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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, Gerhard Hummer, Lukas S. Stelzl, Nicole Erlenbach, Thomas F. Prisner, 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, Thomas F. Prisner, Burkhard Endeward, 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, Nicole Erlenbach, Marcel Heinz, Thomas F. Prisner, 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.
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.
John H Enemark - One of the best experts on this subject based on the ideXlab platform.
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determination of the distance between the mo v and fe iii heme centers of wild type human sulfite oxidase by pulsed epr spectroscopy
Journal of Physical Chemistry B, 2012Co-Authors: Andrei V Astashkin, Asha Rajapakshe, Matthew J Cornelison, Kayunta Johnsonwinters, John H EnemarkAbstract:Intramolecular electron transfer (IET) between the molybdenum and heme centers of vertebrate sulfite oxidase (SO) is proposed to be a key step in the catalytic cycle of the enzyme. However, the X-ray crystallographic distance between these centers, RMoFe = 32.3 A, appears to be too long for the rapid IET rates observed in liquid solution. The Mo and heme domains are linked by a flexible tether, and it has been proposed that dynamic interdomain motion brings the two metal centers closer together and thereby facilitates rapid IET. To date, there have been no direct distance measurements for SO in solution that would support or contradict this model. In this work, pulsed electron–electron Double Resonance (ELDOR) and relaxation induced dipolar modulation enhancement (RIDME) techniques were used to obtain information about RMoFe in the Mo(V)Fe(III) state of wild type recombinant human SO in frozen glassy solution. Surprisingly, the data obtained suggest a fixed structure with RMoFe = 32 A, similar to that det...
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determination of the distance between the mo v and fe iii heme centers of wild type human sulfite oxidase by pulsed epr spectroscopy
The Journal of Physical Chemistry, 2012Co-Authors: Andrei V Astashkin, Asha Rajapakshe, Matthew J Cornelison, Kayunta Johnsonwinters, John H EnemarkAbstract:Intramolecular electron transfer (IET) between the molybdenum and heme centers of vertebrate sulfite oxidase (SO) is proposed to be a key step in the catalytic cycle of the enzyme. However, the X-ray crystallographic distance between these centers, RMₒFₑ = 32.3 A, appears to be too long for the rapid IET rates observed in liquid solution. The Mo and heme domains are linked by a flexible tether, and it has been proposed that dynamic interdomain motion brings the two metal centers closer together and thereby facilitates rapid IET. To date, there have been no direct distance measurements for SO in solution that would support or contradict this model. In this work, pulsed electron–electron Double Resonance (ELDOR) and relaxation induced dipolar modulation enhancement (RIDME) techniques were used to obtain information about RMₒFₑ in the Mo(V)Fe(III) state of wild type recombinant human SO in frozen glassy solution. Surprisingly, the data obtained suggest a fixed structure with RMₒFₑ = 32 A, similar to that determined by X-ray crystallography for chicken SO, although the orientation of the RMₒFₑ radius-vector with respect to the heme center was found to be somewhat different. The implications of these findings for the flexible tether model are discussed.
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pulsed eldor spectroscopy of the mo v fe iii state of sulfite oxidase prepared by one electron reduction with ti iii citrate
Journal of Biological Inorganic Chemistry, 2002Co-Authors: Rachel Codd, Andrei V Astashkin, Andrew Pacheco, Arnold M Raitsimring, John H EnemarkAbstract:The titration of chicken liver sulfite oxidase (SO) with the one-electron reductant Ti(III) citrate, at pH 7.0, results in nearly quantitative selective reduction of the Mo(VI) center to Mo(V), while the b-type heme center remains in the fully oxidized Fe(III) state. The selective reduction of the Mo(VI/V) couple has been established from electronic and EPR spectra. The elec- tronic spectrum of the Fe(III) heme center is essentially unchanged during the titration, and the continuous wave (CW)-EPR spectrum shows the appearance of the well-known Mo(V) signal due to the low pH (lpH) form of SO. Further confirmation of the selective formation of the Mo(V)/Fe(III) form of SO is provided by the � 1:1 ratio of the integrated intensities of the Mo(V) and low- spin Fe(III) EPR signals after addition of one equivalent of Ti(III). The selective generation of the Mo(V)/Fe(III) form of SO is unexpected, considering that previous microcoulometry and flash photolysis investigations have indicated that the Mo(VI/V) and Fe(III/II) couples of SO have similar reduction potentials at pH 7. The nearly quantitative preparation of the one-electron re- duced Mo(V)/Fe(III) form of SO by reduction with Ti(III) has enabled the interaction between these two paramagnetic metalcenters, which are l inked by a flex- ible loop with no secondary structure, to be investigated for the first time by variable-frequency pulsed electron- electron Double Resonance (ELDOR) spectroscopy. The ELDOR kinetics were obtained from frozen solutions at 4.2 K at severalmicrowave frequencies by pumping on the narrow Mo(V) signaland observing the effect on the Fe(III) primary echo at both higher and lower fre- quencies within the microwave C-band region. The ELDOR data indicate that freezing the solution of one- electron reduced SO produces localized regions where the concentration of SO approaches that in the crystal structure, which results in the interpair interactions be- ing the dominant dipolar interaction. However, thor- ough analysis of the ELDOR decay curves and simulations suggests a distribution of intramolecular Mo ... Fe distances, consistent with the proposalof mul - tiple conformations in solution for the flexible loop that connects the Mo and heme domains of SO.
Gerhard Hummer - 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, Gerhard Hummer, Lukas S. Stelzl, Nicole Erlenbach, Thomas F. Prisner, 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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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, Nicole Erlenbach, Marcel Heinz, Thomas F. Prisner, 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.
Olav Schiemann - One of the best experts on this subject based on the ideXlab platform.
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site directed spin labeling of rna with a gem diethylisoindoline spin label peldor relaxation and reduction stability
Molecules, 2019Co-Authors: Christine Wuebben, Dinar Abdullin, Simon Blume, Dominik Brajtenbach, Florian R Haege, Stephanie Kathschorr, Olav SchiemannAbstract:Ribonucleic acid function is governed by its structure, dynamics, and interaction with other biomolecules and influenced by the local environment. Thus, methods are needed that enable one to study RNA under conditions as natural as possible, possibly within cells. Site-directed spin-labeling of RNA with nitroxides in combination with, for example, pulsed electron–electron Double Resonance (PELDOR or DEER) spectroscopy has been shown to provide such information. However, for in-cell measurements, the usually used gem-dimethyl nitroxides are less suited, because they are quickly reduced under in-cell conditions. In contrast, gem-diethyl nitroxides turned out to be more stable, but labeling protocols for binding these to RNA have been sparsely reported. Therefore, we describe here the bioconjugation of an azide functionalized gem-diethyl isoindoline nitroxide to RNA using a copper (I)-catalyzed azide–alkyne cycloaddition (“click”-chemistry). The labeling protocol provides high yields and site selectivity. The analysis of the orientation selective PELDOR data show that the gem-diethyl and gem-dimethyl labels adopt similar conformations. Interestingly, in deuterated buffer, both labels attached to RNA yield TM relaxation times that are considerably longer than observed for the same type of label attached to proteins, enabling PELDOR time windows of up to 20 microseconds. Together with the increased stability in reducing environments, this label is very promising for in-cell Electron Paramagnetic Resonance (EPR) studies.
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determination of nitroxide spin label conformations via peldor and x ray crystallography
Physical Chemistry Chemical Physics, 2016Co-Authors: Dinar Abdullin, Gregor Hagelueken, Olav SchiemannAbstract:Pulsed electron–electron Double Resonance (PELDOR or DEER) in combination with site-directed spin labelling has emerged as an important method for measuring nanometer distance constraints that are used to obtain coarse-grained structures of biomolecules or to follow their conformational changes. Translating measured spin–spin distances between spin labels into structural information requires taking the conformational flexibility of spin label side chains into account. Here, we present an analysis of orientation selective PELDOR data recorded on six singly MTSSL-labelled azurin mutants. The analysis yielded conformational MTSSL ensembles, which are considerably narrower than those predicted using in silico spin labeling methods but match well with spin label conformations found in the corresponding crystal structures. The possible reasons and consequences for predicting spin label conformers in the fold of biomolecules are discussed.
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single and Double nitroxide labeled bis terpyridine copper ii influence of orientation selectivity and multispin effects on peldor and ridme
Physical Chemistry Chemical Physics, 2016Co-Authors: Andreas J Meyer, Dinar Abdullin, Gregor Schnakenburg, Olav SchiemannAbstract:A rigid, nitroxide substituted terpyridine ligand has been used to synthesize hetero- and homoleptic bis-terpyridine complexes of copper(II). The homoleptic complex represents a three-spin system, while the metal ion in the heteroleptic complex is in average bound to one nitroxide bearing ligand. Both complexes are used as model systems for EPR distance measurements using pulsed electron–electron Double Resonance (PELDOR or DEER) and relaxation induced dipolar modulation enhancement (RIDME) sequences. The results of both methods are analyzed using detailed geometric data obtained from the crystal structure of the homoleptic complex as well as information concerning ligand scrambling and the electronic structure of the copper center. In addition, both methods are compared with respect to their sensitivity, the extent of orientation selectivity and the influence of multispin effects.
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conformational state of the mscs mechanosensitive channel in solution revealed by pulsed electron electron Double Resonance peldor spectroscopy
Proceedings of the National Academy of Sciences of the United States of America, 2012Co-Authors: Christos Pliotas, Gregor Hagelueken, Olav Schiemann, Richard J Ward, Emma Branigan, Akiko Rasmussen, H Huang, Susan S Black, Ian R Booth, James H NaismithAbstract:The heptameric mechanosensitive channel of small conductance (MscS) provides a critical function in Escherichia coli where it opens in response to increased bilayer tension. Three approaches have defined different closed and open structures of the channel, resulting in mutually incompatible models of gating. We have attached spin labels to cysteine mutants on key secondary structural elements specifically chosen to discriminate between the competing models. The resulting pulsed electron–electron Double Resonance (PELDOR) spectra matched predicted distance distributions for the open crystal structure of MscS. The fit for the predictions by structural models of MscS derived by other techniques was not convincing. The assignment of MscS as open in detergent by PELDOR was unexpected but is supported by two crystal structures of spin-labeled MscS. PELDOR is therefore shown to be a powerful experimental tool to interrogate the conformation of transmembrane regions of integral membrane proteins.
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counting the monomers in nanometer sized oligomers by pulsed electron electron Double Resonance
Journal of the American Chemical Society, 2007Co-Authors: Bela E Bode, Thomas F. Prisner, Jorn Plackmeyer, Dominik Margraf, Gerd Durner, Olav SchiemannAbstract:In a lot of cases active biomolecules are complexes of higher order, thus methods capable of counting the number of building blocks and elucidating their geometric arrangement are needed. Therefore, we experimentally validate here spin-counting via 4-pulse Electron-Electron Double Resonance (PELDOR) on well-defined test samples. Two biradicals, a symmetric and an asymmetric triradical, and a tetraradical were synthesized in a convergent reaction scheme via palladium-catalyzed cross-coupling reactions. PELDOR was then used to obtain geometric information and the number of spin centers per molecule in a single experiment. The measurement yielded the expected distances (2.2-3.8 nm) and showed that different spin-spin distances in one molecule can be resolved even if the difference amounts to only 5 A. The number of spins n has been determined to be 2.1 in both biradicals, to 3.1 and 3.0 in the symmetric and asymmetric triradicals, respectively, and to 3.9 in the tetraradical. The overall error of PELDOR spin-counting was found to be 5% for up to four spins. Thus, this method is a valuable tool to determine the number of constituting spin-bearing monomers in biologically relevant homo- and heterooligomers and how their oligomerization state and geometric arrangement changes during function.