New pulsed EPR methods and their application to characterize mitochondrial complex I

Electron Paramagnetic Resonance (EPR) spectroscopy is the method of choice to study paramagnetic cofactors that often play an important role as active centers in electron transfer processes in biological systems. However, in many cases more than one paramagnetic species is contributing to the observed EPR spectrum, making the analysis of individual contributions difficult and in some cases impossible. With time-domain techniques it is possible to exploit differences in the relaxation behavior of different paramagnetic species to distinguish between them and separate their individual spectral contribution. Here we give an overview of the use of pulsed EPR spectroscopy to study the iron–sulfur clusters of NADH:ubiquinone oxidoreductase (complex I). While FeS cluster N1 can be studied individually at a temperature of 30 K, this is not possible for FeS cluster N2 due to its severe spectral overlap with cluster N1. In this case Relaxation Filtered Hyperfine (REFINE) spectroscopy can be used to separate the overlapping spectra based on differences in their relaxation behavior.Collaborative Research Centre 472 (Project P2)Collaborative Research Centre 472 (Project P15)Goethe University in Frankfurt/Main. Center for Biomolecular Magnetic Resonanc

Molecular simulations for dynamic nuclear polarization in liquids: a case study of TEMPOL in acetone and DMSO

A computational strategy for calibrating, validating and analyzing molecular dynamics (MD) simulations to predict dynamic nuclear polarization (DNP) coupling factors and relaxivities of proton spins is presented. Simulations of the polarizing agent TEMPOL in liquid acetone and DMSO are conducted at low (infinite dilution) and high (1 M) concentrations of the free radical. Because DNP coupling factors and relaxivities are sensitive to the time scales of the molecular motions, the MD simulations are calibrated to reproduce the bulk translational diffusion coefficients of the pure solvents. The simulations are then validated by comparing with experimental dielectric relaxation spectra, which report on the rotational dynamics of the molecular electric dipole moments. The analysis consists of calculating spectral density functions (SDFs) of the magnetic dipole–dipole interaction between the electron spin of TEMPOL and nuclear spins of the solvent protons. Here, MD simulations are used in combination with an analytically tractable model of molecular motion. While the former provide detailed information at relatively short spin–spin distances, the latter includes contributions at large separations, all the way to infinity. The relaxivities calculated from the SDFs of acetone and DMSO are in excellent agreement with experiments at 9.2 T. For DMSO we calculate a coupling factor in agreement with experiment while for acetone we predict a value that is larger by almost 50%, suggesting a possibility for experimental improvement

1,3,5,7-Tetra­kis(4-iodo­phen­yl)adamantane benzene tetra­solvate

The title mol­ecule, C34H28I4·4C6H6, has crystallographic 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

4,4′,4′′-(Methane­triyl)triphenyl tris­(2,2,5,5-tetra­methyl-1-oxyl-3-pyrroline-3-carboxyl­ate) benzene tris­olvate

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

Noncovalent spin-labeling of RNA: the aptamer approach

Post-print (lokagerð höfundar)In the first example of site-directed spin-labeling of unmodified RNA, a pyrrolidine-nitroxide derivative of tetramethylrosamine (TMR) was shown to bind with high affinity to the malachite green (MG) aptamer, as determined by continuous-wave (CW) electron paramagnetic resonance (EPR), pulsed electron–electron double resonance (PELDOR) and fluorescence spectroscopies.The authors acknowledge financial support by the IcelandicResearch Fund (141062051). S. S. gratefully acknowledgesa doctoral fellowship provided by the University of Iceland.T. H. and T. F. P. would like to acknowledge the CollaborativeResearch Center 902 ‘Molecular Principles of RNA-basedRegulation’ of the German Research Foundation for funding.The authors thank Dr S. Jonsdottir for assistance with collectinganalytical data for structural characterization of the compoundsprepared, K. R. Oskarsson for his assistance with collectingfluorescence data and Dr T. Halbritter for his assistance ingenerating the molecular models. The authors thank membersof the Sigurdsson research group for critical reading of themanuscript and for helpful discussionsPeer reviewe

Noncovalent and site-directed spin labeling of duplex RNA

Post-print (lokagerð höfundar)An isoindoline-nitroxide derivative of guanine (G, "G-spin") was shown to bind specifically and effectively to abasic sites in duplex RNAs. Distance measurements on a G-labeled duplex RNA with PELDOR (DEER) showed a strong orientation dependence. Thus, G is a readily synthesized, orientation-selective spin label for "mix and measure" PELDOR experiments.This work was supported by the Deutsche Forschungsgemeinschaft DFG (SFB 902 Molecular Principles of RNA-based Regulation), the Icelandic Research Fund (141062051) and by a doctoral fellowship to N. R. K. from the University of Iceland Research Fund. We thank Dr S. Jonsdottir for assistance in collecting analytical data for structural characterization of the compounds.Peer reviewe

Biphenyl-4,4′-diyl bis­(2,2,5,5-tetra­methyl-1-oxyl-3-pyrroline-3-carboxyl­ate)

In the title compound, C30H34N2O6, the complete molecule is generated by a crystallographic 2/m symmetry operation. The 1-oxyl-3-pyrroline-3-carboxyl­ate group lies on a mirror plane. The dihedral angle between the ring planes of the biphenyl fragment is constrained by symmetry to be zero, resulting in rather short intramolecular H⋯H contact distances of 2.02 Å. In the crystal, molecules are connected along the a-axis direction by very weak intermolecular methyl–phenyl C—H⋯π interactions. The C—H bond is not directed to the center of the benzene ring, but mainly to one C atom [C—H⋯C(x − 1, y, z): H⋯C = 2.91 Å and C—H⋯C = 143°]

Flexibility and conformation of the cocaine aptamer studied by PELDOR

Publisher's version (útgefin grein)The cocaine aptamer is a DNA three-way junction that binds cocaine at its helical junction. We studied the global conformation and overall flexibility of the aptamer in the absence and presence of cocaine by pulsed electron-electron double resonance (PELDOR) spectroscopy, also called double electronelectron resonance (DEER). The rigid nitroxide spin label C was incorporated pairwise into two helices of the aptamer. Multi-frequency 2D PELDOR experiments allow the determination of the mutual orientation and the distances between two Cs. Since C is rigidly attached to double-stranded DNA, it directly reports on the aptamer dynamics. The cocaine-bound and the non-bound states could be differentiated by their conformational flexibility, which decreases upon binding to cocaine. We observed a small change in the width and mean value of the distance distribution between the two spin labels upon cocaine binding. Further structural insights were obtained by investigating the relative orientation between the two spin-labeled stems of the aptamer. We determined the bend angle between this two stems. By combining the orientation information with a priori knowledge about the secondary structure of the aptamer, we obtained a molecular model describing the global folding and flexibility of the cocaine aptamer.This work was supported by the Deutsche Forschungsgemeinschaft DFG (SFB 902 Molecular Principles of RNA-based Regulation, Cluster of Excellence Frankfurt Macromolecular Complexes) and the Icelandic Research Fund [120001022]. T. F. Prisner and C. M. Grytz gratefully acknowledge support by the Fond der Chemischen Industrie. We thank Dnyaneshwar B. Gophane for help in sample preparation.Peer reviewe

Pulsed EPR dipolar spectroscopy at Q- and G-band on a trityl biradical

Post-print (lokagerð höfundar)Pulsed electron paramagnetic resonance (EPR) spectroscopy is a valuable technique for the precise determination of distances between paramagnetic spin labels that are covalently attached to macromolecules. Nitroxides have commonly been utilised as paramagnetic tags for biomolecules, but trityl radicals have recently been developed as alternative spin labels. Trityls exhibit longer electron spin relaxation times and higher stability than nitroxides under in vivo conditions. So far, trityl radicals have only been used in pulsed EPR dipolar spectroscopy (PDS) at X-band (9.5 GHz), K-u-band (17.2 GHz) and Q-band (34 GHz) frequencies. In this study we investigated a trityl biradical by PDS at Q-band (34 GHz) and G-band (180 GHz) frequencies. Due to the small spectral width of the trityl (30 MHz) at Q-band frequencies, single frequency PDS techniques, like double-quantum coherence (DQC) and single frequency technique for refocusing dipolar couplings (SIFTER), work very efficiently. Hence, Q-band DQC and SIFTER experiments were performed and the results were compared; yielding a signal to noise ratio for SIFTER four times higher than that for DQC. At G-band frequencies the resolved axially symmetric g-tensor anisotropy of the trityl exhibited a spectral width of 130 MHz. Thus, pulsed electron electron double resonance (PELDOR/DEER) obtained at different pump-probe positions across the spectrum was used to reveal distances. Such a multi-frequency approach should also be applicable to determine structural information on biological macromolecules tagged with trityl spin labels.The authors acknowledge Dr Vasyl Denysenkov for the technical support with the G-band EPR spectrometer and Dr Alice Bowen for useful discussions and for proof reading the manuscript. This work is supported by SPP 1601 New Frontiers in Sensitivity for EPR Spectroscopy: from Biological Cells to Nano Materials from the German Research Society DFG, the Cluster of Excellence Frankfurt (CEF) Macromolecular Complexes and the Icelandic Research Fund (120001021), which are all gratefully acknowledged.Peer reviewe