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ABSTRACT: We have performed liquid state ("Overhauser") Dynamic Nuclear Polarization (DNP) experiments at high magnetic field (9.2 T, corresponding to 260 GHz EPR and 400 MHz (1)H-NMR resonance frequency) on aqueous solutions of (14)N-TEMPOL nitroxide radicals. Integrated signal enhancements exceeding -80 were observed for the water protons at microwave superheated temperatures (160 °C) and still -14 at ambient temperatures (45 °C) relevant to biological applications. Different contributions contributing to the DNP enhancement such as saturation factor, leakage factor and sample temperature under microwave irradiation could be determined independently for a high spin concentration of 1 M, allowing the calculation of the coupling factors as a function of temperature and a quantitative comparison of this parameter with values derived from field dependent relaxation measurements or predictions from MD simulation.
Physical Chemistry Chemical Physics 03/2013; · 3.57 Impact Factor
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ABSTRACT: Distance and spins: A new broadband, frequency-selective inversion pulse has been devised for pulsed electron-electron double resonance (PELDOR) spectroscopy, a unique method used to determine distance distributions between two or more paramagnetic centers 2-10 nm apart. The PELDOR modulation depth can be increased by substituting the rectangular pump pulse by a shaped pulse.
Angewandte Chemie International Edition 02/2013; · 13.45 Impact Factor
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ABSTRACT: Pulsed Electron-electron Double Resonance (PELDOR) is a method frequently used to determine the distances between paramagnetic centers in biomacromolecules on the nanometer scale. A standard algorithm for determination of distances from the experimental data assumes that all possible mutual orientations of the spin labels are equally probable. However, in many applications the mobility of the spin labels attached to large molecules can be significantly restricted. In order to determine the total PELDOR signal in this case, the individual contributions of each rigid biradical should be explicitly calculated for the given frequencies of the probe and pump pulses. The solution of the inverse problem of determination of the ensemble of molecular structures that fit the experimental PELDOR data acquired at multiple microwave frequencies and magnetic fields has proven to be a non-trivial task, especially, when no information about the molecular structure under study is available. In this work we present a fitting algorithm that reconstructs experimental data by searching for an optimal combination of presimulated PELDOR time traces for nitroxide biradicals with all relative orientations and with inter-spin distances in the experimentally accessible range. The generated library of PELDOR time traces has been employed to excellently fit experimental data containing orientation selection effects gathered on model biradical systems.
Physical Chemistry Chemical Physics 11/2012; · 3.57 Impact Factor
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ABSTRACT: A 1 ns resolution pulse shaping unit has been developed for pulsed EPR spectroscopy to enable 14-bit amplitude and phase modulation. Shaped broadband excitation pulses designed using optimal control theory (OCT) have been tested with this device at X-band frequency (9 GHz). FT-EPR experiments on organic radicals in solution have been performed with the new pulses, designed for uniform excitation over a significantly increased bandwidth compared to a classical rectangular π/2 pulse of the same B(1) amplitude. The concept of a dead-time compensated prefocused pulse has been introduced to EPR with a self-refocusing of 200 ns after the end of the pulse. Echo-like refocused signals have been recorded and compared to the performance of a classical Hahn-echo sequence. The impulse response function of the microwave setup has been measured and incorporated into the algorithm for designing OCT pulses, resulting in further significant improvements in performance. Experimental limitations and potential new applications of OCT pulses in EPR spectroscopy will be discussed.
Journal of Magnetic Resonance 05/2012; 218:49-58. · 2.14 Impact Factor
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ABSTRACT: Members of the ATP binding cassette (ABC) transporter superfamily translocate various types of molecules across the membrane at the expense of ATP. This requires cycling through a number of catalytic states. Here, we report conformational changes throughout the catalytic cycle of LmrA, a homodimeric multidrug ABC transporter from L. lactis. Using site-directed spin labeling and pulsed electron-electron double resonance (PELDOR/DEER) spectroscopy, we have probed the reorientation of the nucleotide binding domains and transmembrane helix 6 which is of particular relevance to drug binding and part of the dimerization interface. Our data show that LmrA samples a very large conformational space in its apo state, which is significantly reduced upon nucleotide binding. ATP binding but not hydrolysis is required to trigger this conformational change, which results in a relatively fixed orientation of both the nucleotide binding domains and transmembrane helices 6. This orientation is maintained throughout the ATP hydrolysis cycle until the protein cycles back to its apo state. Our data present strong evidence that switching between two dynamically and structurally distinct states is required for substrate translocation.
Journal of the American Chemical Society 03/2012; 134(13):5857-62. · 9.91 Impact Factor
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ABSTRACT: We have used liquid state ("Overhauser") Dynamic Nuclear Polarization (DNP) to significantly enhance the signal to noise ratio (SNR) of Magnetic Resonance Imaging (MRI). For the first time this was achieved by hyperpolarizing directly in the MRI-scanner field of 1.5 T in continuous flow mode and immediately delivering the hyperpolarized substance to the imaging site to ensure maximum contrast between hyperpolarized sample and sample at thermal polarization. We achieve a maximum absolute signal enhancement factor of 98; while the hyperpolarized sample is transported at a flow rate of up to 30 ml/h yielding an average flow speed up to 470 mm/s over a distance of approximately 80 mm. A spatial imaging resolution of 100 μm with a signal to noise ratio of 25 was achieved on the flowing sample. Application to MRI contrast enhancement or microfluidic imaging can be envisaged immediately.
Journal of Magnetic Resonance 02/2012; 215:94-9. · 2.14 Impact Factor
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ABSTRACT: Escherichia coli nitrate reductase A (NarGHI) is a membrane-bound enzyme that couples quinol oxidation at a periplasmically oriented Q-site (Q(D)) to proton release into the periplasm during anaerobic respiration. To elucidate the molecular mechanism underlying such a coupling, endogenous menasemiquinone-8 intermediates stabilized at the Q(D) site (MSQ(D)) of NarGHI have been studied by high-resolution pulsed EPR methods in combination with (1)H2O/2H2O exchange experiments. One of the two non-exchangeable proton hyperfine couplings resolved in hyperfine sublevel correlation (HYSCORE) spectra of the radical displays characteristics typical from quinone methyl protons. However, its unusually small isotropic value reflects a singularly low spin density on the quinone carbon α carrying the methyl group, which is ascribed to a strong asymmetry of the MSQ(D) binding mode and consistent with single-sided hydrogen bonding to the quinone oxygen O1. Furthermore, a single exchangeable proton hyperfine coupling is resolved, both by comparing the HYSCORE spectra of the radical in 1H2O and 2H2O samples and by selective detection of the exchanged deuterons using Q-band 2H Mims electron nuclear double resonance (ENDOR) spectroscopy. Spectral analysis reveals its peculiar characteristics, i.e. a large anisotropic hyperfine coupling together with an almost zero isotropic contribution. It is assigned to a proton involved in a short ∼1.6 Å in-plane hydrogen bond between the quinone O1 oxygen and the Nδ of the His-66 residue, an axial ligand of the distal heme b(D). Structural and mechanistic implications of these results for the electron-coupled proton translocation mechanism at the Q(D) site are discussed, in light of the unusually high thermodynamic stability of MSQ(D).
Journal of Biological Chemistry 12/2011; 287(7):4662-70. · 4.77 Impact Factor
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ABSTRACT: In this chapter we describe the application of CW and pulsed EPR methods for the investigation of structural and dynamical properties of RNA and DNA molecules and their interaction with small molecules and proteins. Special emphasis will be given to recent applications of dipolar spectroscopy on nucleic acids.
Topics in current chemistry 12/2011; 321:159-98. · 4.29 Impact Factor
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ABSTRACT: Pulsed electron-electron double resonance (PELDOR) spectroscopy is a powerful tool for measuring nanometer distances in spin-labeled systems. A common approach is doubly covalent spin-labeling of a macromolecule and measurement of the inter-spin distance, or to use singly-labeled components of a system that forms aggregates or oligomers. This situation has been described as a spin-cluster. The PELDOR signal, however, does not only contain the desired dipolar coupling between the spin-labels of the molecule or cluster under study. In samples of finite concentration the dipolar coupling between the spin-labels of the randomly distributed molecules or spin-clusters also contributes significantly. In homogeneous frozen solutions or lipid vesicle membranes this second contribution can be considered to be an exponential or stretched exponential decay, respectively. In this study, we show that this assumption is not valid in detergent micelles. Spin-labeled fatty acids that are randomly partitioned into different detergent micelles give rise to PELDOR time traces which clearly deviate from stretched exponential decays. The obtained signals can be modeled quantitatively based on the size of the micelles, their aggregation number, the spin-label concentration and the degree of spin-labeling. As a main conclusion a PELDOR signal deviating from a stretched exponential decay does not necessarily prove the observation of specific distance information on the molecule or cluster. These results are important for the interpretation of PELDOR experiments on membrane proteins or lipophilic peptides solubilized in detergent micelles or small vesicles, which often do not show pronounced dipolar oscillations in their time traces.
Journal of Magnetic Resonance 07/2011; 211(1):11-7. · 2.14 Impact Factor
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ABSTRACT: Pulsed Electron-Electron Double Resonance (PELDOR) on double-stranded DNA (ds-DNA) was used to investigate the conformational flexibility of helical DNA. Stretching, twisting, and bending flexibility of ds-DNA was determined by incorporation of two rigid nitroxide spin labels into a series of 20 base pair (bp) DNA duplexes. Orientation-selective PELDOR experiments performed at both X-band (9 GHz/0.3 T) and G-band (180 GHz/6.4 T) with spin label distances in the range of 2-4 nm allowed us to differentiate between different simple models of DNA dynamics existing in the literature. All of our experimental results are in full agreement with a dynamic model for ds-DNA molecules, where stretching of the molecule leads to a slightly reduced radius of the helix induced by a cooperative twist-stretch coupling.
Journal of the American Chemical Society 06/2011; 133(34):13375-9. · 9.91 Impact Factor
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Angewandte Chemie International Edition 05/2011; 50(22):5070-4. · 13.45 Impact Factor
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ABSTRACT: A or B: RNA-DNA hybrids, key intermediates in gene regulation, were classified by pulsed electron-electron double resonance (PELDOR) in combination with CD spectroscopy into two classes, interpreted as A- and B-like structures. RNase H1 cleavage of these hybrids is in full agreement with these assignments, cleaving the hybrids with A-like geometry preferentially. This combined analytical approach allows the interpretation and eventually the design of more easily cleavable hybrids as needed for the antisense technology.
Molecular BioSystems 02/2011; 7(4):1050-2. · 3.53 Impact Factor
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ABSTRACT: Pulsed electron-electron double resonance (PELDOR) spectroscopy is increasingly applied to spin-labeled membrane proteins. However, after reconstitution into liposomes, spin labels often exhibit a much faster transversal relaxation (T(m)) than in detergent micelles, thus limiting application of the method in lipid bilayers. In this study, the main reasons for enhanced transversal relaxation in phospholipid membranes were investigated systematically by use of spin-labeled derivatives of stearic acid and phosphatidylcholine as well as spin-labeled derivatives of the channel-forming peptide gramicidin A under the conditions typically employed for PELDOR distance measurements. Our results clearly show that dephasing due to instantaneous diffusion that depends on dipolar interaction among electron spins is an important contributor to the fast echo decay in cases of high local concentrations of spin labels in membranes. The main difference between spin labels in detergent micelles and membranes is their local concentration. Consequently, avoiding spin clustering and suppressing instantaneous diffusion is the key step for maximizing PELDOR sensitivity in lipid membranes. Even though proton spin diffusion is an important relaxation mechanism, only in samples of low local concentrations does deuteration of acyl chains and buffer significantly prolong T(m). In these cases, values of up to 7 μs have been achieved. Furthermore, our study revealed that membrane composition and labeling position in the membrane can also affect T(m), either by promoting the segregation of spin-labeled species or by altering their exposure to matrix protons. Effects of other experimental parameters including temperature (<50 K), presence of oxygen, and cryoprotectant type are negligible under our experimental conditions.
The Journal of Physical Chemistry B 10/2010; 114(42):13507-16. · 3.70 Impact Factor
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ABSTRACT: Dynamic nuclear polarization (DNP) at high magnetic fields (9.2 T, 400 MHz (1)H NMR frequency) requires high microwave power sources to achieve saturation of the EPR transitions. Here we describe the first high-field liquid-state DNP results using a high-power gyrotron microwave source (20 W at 260 GHz). A DNP enhancement of -29 on water protons was obtained for an aqueous solution of Fremy's Salt; in comparison the previous highest value was -10 using a solid-state microwave power source (maximum power 45 mW). The increased enhancements are partly due to larger microwave saturation and elevated sample temperature. These experimentally observed DNP enhancements, which by far exceed the predicted values extrapolated from low-field DNP experiments, demonstrate experimentally that DNP is possible in the liquid state also at high magnetic fields.
Physical Chemistry Chemical Physics 06/2010; 12(22):5786-90. · 3.57 Impact Factor
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ABSTRACT: Pulsed electron double resonance (PELDOR) spectroscopy reveals a prearranged tertiary structure of the 27 nucleotides long engineered neomycin-responsive riboswitch. Measured distances between spin labels at positions U4-U14, U4-U15, U14-U26, and U15-U26 were unchanged upon neomycin binding which implies that the global stem-loop architecture is preserved in the absence and presence of the ligand. On the basis of our results, we infer that low-temperature PELDOR data unambiguously demonstrate the existence of an enthalpically favorable set of RNA conformations ready to bind the ligand without major global rearrangement.
Journal of the American Chemical Society 02/2010; 132(5):1454-5. · 9.91 Impact Factor
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ABSTRACT: We demonstrate the ability of pulsed electron double resonance (PELDOR) experiments to determine the orientation of spin labels in biological macromolecules. Thus, the distance information usually obtained from PELDOR data can be complemented by the mutual orientation of macromolecular domains. A method to determine the angle beta between the spin label normal and the interspin axis is proposed and analyzed mathematically. The obtained analytical expression allows extraction of angles beta without a fitting procedure if these angles are equal for both nitroxide of biradical. The method was applied to the experimental data gathered on ten spin-labeled DNA samples. The angles estimated from the PELDOR data are in excellent agreement with literature values.
Physical Review E 02/2010; 81(2 Pt 1):021911. · 2.26 Impact Factor
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ABSTRACT: In the asymmetric unit of the title compound, C46H52N3O9·3C6H6, two of the benzene solvent molecules are located in general positions and two are disposed about inversion centers. One of the benzene molecules on an inversion center was grossly disordered and was excluded using the SQUEEZE subroutine in PLATON [Spek (2009). Acta Cryst. D65, 148–155]. In addition, one of the 2,2,5,5-tetramethyl-1-oxyl-3-pyrrolin-3-ylcarbonyl groups is disordered over two orientations with refined occupancies of 0.506 (2) and 0.494 (2). The 1-oxyl-3-pyrroline-3-carboxylate groups are essentially planar, with mean deviations from the planes of 0.026 (2), 0.012 (2), 0.034 (4) and 0.011 (4) Å. In the crystal structure, molecules are connected by five weak intermolecular C—H...O and four weak intermolecular C—H...π(benzene) interactions.
Acta Crystallographica Section E. 01/2010;
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ABSTRACT: The title molecule, C34H28I4·4C6H6, has crystallographic overline{4} symmetry and crystallizes with four symmetry-related benzene solvent molecules. The phenyl group is eclipsed with one of the adamantane C—C bonds. The tetraphenyladamantane units and the benzene solvent molecules are connected by weak intermolecular phenyl–benzene C—H...π and benzene–benzene C—H...π interactions. In the crystal, molecules are linked along the c-axis direction via the iodophenyl groups by a combination of weak intermolecular I...I [3.944 (1) Å] and I...π(phenyl) [3.608 (6) and 3.692 (5) Å] interactions.
Acta Crystallographica Section E. 01/2010;
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ABSTRACT: In the asymmetric unit of the title compound, C(46)H(52)N(3)O(9)·3C(6)H(6), two of the benzene solvent mol-ecules are located in general positions and two are disposed about inversion centers. One of the benzene mol-ecules on an inversion center was grossly disordered and was excluded using the SQUEEZE subroutine in PLATON [Spek (2009 ▶). Acta Cryst. D65, 148-155]. In addition, one of the 2,2,5,5-tetra-methyl-1-oxyl-3-pyrrolin-3-ylcarbonyl groups is disordered over two orientations with refined occupancies of 0.506 (2) and 0.494 (2). The 1-oxyl-3-pyrroline-3-carboxyl-ate groups are essentially planar, with mean deviations from the planes of 0.026 (2), 0.012 (2), 0.034 (4) and 0.011 (4) Å. In the crystal structure, mol-ecules are connected by five weak inter-molecular C-H⋯O and four weak inter-molecular C-H⋯π(benzene) inter-actions.
Acta Crystallographica Section E Structure Reports Online 01/2010; 66(Pt 4):o729-30. · 0.35 Impact Factor
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ABSTRACT: The title mol-ecule, C(34)H(28)I(4)·4C(6)H(6), has crystallographic symmetry and crystallizes with four symmetry-related benzene solvent mol-ecules. The phenyl group is eclipsed with one of the adamantane C-C bonds. The tetra-phenyl-adamantane units and the benzene solvent mol-ecules are connected by weak inter-molecular phen-yl-benzene C-H⋯π and benzene-benzene C-H⋯π inter-actions. In the crystal, mol-ecules are linked along the c-axis direction via the iodo-phenyl groups by a combination of weak inter-molecular I⋯I [3.944 (1) Å] and I⋯π(phen-yl) [3.608 (6) and 3.692 (5) Å] inter-actions.
Acta Crystallographica Section E Structure Reports Online 01/2010; 66(Pt 7):o1636. · 0.35 Impact Factor
