Biomedical subjects

T F Prisner

Publications and source records attributed to T F Prisner.

7 recordsLinked to original sources

PubMed2006

High-field pulsed electron-electron double resonance spectroscopy to determine the orientation of the tyrosyl radicals in ribonucleotide reductase.

Class I ribonucleotide reductases (RNRs) are composed of two subunits, R1 and R2. The R2 subunit contains the essential diferric cluster-tyrosyl radical (Y.) cofactor, and R1 is the site of the conversion of nucleoside diphosphates to 2'-deoxynucleoside diphosphates. It has been proposed that the function of the tyrosyl radical in R2 is to generate a transient thiyl radical (C439.) in R1 over a distance of 35 A, which in turn initiates the reduction process. EPR distance measurements provide a tool with which to study the mechanism of radical initiation in class I RNRs. These types of experiments at low magnetic fields and frequencies (0.3 T, 9 GHz) give insight into interradical distances and populations. We present a pulsed electron-electron double resonance (PELDOR) experiment at high EPR frequency (180-GHz electron Larmor frequency) that detects the dipolar interaction between the Y.s in each protomer of RNR R2 from Escherichia coli. We observe a correlation between the orientation-dependent dipolar interaction and their resolved g-tensors. This information has allowed us to define the relative orientation of two radicals embedded in the active homodimeric protein in solution. This experiment demonstrates that high-field PELDOR spectroscopy is a powerful tool with which to study the assembly of proteins that contain multiple paramagnetic centers.

Dimerization↗

PubMed2006

High-frequency 94 GHz ENDOR characterization of the metal binding site in wild-type Ras x GDP and its oncogenic mutant G12V in frozen solution.

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.

Binding Sites↗

PubMed2005

Pulsed 180-GHz EPR/ENDOR/PELDOR spectroscopy.

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.

Cyclic N-Oxides↗

PubMed2004

Relaxation filtered hyperfine spectroscopy (REFINE).

A simple and general applicable method to separate spectrally overlapping hyperfine spectra of two paramagnetic compounds is presented. Overlapping spectral contributions from different paramagnetic species are a common situation in electron paramagnetic resonance (EPR) spectroscopy, resulting in complicate EPR spectra of metal enzymes, organic radicals or in the field of material sciences. On the other hand, the longitudinal relaxation times T1 of these species contributing to the overall EPR signal can vary by several orders of magnitude, depending on the paramagnetic component under study. These differences can be used to selectively study individual species by using an inversion-recovery preparation sequence as a filter. Here, we demonstrate the possibility to separate hyperfine spectra of two spectrally overlapping paramagnetic species by combining an inversion-recovery based relaxation filter together with ESEEM or ENDOR hyperfine spectroscopy (REFINE). The feasibility of the presented method is demonstrated on model compounds and the necessary requirements are discussed.

Allyl Compounds↗

PubMed2003

Asymmetric binding of the high-affinity Q(H)(*)(-) ubisemiquinone in quinol oxidase (bo3) from Escherichia coli studied by multifrequency electron paramagnetic resonance spectroscopy.

Ubiquinone-2 (UQ-2) selectively labeled with (13)C (I =(1)/(2)) at either the position 1- or the 4-carbonyl carbon is incorporated into the ubiquinol oxidase bo(3) from Escherichia coli in which the native quinone (UQ-8) has been previously removed. The resulting stabilized anion radical in the high-affinity quinone-binding site (Q(H)(*)(-)) is investigated using multifrequency (9, 34, and 94 GHz) electron paramagnetic resonance (EPR) spectroscopy. The corresponding spectra reveal dramatic differences in (13)C hyperfine couplings indicating a strongly asymmetric spin density distribution over the quinone headgroup. By comparison with previous results on labeled ubisemiquinones in proteins as well as in organic solvents, it is concluded that Q(H)(*)(-) is most probably bound to the protein via a one-sided hydrogen bond or a strongly asymmetric hydrogen-bonding network. This observation is discussed with regard to the function of Q(H) in the enzyme and contrasted with the information available on other protein-bound semiquinone radicals.

Binding Sites↗

PubMed2001

Structure of the metal-water complex in Ras x GDP studied by high-field EPR spectroscopy and 31P NMR spectroscopy.

The small GTPase Ras plays a key role as a molecular switch in the intercellular signal transduction. On Mg(2+) --> Mn(2+) substituted samples, the first ligand sphere of the metal ion in the inactive, GDP-bound Ras has been studied by continuous wave EPR at 94 GHz (W-band). Via replacement of normal water with (17)O-enriched water, the (17)O--(55)Mn superhyperfine coupling was used to determine the number of water ligands bound to the metal ion. In contrast to EPR data on frozen solutions and X-ray data from single crystals where four direct ligands to the metal ion are found, the wild-type protein has only three water ligands bound in solution at room temperature. The same number of water ligands is found for the mutant Ras(T35S). However, for the alanine mutant in position 35 Ras(T35A) as well as for the oncogenic mutant Ras(G12V), four water ligands can be observed in liquid solution. The EPR studies were supplemented by (31)P NMR studies on the Mg(2+) x GDP complexes of the wild-type protein and the three mutants. Ras(T35A) exists in two conformational states (1 and 2) with an equilibrium constant K(1)(1,2) of approximately 0.49 and rate constants k(1--1) which are much smaller than 40 s(-1) at 298 K. For wild-type Ras and Ras(T35S), the two states can also be observed with equilibrium constants K(1)(1,2) of approximately 0.31 and 0.21, respectively. In Ras(G12V), only one conformational state could be detected.

Amino Acid Substitution↗

PubMed1993

Measurement of the g-tensor of the P700+. signal from deuterated cyanobacterial photosystem I particles.

We report high-field continuous wave EPR spectra of P700+. in preparations obtained from deuterated cyanobacteria (Synechococcus lividus). Measurements were performed with photosystem I (PS-I) preparations, whole cells from cyanobacteria grown in 2H2O, and photosystem II (PS-II) preparations, as well as with protonated PS-I preparations. Because of the significantly improved resolution of our 140-GHz spectrometer (as compared with X- or Q-band EPR) the principal values of the g-tensor of the primary donor P700+. could be resolved and measured with high accuracy as g11 = 2.00304, g22 = 2.00262, and g33 = 2.00232. Other signals arising from Mn2+ and a dark signal from PS-II at g approximately 2.00266 are distinguished from the P700+. g-tensor powder pattern. The measured g values are compared with those of several bacterial reaction center donors.

Cyanobacteria↗