the protein ribonucleotide reductase (RNR) subunit R2 from Escherichia coli, which contains two tyrosyl radicals at a 33 angstroms distance. At 180 GHz, orientation selectivity is observed and the modulation frequency is found to be in good agreement with theoretical predictions.

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 31P 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 17O ENDOR spectra of the water ligands, and we have accurately determined the 17O 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 31P ENDOR spectra, and their hyperfine tensors lead to distances in agreement with the X-ray structures. Finally, 13C ENDOR spectra of uniformly 13C-labeled Ras(wt) x GDP and Ras(G12V) x GDP complexes as well as of the Ras(wt) x GppNHp and the selectively 1,4-13C-Asp labeled Ras(wt) x 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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Ras is a central constituent of the intracellular signal transduction that switches between its inactive state with GDP bound and its active state with GTP bound. A number of different X-ray structures are available. Different magnetic resonance techniques were used to characterise the conformational states of the protein and are summarised here. 31P NMR spectroscopy was used as probe for the environment of the phosphate groups of the bound nucleotide. It shows that in liquid solution additional conformational states in the GDP as well as in the GTP forms coexist which are not detected by X-ray crystallography. Some of them can also be detected by solid-state NMR in the micro crystalline state. EPR and ENDOR spectroscopy were used to probe the environment of the divalent metal ion (Mg2+ was replaced by Mn2+) bound to the nucleotide in the protein. Here again different states could be observed. Substitution of normal water by 17O-enriched water allowed the determination of the number of water molecules in the first coordination sphere of the metal ion. In liquid solution, they indicate again the existence of different conformational states. At low temperatures in the frozen state ENDOR spectroscopy suggests that only one state exists for the GDP- and GTP-bound form of Ras, respectively.