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Thomas F Prisner - One of the best experts on this subject based on the ideXlab platform.
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high resolution epr distance measurements on rna and dna with the non covalent ǵ spin label
Nucleic Acids Research, 2020Co-Authors: Marcel Heinz, Thomas F Prisner, Gerhard Hummer, Lukas S. Stelzl, Nicole Erlenbach, Snorri Th Sigurdsson, Grace Thierolf, Nilesh R KambleAbstract:Pulsed Electron paramagnetic Resonance (EPR) experiments, among them most prominently pulsed Electron-Electron Double Resonance experiments (PELDOR/DEER), resolve the conformational dynamics of nucleic acids with high resolution. The wide application of these powerful experiments is limited by the synthetic complexity of some of the best-performing spin labels. The recently developed $\bf\acute{G}$ (G-spin) label, an isoindoline-nitroxide derivative of guanine, can be incorporated non-covalently into DNA and RNA duplexes via Watson-Crick base pairing in an abasic site. We used PELDOR and molecular dynamics (MD) simulations to characterize $\bf\acute{G}$, obtaining excellent agreement between experiments and time traces calculated from MD simulations of RNA and DNA Double helices with explicitly modeled $\bf\acute{G}$ bound in two abasic sites. The MD simulations reveal stable hydrogen bonds between the spin labels and the paired cytosines. The abasic sites do not significantly perturb the helical structure. $\bf\acute{G}$ remains rigidly bound to helical RNA and DNA. The distance distributions between the two bound $\bf\acute{G}$ labels are not substantially broadened by spin-label motions in the abasic site and agree well between experiment and MD. $\bf\acute{G}$ and similar non-covalently attached spin labels promise high-quality distance and orientation information, also of complexes of nucleic acids and proteins.
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a semi rigid isoindoline derived nitroxide spin label for rna
Organic and Biomolecular Chemistry, 2018Co-Authors: Dnyaneshwar B Gophane, Burkhard Endeward, Thomas F Prisner, Snorri Th SigurdssonAbstract:A new isoindoline-derived benzimidazole nitroxide spin label, ImUm, was synthesized and incorporated into RNA oligoribonucleotides. ImUm is the first example of a conformationally unambiguous spin label for RNA, in which the nitroxide N-O bond lies on the same axis as the single bond used to attach the rigid isoindoline-based spin label to a uridine base. This results in minimal displacement of the nitroxide upon rotation of this single bond, which is a useful property for a label to be used for distance measurements. Continuous-wave (CW) EPR measurements of RNA duplexes containing ImUm indicate a restricted rotation around this single bond, presumably due to an intramolecular hydrogen bond between the benzimidazole N-H and O4 of the uracil. Orientation-selective pulsed Electron-Electron Double Resonance (PELDOR, also called Double Electron-Electron Resonance, or DEER) distance measurements between two spin labels in two RNA duplexes showed in one case a strong orientation dependence, further confirming the restricted motion of the spin labels in RNA duplexes.
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conformational coupling and trans inhibition in the human antigen transporter ortholog tmrab resolved with dipolar epr spectroscopy
Journal of the American Chemical Society, 2018Co-Authors: Katja Barth, Thomas F Prisner, Susanne Hank, Philipp E Spindler, Robert Tampe, Benesh JosephAbstract:ATP-binding cassette (ABC) exporters actively move chemically diverse substrates across biological membranes. Their malfunction leads to human diseases. Many ABC exporters encompass asymmetric nucleotide-binding sites (NBSs), and some of them are inhibited by the transported substrate. The functional relevance of the catalytic asymmetry or the mechanism for trans-inhibition remains elusive. Here, we investigated TmrAB, a functional homologue of the human antigen translocation complex TAP using advanced Electron–Electron Double Resonance spectroscopy. In the presence of ATP, the heterodimeric ABC exporter exists in a tunable equilibrium between inward- and outward-facing conformations. The two NBSs exhibit pronounced asymmetry in the open-to-close equilibrium. The closed conformation is more favored at the degenerate NBS, and closure of either of the NBS is sufficient to open the extracellular gate. We define the mechanistic basis for trans-inhibition, which operates by a reverse transition from the outwar...
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Resolving the Conformational Dynamics of DNA with Ångstrom Resolution by Pulsed Electron-Electron Double Resonance and Molecular Dynamics.
Journal of the American Chemical Society, 2017Co-Authors: Lukas S. Stelzl, Thomas F Prisner, Nicole Erlenbach, Marcel Heinz, Gerhard HummerAbstract:Pulsed Electron–Electron Double Resonance (PELDOR/DEER) experiments of nucleic acids with rigid spin labels provide highly accurate distance and orientation information. Here we combine PELDOR experiments with molecular dynamics (MD) simulations to arrive at an atomistic view of the conformational dynamics of DNA. The MD simulations closely reproduce the PELDOR time traces, and demonstrate that bending, in addition to twist-stretch motions, underpin the sub-μs dynamics of DNA. PELDOR experiments correctly rank DNA force fields and resolve subtle differences in the conformational ensembles of nucleic acids, on the order of 1–2 A. Long-range distance and angle measurements with rigid spin labels provide critical input for the refinement of computer models and the elucidation of the structure and dynamics of complex biomolecules.
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synthesis of a cytidine phosphoramidite with protected nitroxide spin label for epr experiments with rna
European Journal of Organic Chemistry, 2017Co-Authors: Timo Weinrich, Thomas F Prisner, Markus Granz, Christian Grunewald, Michael W GobelAbstract:Spin labeling of oligonucleotides with nitroxides is hampered by their intrinsic instability under conditions of solid-phase synthesis and enzymatic ligation. Although nitroxide decomposition can be avoided in some cases by postsynthetic introduction or by special reaction conditions, a more general solution would be reversible protection of the radical. We have recently developed such a method based on photolabile protection groups for DNA oligonucleotides and demonstrated their application in EPR spectroscopy. Here, we extend this method to RNA oligonucleotides. By improving the synthetic procedures, the yield of the coumarin-protected phosphoramidite could be increased by a factor of 12. Effective recovery of the nitroxides on a duplex RNA enables pulsed EPR experiments to be performed directly after irradiation and air oxidation. Data at Q-band frequency is shown and distances measured with PELDOR (pulsed Electron-Electron Double Resonance) spectroscopy agree well with the calculated values.
Burkhard Endeward - One of the best experts on this subject based on the ideXlab platform.
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a semi rigid isoindoline derived nitroxide spin label for rna
Organic and Biomolecular Chemistry, 2018Co-Authors: Dnyaneshwar B Gophane, Burkhard Endeward, Thomas F Prisner, Snorri Th SigurdssonAbstract:A new isoindoline-derived benzimidazole nitroxide spin label, ImUm, was synthesized and incorporated into RNA oligoribonucleotides. ImUm is the first example of a conformationally unambiguous spin label for RNA, in which the nitroxide N-O bond lies on the same axis as the single bond used to attach the rigid isoindoline-based spin label to a uridine base. This results in minimal displacement of the nitroxide upon rotation of this single bond, which is a useful property for a label to be used for distance measurements. Continuous-wave (CW) EPR measurements of RNA duplexes containing ImUm indicate a restricted rotation around this single bond, presumably due to an intramolecular hydrogen bond between the benzimidazole N-H and O4 of the uracil. Orientation-selective pulsed Electron-Electron Double Resonance (PELDOR, also called Double Electron-Electron Resonance, or DEER) distance measurements between two spin labels in two RNA duplexes showed in one case a strong orientation dependence, further confirming the restricted motion of the spin labels in RNA duplexes.
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carr purcell pulsed Electron Double Resonance with shaped inversion pulses
Journal of Physical Chemistry Letters, 2015Co-Authors: Philipp E Spindler, Burkhard Endeward, Jorn Plackmeyer, Christine Ziegler, Izabela Waclawska, Thomas F PrisnerAbstract:Pulsed Electron paramagnetic Resonance (EPR) spectroscopy allows the determination of distances, in the range of 1.5–8 nm, between two spin-labels attached to macromolecules containing protons. Unfortunately, for hydrophobic lipid-bound or detergent-solubilized membrane proteins, the maximum distance accessible is much lower, because of a strongly reduced coherence time of the Electron spins. Here we introduce a pulse sequence, based on a Carr–Purcell decoupling scheme on the observer spin, where each π-pulse is accompanied by a shaped sech/tanh inversion pulse applied to the second spin, to overcome this limitation. This pump/probe excitation scheme efficiently recouples the dipolar interaction, allowing a substantially longer observation time window to be achieved. This increases the upper limit and accuracy of distances that can be determined in membrane protein complexes. We validated the method on a bis-nitroxide model compound and applied this technique to the trimeric betaine transporter BetP. Inte...
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pulsed Electron Electron Double Resonance spectroscopy between a high spin mn2 ion and a nitroxide spin label
Physical Chemistry Chemical Physics, 2015Co-Authors: Dmitry Akhmetzyanov, Burkhard Endeward, Jorn Plackmeyer, Vasyl Denysenkov, Thomas F PrisnerAbstract:Pulsed Electron–Electron Double Resonance (PELDOR) has attracted considerable attention for biomolecular applications, as it affords precise measurements of distances between pairs of spin labels in the range of 1.5–8 nm. Usually nitroxide moieties incorporated by site-directed spin labelling with cysteine residues are used as spin probes in protein systems. Recently, naturally occurring cofactors and metal ions have also been explored as paramagnetic spin species for such measurements. In this work we investigate the performance of PELDOR between a nitroxide spin label and a high-spin Mn2+ ion in a synthetic model compound at Q-band (34 GHz) and G-band (180 GHz). We demonstrate that the distances obtained with high-frequency PELDOR are in good agreement with structural predictions. At Q-band frequencies experiments have been performed by probing either the high-spin Mn2+ ion or the nitroxide spin label. At G-band frequencies we have been able to detect changes in the dipolar oscillation frequency, depending on the pump–probe positions across the g-tensor resolved nitroxide EPR spectrum. These changes result from the restricted mobility of the nitroxide spin label in the model compound. Our results demonstrate that the high-spin Mn2+ ion can be used for precise distance measurements and open the doors for many biological applications, as naturally occurring Mg2+ sites can be readily exchanged for Mn2+.
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Carr–Purcell Pulsed Electron Double Resonance with Shaped Inversion Pulses
2015Co-Authors: Philipp E. Spindler, Burkhard Endeward, Jorn Plackmeyer, Christine Ziegler, Izabela Waclawska, Thomas F PrisnerAbstract:Pulsed Electron paramagnetic Resonance (EPR) spectroscopy allows the determination of distances, in the range of 1.5–8 nm, between two spin-labels attached to macromolecules containing protons. Unfortunately, for hydrophobic lipid-bound or detergent-solubilized membrane proteins, the maximum distance accessible is much lower, because of a strongly reduced coherence time of the Electron spins. Here we introduce a pulse sequence, based on a Carr–Purcell decoupling scheme on the observer spin, where each π-pulse is accompanied by a shaped sech/tanh inversion pulse applied to the second spin, to overcome this limitation. This pump/probe excitation scheme efficiently recouples the dipolar interaction, allowing a substantially longer observation time window to be achieved. This increases the upper limit and accuracy of distances that can be determined in membrane protein complexes. We validated the method on a bis-nitroxide model compound and applied this technique to the trimeric betaine transporter BetP. Interprotomer distances as long as 6 nm could be reliably determined, which is impossible with the existing methods
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conformationally restricted isoindoline derived spin labels in duplex dna distances and rotational flexibility by pulsed Electron Electron Double Resonance spectroscopy
Chemistry: A European Journal, 2014Co-Authors: Dnyaneshwar B Gophane, Burkhard Endeward, Thomas F Prisner, Snorri Th SigurdssonAbstract:Three structurally related isoindoline-derived spin labels that have different mobilities were incorporated into duplex DNA to systematically study the effect of motion on orientation-dependent pulsed Electron–Electron Double Resonance (PELDOR) measurements. To that end, a new nitroxide spin label, ExImU, was synthesized and incorporated into DNA oligonucleotides. ExImU is the first example of a conformationally unambiguous spin label for nucleic acids, in which the nitroxide NO bond lies on the same axis as the three single bonds used to attach the otherwise rigid isoindoline-based spin label to a uridine base. Continuous-wave (CW) EPR measurements of ExImU confirm a very high rotational mobility of the spin label in duplex DNA relative to the structurally related spin label ImU, which has restricted mobility due to an intramolecular hydrogen bond. The X-band CW-EPR spectra of ExImU can be used to identify mismatches in duplex DNA. PELDOR distance measurements between pairs of the spin labels ImU, OxU, and ExImU in duplex DNA showed a strong angular dependence for ImU, a medium dependence for OxU, and no orientation effect for ExImU. Thus, precise distances can be extracted from ExImU without having to take orientational effects into account.
Daniella Goldfarb - One of the best experts on this subject based on the ideXlab platform.
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high sensitivity in cell epr distance measurements on proteins using an optimized gd iii spin label
Journal of Physical Chemistry Letters, 2018Co-Authors: Yin Yang, Feng Yang, Yanjun Gong, Thorsten Bahrenberg, Akiva Feintuch, Daniella GoldfarbAbstract:Distance measurements by Electron–Electron Double Resonance (DEER) carried out on spin-labeled proteins delivered into cells provide new insights into the conformational states of proteins in their native environment. Such measurements depend on spin labels that exhibit high redox stability and high DEER sensitivity. Here we present a new Gd(III)-based spin label, BrPSPy-DO3A-Gd(III), which was derived from an earlier label, BrPSPy-DO3MA-Gd(III), by removing the methyl group from the methyl acetate pending arms. The small chemical modification led to a reduction in the zero-field splitting and to a significant increase in the phase memory time, which together culminated in a remarkable improvement of in-cell DEER sensitivity, while maintaining the high distance resolution. The excellent performance of BrPSPy-DO3A-Gd(III) in in-cell DEER measurements was demonstrated on doubly labeled ubiquitin and GB1 delivered into HeLa cells by electroporation.
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thiolate spin population of type i copper in azurin derived from 33s hyperfine coupling
Inorganic Chemistry, 2017Co-Authors: Marie Ramirez Cohen, Akiva Feintuch, Ilia Kaminker, Netanel Mendelman, Marina Radoul, Tiffany D Wilson, Masha G Savelieff, Herbert Zimmermann, Yi Lu, Daniella GoldfarbAbstract:The Electron transfer mediating properties of type I copper proteins stem from the intricate ligand coordination sphere of the Cu ion in their active site. These redox properties are in part due to unusual cysteine thiol coordination, which forms a highly covalent copper–sulfur (Cu–S) bond. The structure and Electronic properties of type I copper have been the subject of many experimental and theoretical studies. The measurement of spin delocalization of the Cu(II) unpaired Electron to neighboring ligands provides an elegant experimental way to probe the fine details of the Electronic structure of type I copper. To date, the crucial parameter of Electron delocalization to the sulfur atom of the cysteine ligand has not been directly determined experimentally. We have prepared 33S-enriched azurin and carried out W-band (95 GHz) Electron paramagnetic Resonance (EPR) and Electron–Electron Double Resonance detected NMR (EDNMR) measurements and, for the first time, recorded the 33S nuclear frequencies, from whi...
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the Electron depolarization during dynamic nuclear polarization measurements and simulations
Physical Chemistry Chemical Physics, 2015Co-Authors: Yonatan Hovav, Akiva Feintuch, Daniella Goldfarb, Ilia Kaminker, Daphna Shimon, Shimon VegaAbstract:Dynamic nuclear polarization is typically explained either using microscopic systems, such as in the solid effect and cross effect mechanisms, or using the macroscopic formalism of spin temperature which assumes that the state of the Electrons can be described using temperature coefficients, giving rise to the thermal mixing mechanism. The distinction between these mechanisms is typically made by measuring the DNP spectrum – i.e. the nuclear enhancement profile as a function of irradiation frequency. In particular, we have previously used the solid effect and cross effect mechanisms to explain temperature dependent DNP spectra. Our past analysis has however neglected the effect of depolarization of the Electrons resulting from the microwave (MW) irradiation. In this work we concentrate on this Electron depolarization process and perform Electron–Electron Double Resonance (ELDOR) experiments on TEMPOL and trityl frozen solutions, using a 3.34 Tesla magnet and at 2.7–30 K, in order to measure the state of the Electron polarization during DNP. The experiments indicate that a significant part of the EPR line is affected by the irradiation due to spectral diffusion. Using a theoretical framework based on rate equations for the polarizations of the different Electron spin packets and for those of the nuclei we simulated the various ELDOR line-shapes and reproduced the MW frequency and irradiation time dependence. The obtained Electron polarization distribution cannot be described using temperature coefficients as required by the classical thermal mixing mechanism, and therefore the DNP mechanism cannot be described by thermal mixing. Instead, the theoretical framework presented here for the analysis of the ELDOR data forms a basis for future interpretation of DNP spectra in combination with EPR measurements.
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increasing sensitivity of pulse epr experiments using echo train detection schemes
Journal of Magnetic Resonance, 2013Co-Authors: Frederic Mentinkvigier, Akiva Feintuch, Ilia Kaminker, Alberto Collauto, V Tarle, Daniella GoldfarbAbstract:Abstract Modern pulse EPR experiments are routinely used to study the structural features of paramagnetic centers. They are usually performed at low temperatures, where relaxation times are long and polarization is high, to achieve a sufficient Signal/Noise Ratio (SNR). However, when working with samples whose amount and/or concentration are limited, sensitivity becomes an issue and therefore measurements may require a significant accumulation time, up to 12 h or more. As the detection scheme of practically all pulse EPR sequences is based on the integration of a spin echo – either primary, stimulated or refocused – a considerable increase in SNR can be obtained by replacing the single echo detection scheme by a train of echoes. All these echoes, generated by Carr–Purcell type sequences, are integrated and summed together to improve the SNR. This scheme is commonly used in NMR and here we demonstrate its applicability to a number of frequently used pulse EPR experiments: Echo-Detected EPR, Davies and Mims ENDOR (Electron-Nuclear Double Resonance), DEER (Electron-Electron Double Resonance|) and EDNMR (Electron–Electron Double Resonance (ELDOR)-Detected NMR), which were combined with a Carr–Purcell–Meiboom–Gill (CPMG) type detection scheme at W-band. By collecting the transient signal and integrating a number of refocused echoes, this detection scheme yielded a 1.6–5 folds SNR improvement, depending on the paramagnetic center and the pulse sequence applied. This improvement is achieved while keeping the experimental time constant and it does not introduce signal distortion.
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determination of the 14n quadrupole coupling constant of nitroxide spin probes by w band eldor detected nmr
Journal of Magnetic Resonance, 2011Co-Authors: Marc Florent, Ilia Kaminker, Vijayasarathi Nagarajan, Daniella GoldfarbAbstract:Abstract Nitroxide spin probe Electron paramagnetic Resonance (EPR) has proven to be a very successful method to probe local polarity and solvent hydrogen bonding properties at the molecular level. The g xx and the 14 N hyperfine A zz principal values are the EPR parameters of the nitroxide spin probe that are sensitive to these properties and are therefore monitored experimentally. Recently, the 14 N quadrupole interaction of nitroxides has been shown to be also highly sensitive to polarity and H-bonding (A. Savitsky et al., J. Phys. Chem. B 112 (2008) 9079). High-field Electron spin echo envelope modulation (ESEEM) was used successfully to determine the P xx and P yy principal components of the 14 N quadrupole tensor. The P zz value was calculated from the traceless character of the quadrupole tensor. We introduce here high-field (W-band, 95 GHz, 3.5 T) Electron–Electron Double Resonance (ELDOR)-detected NMR as a method to obtain the 14 N P zz value directly, together with A zz . This is complemented by W-band hyperfine sublevel correlation (HYSCORE) measurements carried out along the g xx direction to determine the principal P xx and P yy components. Through measurements of TEMPOL dissolved in solvents of different polarities, we show that A zz increases, while | P zz | decreases with polarity, as predicted by Savitsky et al.
Wolfgang Lubitz - One of the best experts on this subject based on the ideXlab platform.
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intersubunit distances in full length dimeric bacterial phytochrome agp1 as measured by pulsed Electron Electron Double Resonance peldor between different spin label positions remain unchanged upon photoconversion
Journal of Biological Chemistry, 2017Co-Authors: Sylwia Kacprzak, Ibrahim Njimona, Anja Renz, Juan Feng, Edward J Reijerse, Wolfgang Lubitz, Norbert Krauss, Patrick Scheerer, Soshichiro Nagano, Tillman LamparterAbstract:Abstract Bacterial phytochromes are dimeric light-regulated histidine kinases that convert red light into signaling events. Light absorption by the N-terminal photosensory core module (PCM) causes the proteins to switch between two spectrally distinct forms, Pr and Pfr, thus resulting in a conformational change that modulates the C-terminal histidine kinase region. To provide further insights into structural details of photoactivation, we investigated the full-length Agp1 bacteriophytochrome from the soil bacterium Agrobacterium fabrum using a combined spectroscopic and modeling approach. We generated seven mutants suitable for spin labeling to enable application of pulsed Electron paramagnetic Resonance (EPR) techniques. The distances between attached spin labels were measured using pulsed Electron-Electron Double Resonance (PELDOR) spectroscopy to probe the arrangement of the subunits within the dimer. We found very good agreement of experimental and calculated distances for the histidine-kinase region when both subunits are in a parallel orientation. However, experimental distance distributions surprisingly showed only limited agreement with either parallel- or antiparallel-arranged dimer structures when spin labels were placed into the PCM region. This observation indicates that the arrangements of the PCM subunits in the full-length protein dimer in solution differ significantly from that in the PCM crystals. The PELDOR data presented here revealed either no or only minor changes of distance distributions upon Pr-to-Pfr photoconversion.
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intersubunit distances in full length dimeric bacterial phytochrome agp1 as measured by pulsed Electron Electron Double Resonance peldor between different spin label positions remain unchanged upon photoconversion
Journal of Biological Chemistry, 2017Co-Authors: Sylwia Kacprzak, Ibrahim Njimona, Anja Renz, Juan Feng, Edward J Reijerse, Wolfgang Lubitz, Norbert Krauss, Patrick Scheerer, Soshichiro Nagano, Tillman LamparterAbstract:Bacterial phytochromes are dimeric light-regulated histidine kinases that convert red light into signaling events. Light absorption by the N-terminal photosensory core module (PCM) causes the proteins to switch between two spectrally distinct forms, Pr and Pfr, thus resulting in a conformational change that modulates the C-terminal histidine kinase region. To provide further insights into structural details of photoactivation, we investigated the full-length Agp1 bacteriophytochrome from the soil bacterium Agrobacterium fabrum using a combined spectroscopic and modeling approach. We generated seven mutants suitable for spin labeling to enable application of pulsed EPR techniques. The distances between attached spin labels were measured using pulsed Electron-Electron Double Resonance spectroscopy to probe the arrangement of the subunits within the dimer. We found very good agreement of experimental and calculated distances for the histidine-kinase region when both subunits are in a parallel orientation. However, experimental distance distributions surprisingly showed only limited agreement with either parallel- or antiparallel-arranged dimer structures when spin labels were placed into the PCM region. This observation indicates that the arrangements of the PCM subunits in the full-length protein dimer in solution differ significantly from that in the PCM crystals. The pulsed Electron-Electron Double Resonance data presented here revealed either no or only minor changes of distance distributions upon Pr-to-Pfr photoconversion.
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high field eldor detected nmr study of a nitroxide radical in disordered solids towards characterization of heterogeneity of microenvironments in spin labeled systems
Journal of Magnetic Resonance, 2014Co-Authors: Anna Irena Nalepa, Wolfgang Lubitz, K Mobius, Anton SavitskyAbstract:The combination of high-field EPR with site-directed spin-labeling (SDSL) techniques employing nitroxide radicals has turned out to be particularly powerful in probing the polarity and proticity characteristics of protein/matrix systems. This information is concluded from the principal components of the nitroxide Zeeman (g), nitrogen hyperfine (A) and quadrupole (P) tensors of the spin labels attached to specific sites. Recent multi-frequency high-field EPR studies underlined the complexity of the problem to treat the nitroxide microenvironment in proteins adequately due to inherent heterogeneities which result in several principal x-components of the nitroxide g-tensor. Concomitant, but distinctly different nitrogen hyperfine components could, however, not be determined from high-field cw EPR experiments owing to the large intrinsic EPR linewidth in fully protonated guest/host systems. It is shown in this work that, using the W-band (95GHz) ELDOR- (Electron-Electron Double Resonance) detected NMR (EDNMR) method, different principal nitrogen hyperfine, Azz, and quadrupole, Pzz, tensor values of a nitroxide radical in glassy 2-propanol matrix can be measured with high accuracy. They belong to nitroxides with different hydrogen-bond situations. The satisfactory resolution and superior sensitivity of EDNMR as compared to the standard ENDOR (Electron-nuclear Double Resonance) method are demonstrated.
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Electron Electron Double Resonance detected nmr to measure metal hyperfine interactions 61ni in the ni b state of the nife hydrogenase of desulfovibrio vulgaris miyazaki f
Journal of the American Chemical Society, 2008Co-Authors: Marco Flores, Aruna Goenka Agrawal, Maurice Van Gastel, Wolfgang Gartner, Wolfgang LubitzAbstract:Electron Double Resonance-detected NMR (EDNMR) is introduced as a powerful technique to directly measure metal hyperfine interactions in the active sites of metalloproteins. Measurement of these quantities by Electron nuclear Double Resonance is usually difficult because of fast relaxation times and large anisotropic (dipolar) hyperfine couplings. In EDNMR, Electron paramagnetic Resonance (EPR) “forbidden” transitions are excited, which become EPR allowed to some extent because of the presence of a large dipolar hyperfine and possibly quadrupole interaction. The usefulness of EDNMR is demonstrated with measurements on 61Ni-enriched hydrogenase of D. vulgaris Miyazaki F in the Ni−B state.
Snorri Th Sigurdsson - One of the best experts on this subject based on the ideXlab platform.
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high resolution epr distance measurements on rna and dna with the non covalent ǵ spin label
Nucleic Acids Research, 2020Co-Authors: Marcel Heinz, Thomas F Prisner, Gerhard Hummer, Lukas S. Stelzl, Nicole Erlenbach, Snorri Th Sigurdsson, Grace Thierolf, Nilesh R KambleAbstract:Pulsed Electron paramagnetic Resonance (EPR) experiments, among them most prominently pulsed Electron-Electron Double Resonance experiments (PELDOR/DEER), resolve the conformational dynamics of nucleic acids with high resolution. The wide application of these powerful experiments is limited by the synthetic complexity of some of the best-performing spin labels. The recently developed $\bf\acute{G}$ (G-spin) label, an isoindoline-nitroxide derivative of guanine, can be incorporated non-covalently into DNA and RNA duplexes via Watson-Crick base pairing in an abasic site. We used PELDOR and molecular dynamics (MD) simulations to characterize $\bf\acute{G}$, obtaining excellent agreement between experiments and time traces calculated from MD simulations of RNA and DNA Double helices with explicitly modeled $\bf\acute{G}$ bound in two abasic sites. The MD simulations reveal stable hydrogen bonds between the spin labels and the paired cytosines. The abasic sites do not significantly perturb the helical structure. $\bf\acute{G}$ remains rigidly bound to helical RNA and DNA. The distance distributions between the two bound $\bf\acute{G}$ labels are not substantially broadened by spin-label motions in the abasic site and agree well between experiment and MD. $\bf\acute{G}$ and similar non-covalently attached spin labels promise high-quality distance and orientation information, also of complexes of nucleic acids and proteins.
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a semi rigid isoindoline derived nitroxide spin label for rna
Organic and Biomolecular Chemistry, 2018Co-Authors: Dnyaneshwar B Gophane, Burkhard Endeward, Thomas F Prisner, Snorri Th SigurdssonAbstract:A new isoindoline-derived benzimidazole nitroxide spin label, ImUm, was synthesized and incorporated into RNA oligoribonucleotides. ImUm is the first example of a conformationally unambiguous spin label for RNA, in which the nitroxide N-O bond lies on the same axis as the single bond used to attach the rigid isoindoline-based spin label to a uridine base. This results in minimal displacement of the nitroxide upon rotation of this single bond, which is a useful property for a label to be used for distance measurements. Continuous-wave (CW) EPR measurements of RNA duplexes containing ImUm indicate a restricted rotation around this single bond, presumably due to an intramolecular hydrogen bond between the benzimidazole N-H and O4 of the uracil. Orientation-selective pulsed Electron-Electron Double Resonance (PELDOR, also called Double Electron-Electron Resonance, or DEER) distance measurements between two spin labels in two RNA duplexes showed in one case a strong orientation dependence, further confirming the restricted motion of the spin labels in RNA duplexes.
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conformationally restricted isoindoline derived spin labels in duplex dna distances and rotational flexibility by pulsed Electron Electron Double Resonance spectroscopy
Chemistry: A European Journal, 2014Co-Authors: Dnyaneshwar B Gophane, Burkhard Endeward, Thomas F Prisner, Snorri Th SigurdssonAbstract:Three structurally related isoindoline-derived spin labels that have different mobilities were incorporated into duplex DNA to systematically study the effect of motion on orientation-dependent pulsed Electron–Electron Double Resonance (PELDOR) measurements. To that end, a new nitroxide spin label, ExImU, was synthesized and incorporated into DNA oligonucleotides. ExImU is the first example of a conformationally unambiguous spin label for nucleic acids, in which the nitroxide NO bond lies on the same axis as the three single bonds used to attach the otherwise rigid isoindoline-based spin label to a uridine base. Continuous-wave (CW) EPR measurements of ExImU confirm a very high rotational mobility of the spin label in duplex DNA relative to the structurally related spin label ImU, which has restricted mobility due to an intramolecular hydrogen bond. The X-band CW-EPR spectra of ExImU can be used to identify mismatches in duplex DNA. PELDOR distance measurements between pairs of the spin labels ImU, OxU, and ExImU in duplex DNA showed a strong angular dependence for ImU, a medium dependence for OxU, and no orientation effect for ExImU. Thus, precise distances can be extracted from ExImU without having to take orientational effects into account.
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orientation selection in distance measurements between nitroxide spin labels at 94 ghz epr with variable dual frequency irradiation
Physical Chemistry Chemical Physics, 2013Co-Authors: Igor Tkach, Snorri Th Sigurdsson, Ulf Diederichsen, Soraya Pornsuwan, Claudia Hobartner, Falk Wachowius, Tatiana Y Baranova, Giuseppe Sicoli, Marina BennatiAbstract:Pulsed Electron–Electron Double Resonance (PELDOR, also known as DEER) has become a method of choice to measure distances in biomolecules. In this work we show how the performance of the method can be improved at high EPR frequencies (94 GHz) using variable dual frequency irradiation in a dual mode cavity in order to obtain enhanced resolution toward orientation selection. Dipolar evolution traces of a representative RNA duplex and an α-helical peptide were analysed in terms of possible bi-radical structures by considering the inherent ambiguity of symmetry-related solutions.