128 research outputs found
High-field pulsed electron-electron double resonance spectroscopy to determine the orientation of the tyrosyl radicals in ribonucleotide reductase
No full text
Studies of dynamic nuclear polarization with nitroxides in aqueous solution.
No full textThe dynamic nuclear polarization effect between the nitroxide radical TEMPO and the H-1 protons of water solution was investigated at an electron pumping frequency of 9.7 GHz for different experimental conditions. In particular, we compared N-14-4-hydroxy-TEMPO (TEMPOL) with 4-oxo-TEMPO and evaluated the effect of H-2 and N-15 isotope labelling. Furthermore, we tested the effect of concomitant irradiation on both electron paramagnetic resonance (EPR) lines with the N-15-labelled compound. Our results show that the reduction in the EPR line width given by the H-1 and N-15 labelling and the collapse in the number of hyperfine lines due to N-15 substitution leads to the highest enhancement of epsilon = -140 ever reported in the literature. Concomitant pumping on two hyperfine lines does not give higher enhancements at our experimental conditions
Base-specific spin-labeling of RNA for structure determination
No full textTo facilitate the measurement of intramolecular distances in solvated RNA systems, a combination of spin-labeling, electron paramagnetic resonance (EPR), and molecular dynamics (MD) simulation is presented. The fairly rigid spin label 2,2,5,5-tetramethyl-pyrrolin-1-yloxyl-3-acetylene (TPA) was base and site specifically introduced into RNA through a Sonogashira palladium catalyzed crosscoupling on column. For this purpose 5-iodouridine, 5-iodo-cytidine and 2-iodo-adenosine phosphoramidites were synthesized and incorporated into RNA-sequences. Application of the recently developed ACE (R) chemistry presented the main advantage to limit the reduction of the nitroxide to an amine during the oligonucleotide automated synthesis and thus to increase substantially the reliability of the synthesis and the yield of labeled oligonucleotides. 4-Pulse Electron Double Resonance (PELDOR) was then successfully used to measure the intramolecular spin–spin distances in six doubly labeled RNA-duplexes. Comparison of these results with our previous work on DNA showed that A- and B-Form can be differentiated. Using an all-atom force field with explicit solvent, MD simulations gave results in good agreement with the measured distances and indicated that the RNA A-Form was conserved despite a local destabilization effect of the nitroxide label. The applicability of the method to more complex biological systems is discussed
A note on periodicity of the 2-distance operator
No full textThe paper solves one problem by E. Prisner concerning the 2-distance operator T₂. This is an operator on the class of all finite undirected graphs. If G is a graph from , then T₂(G) is the graph with the same vertex set as G in which two vertices are adjacent if and only if their distance in G is 2. E. Prisner asks whether the periodicity ≥ 3 is possible for T₂. In this paper an affirmative answer is given. A result concerning the periodicity 2 is added
Dynamic nuclear polarization at high magnetic fields in liquids
No full textHigh field dynamic nuclear polarization spectrometer for liquid samples have
been constructed. ► The field dependence of the Overhauser DNP efficiency has
been measured for the first time up to 9.2 T. ► High DNP enhancements for
liquid samples have been observed at high magnetic fields. ► The enhancements
have been compared with results from NMRD, MD and theoretical models. ►
Coherent and relaxation effects within fast magnetic field changes have been
analyzed
High-frequency 94 GHz ENDOR characterization of the metal binding site in wild type Ras. GDP and its oncogenic mutant G12V in frozen solution
No full textThe 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 aiso = -0.276 mT, supporting the results of previous line shape analysis in solution. Further, the distinct resonances of the -, -, and -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)·GDP and Ras(G12V)·GDP complexes as well as of the Ras(wt)·GppNHp and the selectively 1,4-13C-Asp labeled Ras(wt)·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
Dynamic nuclear polarization of ¹³C nuclei in the liquid state over a 10 Tesla field range.
No full textNuclear magnetic resonance (NMR) techniques play an essential role in natural science and medicine. In spite of the tremendous utility associated with the small energies detected, the most severe limitation is the low signal-to-noise ratio. Dynamic nuclear polarization (DNP), a technique based on transfer of polarization from electron to nuclear spins, has emerged as a tool to enhance sensitivity of NMR. However, the approach in liquids is still facing several challenges. Here we report the observation of room temperature, liquid DNP ¹³C signal enhancements in organic small molecules as high as 600 at 9.4 Tesla and 800 at 1.2 Tesla. A mechanistic investigation of the ¹³C-DNP field dependence shines light on parameters governing the underlying scalar DNP, indicating that DNP efficiency is raised by proper choice of the polarizing agent (paramagnetic center) and by halogen atoms as mediators of scalar hyperfine interaction. Observation of sizable DNP of ¹³CH₂ and ¹³CH₃ groups in organic molecules at 9.4 T opens up perspective for a broader application of this method
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