Pulsed Electron Paramagnetic Resonance Techniques Research Articles

Exploring the conformational landscapes of protein kinases: perspectives from FRET and DEER.

Conformational changes of catalytically-important structural elements are a key feature of the regulation mechanisms of protein kinases and are important for dictating inhibitor binding modes and affinities. The lack of widely applicable methods for tracking kinase conformational changes in solution has hindered our understanding of kinase regulation and our ability to design conformationally selective inhibitors. Here we provide an overview of two recently developed methods that detect conformational changes of the regulatory activation loop and αC-helix of kinases and that yield complementary information about allosteric mechanisms. An intramolecular Förster resonance energy transfer-based approach provides a scalable platform for detecting and classifying structural changes in high-throughput, as well as quantifying ligand binding cooperativity, shedding light on the energetics governing allostery. The pulsed electron paramagnetic resonance technique double electron-electron resonance provides lower throughput but higher resolution information on structural changes that allows for unambiguous assignment of conformational states and quantification of population shifts. Together, these methods are shedding new light on kinase regulation and drug interactions and providing new routes for the identification of novel kinase inhibitors and allosteric modulators.

Interaction of barley β-glucan with food dye molecules – An insight from pulse dipolar EPR spectroscopy

The interactions between dietary fibers (DFs) and small molecules are of great interest to food chemistry and nutrition science. However, the corresponding interaction mechanisms and structural rearrangements of DFs at the molecular level are still opaque due to the usually weak binding and the lack of appropriate techniques to determine details of conformational distributions in such weakly organized systems. By combining our previously established methodology on stochastic spin-labelling of DFs with the appropriately revised set of pulse electron paramagnetic resonance techniques, we present here a toolkit to determine the interactions between DFs and small molecules, using barley β-glucan as an example for neutral DF and a selection of food dye molecules as examples for small molecules. The proposed here methodology allowed us to observe subtle conformational changes of β-glucan by detecting multiple details of the local environment of the spin labels. Substantial variations of binding propensities were detected for different food dyes.

Maximizing the applicability of continuous wave (CW) Electron Paramagnetic Resonance (EPR): what more can we do after a century?

Electron Paramagnetic Resonance/Electron Spin Resonance (EPR/ESR) spectroscopy is a powerful experimental approach solving challenging problems that are inaccessible to other experimental techniques. While pulsed EPR techniques and their applications have been the central focus of the field nowadays, methodology development and application of continuous wave (CW) EPR have never been undervalued. Although the importance of EPR in research has been recognized in many areas as indicated by the increasing number of EPR research articles published every year, the growth in the number of EPR research teams seems to not match the former, likely caused by the relatively high cost of modern pulsed EPR spectrometers. In this perspective, we aim to discuss the new areas and new directions of the application of CW EPR in combination with spin labeling that are slightly different from the classic/traditional applications of the CW EPR technique. Based on our experience, we show example applications of CW EPR on the interfaces of protein-confinement materials, protein-inorganic crystal lattices, protein-nanoparticles, and protein-polymers. Various structure and dynamics information can be resolved from the data analysis of CW EPR, leading to enhanced understanding of protein biophysics upon interaction with the synthetic nanostructures as well as possible guidance of hybrid materials development based on the combination of proteins and nanostructures. We also discussed the possibilities of even broadening the application spectrum of EPR spectroscopy. The relatively high affordability of CW EPR spectrometers (as compared to the pulsed ones) also facilitates the growth in EPR research groups worldwide.

Study of Electron-Nuclear Interactions in Doped Calcium Phosphates by Various Pulsed EPR Spectroscopy Techniques.

Substituted calcium phosphates (CaPs) are vital materials for the treatment of bone diseases and repairing and replacement of defects in human hard tissues. In this paper, we present some applications of the rarely used pulsed electron paramagnetic resonance (EPR) and hyperfine interaction spectroscopy approaches [namely, electron spin-echo envelope modulation (ESEEM) and electron–electron double-resonance detected nuclear magnetic resonance (EDNMR)] to investigate synthetic CaPs (hydroxyapatite, tricalcium, and octacalcium phosphate) doped with various cations (Li+, Na+, Mn2+, Cu2+, Fe3+, and Ba2+). These resonance techniques provide reliable tools to obtain unique information about the presence and localization of impurity centers and values of hyperfine and quadrupole tensors. We show that revealed in CaPs by EPR techniques, radiation-induced stable nitrogen-containing species and carbonate radicals can serve as sensitive paramagnetic probes to follow CaPs’ structural changes caused by cation doping. The most pulsed EPR, ESEEM, and EDNMR spectra can be detected at room temperature, reducing the costs of the measurements and facilitating the usage of pulsed EPR techniques for CaP characterization.

Application of pulsed and high-frequency electron paramagnetic resonance techniques to study petroleum disperse systems

The spectral and relaxation characteristics of “free” organic radicals (FR) and vanadyl-porphyrin (VP) complexes in various petroleum disperse systems (PDS) like bitumen, petroleum, their high-molecular components and solutions were studied using stationary (conventional) and pulsed electron paramagnetic resonance (EPR) techniques in two frequency ranges (X- and W-bands, with the microwave radiation frequencies of about 9 GHz and 95 GHz, respectively). The features of the pulsed approaches (electron spin echo, modulation of the electron spin echo signal decay, electronic relaxation times) and high-frequency EPR for PDS investigations were examined. W-band EPR allows to resolve spectrally the lines from the different paramagnetic centers and more accurately determine their spectral characteristics. It is shown that the electron spin echo can be observed at room temperatures even at high magnetic fields of 3.4 T demonstrating the potential of application of pulsed EPR techniques for the low-cost oilfield measurements. Analysis of the VP transverse magnetization decay curve permits to identify electron-nuclear interactions with the 14N and 1H nuclei in situ while in the EPR spectra these hyperfine interactions usually cannot be detected. It is found from the W-band EPR measurements that FR lineshape cannot be fitted with isotropic parameters in contrast to the established X-band results. The observed effect of increasing the rates of electronic transverse relaxation in asphaltenes is described in the framework of a model of spectral diffusion between the fast- and slow-relaxing paramagnetic centers in supramolecular complexes of asphaltenes.

Probing the Y2 Receptor on Transmembrane, Intra- and Extra-Cellular Sites for EPR Measurements

The function of G protein-coupled receptors is intrinsically linked to their conformational dynamics. In conjugation with site-directed spin labeling, electron paramagnetic resonance (EPR) spectroscopy provides powerful tools to study the highly dynamic conformational states of these proteins. Here, we explored positions for nitroxide spin labeling coupled to single cysteines, introduced at transmembrane, intra- and extra-cellular sites of the human neuropeptide Y2 receptor. Receptor mutants were functionally analyzed in cell culture system, expressed in Escherichia coli fermentation with yields of up to 10 mg of purified protein per liter expression medium and functionally reconstituted into a lipid bicelle environment. Successful spin labeling was confirmed by a fluorescence assay and continuous wave EPR measurements. EPR spectra revealed mobile and immobile populations, indicating multiple dynamic conformational states of the receptor. We found that the singly mutated positions by MTSL ((1-oxyl-2,2,5,5-tetramethyl-2,5-dihydro-1H-pyrrol-3-yl) methyl methanesulfonothioate) have a water exposed immobilized conformation as their main conformation, while in case of the IDSL (bis(1-oxyl-2,2,5,5-tetramethyl-3-imidazolin-4-yl) disulfide) labeled positions, the main conformation are mainly of hydrophobic nature. Further, double cysteine mutants were generated and examined for potential applications of distance measurements by double electron–electron resonance (DEER) pulsed EPR technique on the receptor.

An Unexpected Species Determined by X-ray Crystallography that May Represent an Intermediate in the Reaction Catalyzed by Quinolinate Synthase

Quinolinic acid is a common intermediate in the biosynthesis of nicotinamide adenine dinucleotide and its derivatives in all organisms that synthesize the molecule de novo. In most prokaryotes, it is formed from the condensation of dihydroxyacetone phosphate (DHAP) and iminoaspartate (IA) by the action of quinolinate synthase (NadA). NadA contains a [4Fe-4S] cluster cofactor with a unique noncysteinyl-ligated iron ion (Fea), which is proposed to bind the hydroxyl group of an intermediate in its reaction to facilitate a dehydration step. However, direct evidence for this role in catalysis has yet to be provided, and the exact chemical mechanism that underlies this transformation remains elusive. Herein, we present a structure of NadA from Pyrococcus horikoshii (PhNadA) in complex with IA and show that a carboxylate group of the molecule is ligated to Fea of the iron-sulfur cluster, occupying the site to which DHAP has been proposed to bind during catalysis. When crystals of PhNadA in complex with IA are soaked briefly in DHAP before freezing, electron density for a new molecule is observed, which we suggest is related to an intermediate in the reaction. Similar, but slightly different, "intermediates" are observed when crystals of a PhNadA Glu198Gln variant are incubated with DHAP, oxaloacetate, and ammonium chloride, conditions under which IA is formed chemically. Continuous-wave and pulse electron paramagnetic resonance techniques are used to verify the binding mode of substrates and proposed intermediates in frozen solution.

Slow spin relaxation of paramagnetic centers in graphene oxide

Paramagnetic centers in graphene oxide (GO) were studied by continuous wave and pulsed electron paramagnetic resonance (EPR) in the temperature range 4.2–300 K. Saturation of the EPR signal indicates long spin relaxation times of the paramagnetic centers and show that the relaxation times increase with lowering temperature as well as with decreasing the number of water molecules by drying. Long spin-lattice and phase memory relaxation times in deeply dried GO are quantitatively studied by the pulsed EPR techniques. The spin-lattice relaxation time is found to decrease from 52 ms at 5 K to 0.153 ms at 240 K and is dominated by the direct process below 100 K. The T5 dependence observed at higher temperatures can result from Raman processes. The phase memory time is about 1 μs at 240 K, changes non-monotonically with lowering temperature, and reaches its maximum of 2.2 μs at 5 K. Molecular motions of the functional groups and the adsorbed water molecules are employed to explain this dependence. The paramagnetic centers are attributed to the unfunctionalized carbons in the highly functionalized regions of GO. Their interactions with protons are confirmed by ESEEM. Slow relaxation extends possible applications of GO for quantum computing and spintronics.

Lattice distortions in hydroxyapatites with size as follows from the electronic relaxation time measurements

Pure and substituted hydroxyapatites (Ca10(PO4)6(OH)2, HAp) have been recently gaining special importance in catalysis for selective reactions where the synergetic functioning of acid–base active site pair facilitates the molecular transformations. Transition to the nanostructured HAp materials and ion incorporation can cause lattice distortions and influence physicochemical properties of HAp. We have investigated powders of HAp synthesized by wet techniques from the nitrogen-containing solutions with the average particle sizes of 30 (nanosized) and 1000 nm (microsized) with pulsed electron paramagnetic resonance (EPR). Electronic relaxation times (T1e) for the stable impurity paramagnetic NO32− centers were measured in the temperature range of 10-300 K. It is revealed that T1e shortens with the size reduction of factor two in the whole temperature range. The results are discussed in the framework of the modified Debye model taking into account non-Debye HAp phonon density. The obtained results show the feasibility of pulsed EPR techniques for characterization HAp based materials.

Gd(III)-Gd(III) Relaxation-Induced Dipolar Modulation Enhancement for In-Cell Electron Paramagnetic Resonance Distance Determination.

In-cell distance determination by electron paramagnetic resonance (EPR) spectroscopy reveals essential structural information about biomacromolecules under native conditions. We demonstrate that the pulsed EPR technique RIDME (relaxation induced dipolar modulation enhancement) can be utilized for such distance determination. The performance of in-cell RIDME has been assessed at Q-band using stiff molecular rulers labeled with Gd(III)-PyMTA and microinjected into Xenopus laevis oocytes. The overtone coefficients are determined to be the same for protonated aqueous solutions and inside cells. As compared to in-cell DEER (double electron–electron resonance, also abbreviated as PELDOR), in-cell RIDME features approximately 5 times larger modulation depth and does not show artificial broadening in the distance distributions due to the effect of pseudosecular terms.

Influence of Mg2+ on the conformational flexibility of a tetracycline aptamer.

The tetracycline-binding RNA aptamer (TC-aptamer) is a synthetic riboswitch that binds the antibiotic tetracycline (TC) with exceptionally high affinity. Although a crystal structure exists of the TC-bound state, little is known about the conformational dynamics and changes upon ligand binding. In this study, pulsed electron paramagnetic resonance techniques for measuring distances (PELDOR) in combination with rigid nitroxide spin labels (Çm spin label) were used to investigate the conformational flexibility of the TC-aptamer in the presence and absence of TC at different Mg2+ concentrations. TC was found to be the essential factor for stabilizing the tertiary structure at intermediate Mg2+ concentrations. At higher Mg2+ concentrations, Mg2+ alone is sufficient to stabilize the tertiary structure. In addition, the orientation of the two spin-labeled RNA helices with respect to each other was analyzed with orientation-selective PELDOR and compared to the crystal structure. These results demonstrate for the first time the unique value of the Çm spin label in combination with PELDOR to provide information about conformational flexibilities and orientations of secondary structure elements of biologically relevant RNAs.

Continuous Wave and Pulse EPR Characterization of Open-Shell Ti3+ Ions Generated in Hybrid SiO2–TiO2 Monoliths

Open-shell Ti3+ ions are generated within hybrid SiO2–TiO2 mesoporous monoliths by reaction with triethylaluminium vapors. Continuous wave and 2D pulse electron paramagnetic resonance techniques are used to investigate the local coordination environment of the Ti3+ species through detection of hyperfine interactions of the unpaired electron with magnetically active nuclei of the matrix, including natural abundant framework 29Si. The results provide evidence for the reducing power of triethylaluminium towards isolated Ti4+ ions through the formation of open-shell Ti3+ ions atomically dispersed and fully incorporated in the SiO2 framework with a local structure similar to that of titanium silicalite and no segregation of TiO2 phases at this level of Ti doping.

Salen Complexes as Fire Protective Agents for Thermoplastic Polyurethane: Deep Electron Paramagnetic Resonance Spectroscopy Investigation.

The contribution of copper complexes of salen-based Schiff bases N, N'-bis(salicylidene)ethylenediamine (C1), N, N'-bis(4-hydroxysalicylidene)ethylenediamine (C2), and N, N'-bis(5-hydroxysalicylidene)ethylenediamine (C3) to the flame retardancy of thermoplastic polyurethane (TPU) is investigated in the context of minimizing the inherent flammability of TPU. Thermal and fire properties of TPU are evaluated. It is observed that fire performances vary depending upon the substitution of the salen framework. Cone calorimetry [mass loss calorimetry (MLC)] results show that, in TPU at 10 wt % loading, C2 and C3 reduce the peak of heat release rate by 46 and 50%, respectively. At high temperature, these copper complexes undergo polycondensation leading to resorcinol-type resin in the condensed phase and thus acting as intumescence reinforcing agents. C3 in TPU is particularly interesting because it delays significantly the time to ignition (MLC experiment). In addition, pyrolysis combustion flow calorimetry shows reduction in the heat release rate curve, suggesting its involvement in gas-phase action. Structural changes of copper complexes and radical formation during thermal treatment as well as their influence on fire retardancy of TPU in the condensed phase are investigated by spectroscopic studies supported by microscopic and powder diffraction studies. Electron paramagnetic resonance (EPR) spectroscopy was fully used to follow the redox changes of Cu(II) ions as well as radical formation of copper complexes/TPU formulations in their degradation pathways. Pulsed EPR technique of hyperfine sublevel correlation spectroscopy reveals evolution of the local surrounding of copper and radicals with a strong contribution of nitrogen fragments in the degradation products. Further, the spin state of radicals was investigated by the two-dimensional technique of phase-inverted echo-amplitude detected nutation experiment. Two different radicals were detected, that is, one monocarbon radical and an oxygen biradical. Thus, the EPR study permits to deeply investigate the mode of action of copper salen complexes in TPU.

Selective Distance Measurements Using Triple Spin Labeling with Gd3+, Mn2+, and a Nitroxide.

Distance measurements by pulse electron paramagnetic resonance techniques, such as double electron-electron resonance (DEER, also called PELDOR), have become an established tool to explore structural properties of biomacromolecules and their assemblies. In such measurements a pair of spin labels provides a single distance constraint. Here we show that by employing three different types of spin labels that differ in their spectroscopic and spin dynamics properties it is possible to extract three independent distances from a single sample. We demonstrate this using the Antennapedia homeodomain orthogonally labeled with Gd3+ and Mn2+ tags in complex with its cognate DNA binding site labeled with a nitroxide.

Novel Pulsed Electron Paramagnetic Resonance Techniques for the Studies of Structure and Dynamics of Photo‐excited Triplet State of Organic Molecules: A Professional Journey

We present a few novel pulsed electron paramagnetic resonance techniques developed in our laboratory for the studies of structure and dynamics of the photo‐excited triplet state of organic molecules. We discuss many aspects of these new techniques and the significances of these measurements: (1) enhancing NMR signal intensity by dynamic nuclear polarization ‐ integrated solid effect, (2) performing magnetic resonance in zero‐field and low‐field by pulsed microwave, (3) mapping molecular motion of organic crystals by pulsed zero‐field and low‐field experiments, (4) probing spin dynamics at level anti‐crossing by fast field switching, (5) measuring hyperfine interaction by electron spin echo envelop modulation and spin‐echo electron nuclear double resonance and (6) detecting spin dynamics, nuclear quantum oscillation, entanglements and new avenues for quantum computer. We have employed the highly electron spin polarized pentacene triplet state as the model system in all of our pulsed EPR experiments. We performed most of our experiments at room temperature. The goals of our studies are aiming to improve spin detectability, to probe molecular dynamics, to determine electronic structures, to measure molecular interaction and motion, and to examine quantum coherence and oscillation which may yield new avenues in the applications of pulsed EPR techniques to quantum computer.

Magnetic resonance spectroscopy.

Since the original observation by Zeeman, that spectral lines can be affected by magnetic fields, 'magnetic spectroscopy' has evolved into the broad arsenal of techniques known as 'magnetic resonance'. This review focuses on nuclear magnetic resonance (NMR), electron paramagnetic resonance (EPR), and muon spin resonance (μSR): methods which have provided unparalleled insight into the structures, reactivity and dynamics of molecules, and thereby contributed to a detailed understanding of important aspects of chemistry, and the materials, biomedical, and environmental sciences. Magnetic resonance imaging (MRI), in vivo magnetic resonance spectroscopy (MRS) and functional magnetic resonance spectroscopy (fMRS) are also described. EPR is outlined as a principal method for investigating free radicals, along with biomedical applications, and mention is given to the more recent innovation of pulsed EPR techniques. In the final section of the article, the various methods known as μSR are collected under the heading 'muon spin resonance', in order to emphasise their complementarity with the more familiar NMR and EPR.

ELDOR-detected NMR at Q-Band

In recent years, electron–electron double resonance detected nuclear magnetic resonance (EDNMR) has gained considerable attention as a pulsed electron paramagnetic resonance technique to probe hyperfine interactions. Most experiments published so far were performed at W-band frequencies or higher, as at lower frequencies detection of weakly coupled low-γ nuclei is hampered by the presence of a central blind spot, which occurs at zero frequency. In this article we show that EDNMR measurements and a meaningful data analysis is indeed possible at intermediate microwave frequencies (Q-band, 34 GHz), once experimental parameters have been optimized. With highly selective detection pulses and Gaussian shaped electron–electron double resonance (ELDOR) pulses it is possible to detect low-γ nuclei coupled to paramagnetic Mn2+. Weakly coupled 14N resonances, which are separated from the zero frequency by only 2.8 MHz, were readily detected. In systems where different spin active nuclei are coupled to the electron spin, particular care has to be taken when using higher powered ELDOR pulses, as combination frequencies from the two nuclei (∆m S = ±1, ∆m I,1 = ±1, ∆m I,2 = ±1) can lead to severe line broadening and complicated EDNMR spectra. We also compare the EDNMR spectra of 13C-labeled Mn–DOTA to 13C-Mims electron–nuclear double resonance to get a better insight into the similarities and differences in the results of the two techniques for 13C hyperfine coupling.

Identification of slow relaxing spin components by pulse EPR techniques in graphene-related materials

Electron Paramagnetic Resonance (EPR) is a powerful technique that is suitable to study graphene-related materials. The challenging ability requested to the spectroscopy is its capability to resolve the variety of structures, relatively similar, that are obtained in materials produced through different methods, but that also coexist inside a single sample. In general, because of the intrinsic inhomogeneity of the samples, the EPR spectra are therefore a superposition of spectra coming from different structures. We show that by pulse EPR techniques (echo-detected EPR, ESEEM and Mims ENDOR) we can identify and characterize species with slow spin relaxing properties. These species are generally called molecular states, and are likely small pieces of graphenic structures of limited dimensions, thus conveniently described by a molecular approach. We have studied commercial reduced graphene oxide and chemically exfoliated graphite, which are characterized by different EPR spectra. Hyperfine spectroscopies enabled us to characterize the molecular components of the different materials, especially in terms of the interaction of the unpaired electrons with protons (number of protons and hyperfine coupling constants). We also obtained useful precious information about extent of delocalization of the molecular states.

Pulsed electron‐nuclear double resonance diagnostics of Ce3+ emitters in scintillating garnets

Pulsed electron paramagnetic resonance (EPR) and pulsed electron‐nuclear double resonance (ENDOR) techniques have been applied to study the environment of luminescent Ce3+ ions in garnet based scintillator powders and ceramics. The presence of aluminum and gallium isotopes with large nuclear magnetic and quadrupole moments in the nearest neighborhood of the Ce3+ ion allows for the use of the hyperfine and quadrupole interactions with these ions for determination of the unpaired electron spatial distribution and the definition of the electric field gradient at the aluminum and gallium sites. Pulsed EPR and ENDOR techniques made it possible to study the coherent properties of the Ce3+ spin system in garnet powders and ceramics, which is important for spin manipulation on Ce3+ centers.

EPR and DFT analysis of biologically relevant chromium(V) complexes with d-glucitol and d-glucose

1,2-diolato ligands, such as carbohydrates and glycoproteins, tend to stabilize chromium(V), thus forming important intermediates that have been implicated in the genotoxicity of Cr(VI). Since many years, room-temperature continuous-wave electron paramagnetic resonance (EPR) at X-band microwave frequencies has been used as a standard characterization tool to study chromium(V) intermediates formed during the reduction of Cr(VI) in the presence of biomolecules. In this work, the added value is tested of using a combination of pulsed and high-field EPR techniques with density functional theory computations to unravel the nature of Cr(V) complexes with biologically relevant chelators, such as carbohydrates. The study focuses on the oxidochromium(V) complexes formed during reduction of potassium dichromate with glutathione in the presence of the monosaccharide d-glucose or the polyalcohol d-glucitol. It is shown that although the presence of a multitude of Cr(V) intermediates may hamper a complete structural determination, the combined EPR and DFT approach reveals unambiguously the effect of freezing on the location of the counterions, the gradual replacement of water ligands by the diols, and the preference of Cr(V) to bind certain conformers.