Sensitivity Of Electron Paramagnetic Resonance Research Articles

Interaction of DWORF with SERCA and PLB as determined by EPR spectroscopy

Insufficient sarco/endoplasmic reticulum calcium ATPase (SERCA) activity significantly contributes to heart failure, which is a leading cause of death worldwide. A characteristic pathology of cardiac disease is the slow and incomplete Ca2+ removal from the myocyte cytoplasm in diastole, which is primarily driven by SERCA, the integral transmembrane Ca2+ pump. Phospholamban (PLB) allosterically inhibits SERCA by reducing its apparent Ca2+ affinity. Recently, the 34-codon novel dwarf open reading frame (DWORF) micropeptide has been identified as a muscle-specific SERCA effector, capable of reversing the inhibitory effects of PLB and independently activating SERCA in the absence of PLB. However, the structural basis for these functions has not yet been determined in a system of defined molecular components. We have used electron paramagnetic resonance (EPR) spectroscopy to investigate the protein-protein interactions of DWORF, co-reconstituted in proteoliposomes with SERCA and spin-labeled PLB. We analyzed the change of PLB rotational mobility in response to varying DWORF concentration, to quantify competitive binding of DWORF and PLB. We determined that DWORF competes with PLB for binding to SERCA at low [Ca2+], although the measured affinity of DWORF for SERCA is an order of magnitude weaker than that of PLB for SERCA, indicating cooperativity. The sensitivity of EPR to structural dynamics, using stereospecifically attached spin labels, allows us to obtain new information needed to refine the molecular model for regulation of SERCA activity, as needed for development of novel therapeutic remedies against cardiac pathologies.

STUDY OF DOSIMETRY POSSIBILITIES USING ELECTRON PARAMAGNETIC RESONANCE ON ALANINE FOR ILU-10 ACCELERATOR INP RK

Experimental studying possibilities of the electron paramagnetic resonance (EPR) method application for the dose control using alanine deteectors in the process of technological irradiations with brake radiation at the accelerator ILU-10 INP RK has been carried out. The EPR signal stability and sensitivity of original alanine detectors have been studied, serviceability in standard modes was shown in the range of small doses of brake irradiation (0,005–1 kGy); for dose estimation the calibration on gamma-irrdiation was used. The obtained data confirmed the promise of alanine EPR dosimetry at the ILU-10 accelerator INP RK.

Simplifying quantitative measurement of free radical species using an X-band EPR spectrometer.

The quantitative measurement of free radicals in liquid using an X-band electron paramagnetic resonance (EPR) was systematized. Quantification of free radicals by EPR requires a standard sample that contains a known spin amount/concentration. When satisfactory reproducibility of the sample material, volume, shape, and positioning in the cavity for EPR measurements can be guaranteed, a sample tested and a standard can be directly compared and the process of quantification can be simplified. The purpose of this study was to simplify manual quantitative EPR measurement. A suitable sample volume for achieving a stable EPR intensity was estimated. The effects of different solvents on the EPR sensitivity were compared. The stability and reproducibility of the EPR intensity of standard nitroxyl radical solutions were compared among different types of sample tubes. When the sample tubes, sample volumes, and/or solvents were the same, the EPR intensity was reproduced with an error of 2% or less for μM samples. The quantified sample and the standard sample in the same solvent and the same volume drawn into the same sample tube was able to be directly compared. The standard sample for quantification should be measured just before or after every daily experiment.

Impact of the Characteristic Impedance of Coaxial Lines on the Sensitivity of a 750-MHz Electronically Tunable EPR Resonator

A 750-MHz electronically tunable resonator was investigated in terms of the sensitivity of electron paramagnetic resonance (EPR) signal detection. The conversion efficiency of the radio-frequency magnetic field was calculated for resonators with 50- and 100-Ω coaxial coupling lines using three-dimensional (3D) microwave field and microwave circuit simulators. Based on the simulation results, two tunable resonators were physically constructed and compared in terms of EPR signal sensitivity using a nitroxyl radical solution. While the resonator with 100-Ω coaxial lines provided 14% greater signal intensity, its signal-to-noise ratio was lower than that of the resonator with 50-Ω lines. To demonstrate the capability of the constructed tunable resonator for EPR imaging experiments, a solution of nitroxyl radical and the leg of a tumor-bearing mouse were visualized.

Probing Gas Adsorption in Metal–Organic Framework ZIF-8 by EPR of Embedded Nitroxides

Metal–organic frameworks (MOFs) are being increasingly considered as promising materials for gas separation and storage, yet specific interactions between gas molecules and the inner surface of pores are still not well understood. In this work, we propose a new approach for investigation of such interactions by Electron Paramagnetic Resonance (EPR). We use stable nitroxide radicals as multifunctional agents embedded into the pores of a MOF prior to the gas sorption. They act as EPR-active reporters, and simultaneously as competitor molecules during the gas adsorption process. We exemplify this approach using a ZIF-8 framework, nitroxide TEMPO ((2,2,6,6-tetramethylpiperidin-1-yl)oxyl), and CO2, N2, and O2 gases. The mobility of nitroxide monitored by continuous wave EPR behaves differently upon adsorption of each of these gases. In particular, a noticeable increase of mobility in the presence of CO2 reveals the weakening of guest–host interactions TEMPO–MOF induced by CO2, which was qualitatively supported by molecular dynamic calculations. The nitroxides can be embedded in MOFs postsynthetically, and their negligible amounts (≤1 per 1000 cells) are required due to a high sensitivity of EPR. Therefore, the proposed approach is sufficiently versatile and might find broad applications for various MOFs.

Bioactivity characterization of 45S5 bioglass using TL, OSL and EPR: Comparison with the case of 58S sol-gel bioactive glass

The current work exploits the effective application of thermoluminescence (TL), optically stimulated luminescence (OSL) and the possibility of applying Electron Paramagnetic Resonance (EPR) for the discrimination between different bioactive responses in the case of the 45S5 bioactive glass (SiO2 45, Na2O 24.5, CaO 24.5, P2O5 6 in wt%), which was synthesized through melting process. These techniques are suggested mainly due to their low spectroscopic detection thresholds. The original 45S5 in grain size range of 20–40μm was immersed in the Simulated Body Fluid (SBF) for various different immersion times ranging over one week. In this work the 110°C TL peak, a specific OSL component and the EPR signal at g=2.013 ascribed to oxygen hole center (OHC) are used due to their sensitivity to the different bioactive responses. For all luminescence and EPR components, the intensity plot versus immersion time yields sharp discontinuities, resulting in effective probes regarding the timescale for both the beginning as well as the end of the procedure of the crystalline HCAp formation respectively. On the contrary to the smooth decreasing pattern of both luminescence entities, the peak to peak amplitude of the EPR signal indicates an initial increase for the initial 16min of immersion, followed by a further decrease throughout the immersion time duration. The discontinuities monitored for both sensitivity of TL, OSL and EPR, in conjunction with the discontinuities monitored for the sensitization of TL and OSL, when plotted versus immersion time, provide an individual time scale for each one of the chemical reactions involved in the five steps of the aforementioned procedure. According to the authors' best knowledge, scarce characterization techniques could provide this time scale frame, while it is the first time that such an application of OSL and EPR is attempted. Finally, the bioactive response of the 45S5 bioglass was compared with that of the 58S sol-gel bioactive glass, in terms of the timescale of these five stages required for the final formation of the HCAp. The techniques of luminescence and EPR which take advantage of trapped charges are proposed as alternative cheap and prompt effective techniques towards discrimination between different bioactive responses in bioactive glasses.

Demystifying EPR: A Rookie Guide to the Application of Electron Paramagnetic Resonance Spectroscopy on Biomolecules

Electron Paramagnetic Resonance (EPR) spectroscopy, also known as Electron Spin Resonance(ESR) especially among physicists, is a strong and versatile spectroscopic method forinvestigation of paramagnetic systems, i.e. systems like free radicals and most transition metalions, which have unpaired electrons. The sensitivity and selectivity of EPR are notable andintriguing as compared to other spectroscopic methods and approaches. As a qualitative method,EPR can detect species down to the nanomolar range. On the other hand, the specificity of themethod stems from spectral features which directly depend on the types, distances, and relativeorientations of the atoms in the neighborhood of the electron spin centers. In addition tostructural information, EPR can be used to elucidate time dependent behavior of the studiedsystem and it is applicable to systems of different size, ranging from small molecules tomacromolecules. The following short review of general EPR methods is intended for anaudience with little prior knowledge about EPR. It includes examples of suitable representativesystems, techniques for the study of short lived paramagnetic species and even diamagneticmolecules, and introduces the reader to tools necessary for making sense of the spectra. Thispaper focuses only on continuous wave (cw) EPR and does not elaborate on the more advancedpulsed EPR methods.

The spatial effect of protein deuteration on nitroxide spin-label relaxation: Implications for EPR distance measurement

Pulsed electron–electron double resonance (PELDOR) coupled with site-directed spin labeling is a powerful technique for the elucidation of protein or nucleic acid, macromolecular structure and interactions. The intrinsic high sensitivity of electron paramagnetic resonance enables measurement on small quantities of bio-macromolecules, however short relaxation times impose a limit on the sensitivity and size of distances that can be measured using this technique. The persistence of the electron spin-echo, in the PELDOR experiment, is one of the most crucial limitations to distance measurement. At a temperature of around 50K one of the predominant factors affecting persistence of an echo, and as such, the sensitivity and measurable distance between spin labels, is the electron spin echo dephasing time (Tm). It has become normal practice to use deuterated solvents to extend Tm and recently it has been demonstrated that deuteration of the underlying protein significantly extends Tm. Here we examine the spatial effect of segmental deuteration of the underlying protein, and also explore the concentration and temperature dependence of highly deuterated systems.

A high-frequency electron paramagnetic resonance spectrometer for multi-dimensional, multi-frequency, and multi-phase pulsed measurements.

We describe instrumentation for a high-frequency electron paramagnetic resonance (EPR) and pulsed electron-electron double resonance (PELDOR) spectroscopy. The instrumentation is operated in the frequency range of 107-120 GHz and 215-240 GHz and in the magnetic field range of 0-12.1 T. The spectrometer consisting of a high-frequency high-power solid-state source, a quasioptical system, a phase-sensitive detection system, a cryogenic-free superconducting magnet, and a (4)He cryostat enables multi-frequency continuous-wave EPR spectroscopy as well as pulsed EPR measurements with a few hundred nanosecond pulses. Here we discuss the details of the design and the pulsed EPR sensitivity of the instrumentation. We also present performance of the instrumentation in unique experiments including PELDOR spectroscopy to probe correlations in an insulating electronic spin system and application of dynamical decoupling techniques to extend spin coherence of electron spins in an insulating solid-state system.

Site-directed spin-labeling of nucleotides and the use of in-cell EPR to determine long-range distances in a biologically relevant environment

Double electron-electron resonance (DEER) is an electron paramagnetic resonance (EPR) technique used to determine distance distributions in the nanometer range between spin labels by measuring their dipole-dipole interactions. Here we describe how in-cell DEER can be applied to spin-labeled DNA sequences to unravel their conformations in living cells by long-range distance measurements in cellula. As EPR detects unpaired electron spins only, diamagnetic molecules provide no background and do not reduce detection sensitivity of the specific signal. Compared with in-cell NMR spectroscopy, low concentrations of spin-labeled molecules can be used owing to the higher sensitivity of EPR per spin. This protocol describes the synthesis of the spin labels, their introduction in DNA strands, the injection of labeled DNA solutions in cells and the performance of in-cell EPR measurements. Completion of the entire protocol takes ~20 d.

Electron paramagnetic resonance probed oxygen deficiency in SrTiO3 with different cap layers

The oxygen vacancies in the SrTiO3 (STO) single crystal with two different cap layers, CoFe2O4 (CFO) and BiFeO3 (BFO), are investigated with the technique of electron paramagnetic resonance (EPR). EPR spectra show the resonance lines of the residual oxygen vacancy in air annealed STO, STO/CFO, and STO/BFO, showing the high sensitivity of EPR to the low concentration defects. The experimental results indicate that the oxide cap layers enhance the oxygen deficiency in bulk STO and the amount of oxygen vacancy is dependent on the electronegativity of metal ion in the cap layer. Accordingly, STO/BFO has a larger amount of oxygen vacancy than that of STO/CFO.

Rotational Diffusion of Membrane Proteins: Characterization of Protein-Protein Interactions in Membranes

It has long been appreciated that the rotational diffusion coefficient (Dr) and hence the rotational correlation time (τr) of a transmembrane protein about its membrane normal axis is predicted to be highly sensitive to its cylindrical radius (e.g., Saffman and Delbruck (1)). Due to the highly viscous nature of a cellular membrane or a membrane bilayer, the correlation times for most transmembrane proteins are predicted to be in the microsecond or longer correlation time range. This time range is not readily accessible to classical spectroscopic techniques like time-resolved or frequency domain fluorescence anisotropy when using conventional fluorescence probes or by continuous-wave electron paramagnetic resonance (EPR) when using nitroxide spin labels. However, as shown by the early work of Hyde and Dalton (2) and in the seminal study by Thomas et al. (3), the range of motional sensitivity of EPR could be extended into the microsecond-to-millisecond time range by saturation transfer EPR (ST-EPR) spectroscopy using conventional nitroxide spin labels. Since its introduction, ST-EPR has been utilized to study the very slow rotational motions of a wide range of membrane proteins and other large protein assemblies.

ChemInform Abstract: Electrically Detected Magnetic Resonance in Dielectric Semiconductor Systems of Current Interest

AbstractReview: 30 refs.

Coarse and Fine Control of the EPR Frequency of a Loop-Gap Resonator Using a Single-Turn Coil with a Varactor Diode Attached

Coarse control and fine control of the resonant frequency of a loop-gap resonator (LGR) operating at an electron paramagnetic resonance (EPR) frequency of ca. 650 MHz were achieved using a single-turn coil with a varactor diode attached (a frequency shift coil). When the distance between the LGR and the frequency shift coil was changed from 15 to 10 mm under the condition of constant voltage to the varactor diode (0 V), a shift of the resonant frequency of the LGR of ca. 20 MHz was observed (coarse frequency control). When the voltage applied to the varactor diode was changed from 0 to 15 V at the same distance between the LGR and the frequency shift coil (10 mm), a shift of the resonant frequency of the LGR of ca. 200 kHz was observed (fine frequency control). There were no significant changes in sensitivity of EPR measurements of a phantom (comprised of agar with a nitroxide radical and physiological saline solution) without and with the frequency shift coil. The EPR sensitivity did not change discernibly when the resonant frequency was shifted by the frequency shift coil. Furthermore, radio-frequency phase adjustment for homodyne detection could be performed by using the frequency shift coil without applying frequency modulation to the carrier wave.

Electron spin resonance in membrane research: protein-lipid interactions from challenging beginnings to state of the art.

Conventional electron paramagnetic resonance (EPR) spectra of lipids that are spin-labelled close to the terminal methyl end of the acyl chains are able to resolve the lipids directly contacting the protein from those in the fluid bilayer regions of the membrane. This allows determination of both the stoichiometry of lipid–protein interaction (i.e., number of lipid sites at the protein perimeter) and the selectivity of the protein for different lipid species (i.e., association constants relative to the background lipid). Spin-label EPR data are summarised for 20 or more different transmembrane peptides and proteins, and 7 distinct species of lipids. Lineshape simulations of the two-component conventional spin-label EPR spectra allow estimation of the rate at which protein-associated lipids exchange with those in the bulk fluid regions of the membrane. For lipids that do not display a selectivity for the protein, the intrinsic off-rates for exchange are in the region of 10 MHz: less than 10× slower than the rates of diffusive exchange in fluid lipid membranes. Lipids with an affinity for the protein, relative to the background lipid, have off-rates for leaving the protein that are correspondingly slower. Non-linear EPR, which depends on saturation of the spectrum at high radiation intensities, is optimally sensitive to dynamics on the timescale of spin-lattice relaxation, i.e., the microsecond regime. Both progressive saturation and saturation transfer EPR experiments provide definitive evidence that lipids at the protein interface are exchanging on this timescale. The sensitivity of non-linear EPR to low frequencies of spin exchange also allows the location of spin-labelled membrane protein residues relative to those of spin-labelled lipids, in double-labelling experiments.

Microstrip resonators for electron paramagnetic resonance experiments

In this article we evaluate the performance of an electron paramagnetic resonance (EPR) setup using a microstrip resonator (MR). The design and characterization of the resonator are described and parameters of importance to EPR and spin manipulation are examined, including cavity quality factor, filling factor, and microwave magnetic field in the sample region. Simulated microwave electric and magnetic field distributions in the resonator are also presented and compared with qualitative measurements of the field distribution obtained by a perturbation technique. Based on EPR experiments carried out with a standard marker at room temperature and a MR resonating at 8.17 GHz, the minimum detectable number of spins was found to be 5 x 10(10) spins/GHz(1/2) despite the low MR unloaded quality factor Q0=60. The functionality of the EPR setup was further evaluated at low temperature, where the spin resonance of Cr dopants present in a GaAs wafer was detected at 2.3 K. The design and characterization of a more versatile MR targeting an improved EPR sensitivity and featuring an integrated biasing circuit for the study of samples that require an electrical contact are also discussed.

On the possible manifestation of harmonic-anharmonic dynamical transition in glassy media in electron paramagnetic resonance of nitroxide spin probes

Continuous wave (cw) electron paramagnetic resonance (EPR) and echo-detected (ED) EPR were applied to study molecular motions of nitroxide spin probes in glassy glycerol and o-terphenyl. A linear decrease with increasing temperature of the total splitting in the cw EPR line shape was observed at low temperatures in both solvents. Above some temperature points the temperature dependencies become sharper. Within the model of molecular librations, this behavior is in qualitative and quantitative agreement with the numerical data on neutron scattering and Mossbauer absorption for molecular glasses and biomolecules, where temperature dependence of the mean-squared amplitude of the vibrational motion was obtained. In analogy with these data the departure from linear temperature dependence in cw EPR may be ascribed to the transition from harmonic to anharmonic motion (this transition is called dynamical transition). ED EPR spectra were found to change drastically above 195 K in glycerol and above 245 K in o-terphenyl, indicating the appearance of anisotropic transverse spin relaxation. This appearance may also be attributed to the dynamical transition as an estimation shows the anisotropic relaxation rates for harmonic and anharmonic librational motions and because these temperature points correspond well to those known from neutron scattering for these solvents. The low sensitivity of ED EPR to harmonic motion and its high sensitivity to the anharmonic one suggests that ED EPR may serve as a sensitive tool to detect dynamical transition in glasses and biomolecules.

From Local Moment EPR in Superconductors to Nanoscale Ferromagnets

Electron paramagnetic resonance (EPR) in metals has contributed a lot to the understanding of the electronic structure and magnetic properties in dilute alloys as well as in concentrated ferromagnets. We recall some pioneering work of the Kazan group and others, studying local moment EPR in superconductors. An SNS Josephson junction has been used as a microwave generator and as an EPR detector at once. EPR was also used to study the Kondo effect in the EPR g-shift and linewidth. Moreover, the high sensitivity of EPR (down to 1010 spins) allows to study single atomic layers of ferromagnets below and above the Curie temperature T C as well as the spin fluctuations at T C. The in situ ferromagnetic resonance (FMR) in ultrahigh vacuum (UHV) offers a unique possibility to study the interlayer exchange coupling (IEC) and spin dynamics of coupled ferromagnetic films. Furthermore, the magnetic resonance enables us to measure basic parameters of nanoscale magnets in absolute energy units (i.e., μeV/spin). The current status of the UHV-FMR in nanoscale ferromagnets will be discussed.

Application of Ferroelectrics in Radiospectroscopy

Our results show that the application of ferroelectrics as ferroelectric resonators can significantly increase the sensitivity of electron paramagnetic resonance, nuclear magnetic resonance methods and provide a higher quality of magnetic resonance imaging. Ferroelectric resonators allow the possibility to observe natural paramagnetic impurities in electron paramagnetic resonance and to record impurities without special doping in nuclear magnetic resonance. Secondly, it is possible to investigate electron paramagnetic resonance in vivo because the ferroelectric resonator can be located outside the cavity. Finally, a ferroelectric resonator made the development of a portable and sensitive electron paramagnetic resonance spectrometer possible.

Synergistic anion-directed coordination of ferric and cupric ions to bovine serum transferrin – an inorganic perspective

A series of new iron(III) and copper(II) complexes of bovine serum transferrin (BTf), with carbonate and/or oxalate as the synergistic anion, are presented. The complexes [Fe 2(CO 3) 2BTf], [Fe 2(C 2O 4) 2BTf], [Cu 2(CO 3) 2BTf] and [Cu(C 2O 4)BTf] were prepared by standard titrimetric techniques. The oxalate derivatives were also obtained from the corresponding carbonate complexes by anion-displacement. The site-preference of the transition metal–oxalate synergism has facilitated the preparation and isolation of the mononuclear complex [Cu(C 2O 4)BTf], the mixed-anion complexes [Cu 2(CO 3)(C 2O 4)BTf] and [Fe 2(CO 3)(C 2O 4)BTf] and the mixed-metal complex [FeCu(C 2O 4) 2BTf]. The sensitivity of electron paramagnetic resonance (EPR) spectroscopy to the nature of the synergistic anions at the specific-binding sites of the transferrins has made this physical technique particularly indispensable to this study. None of the other members of the transferrin family of proteins has ever been demonstrated to bind the ferric and cupric ions one after the other, each occupying a separate specific-binding site of the same transferrin molecule, as a response to the coordination restrictions imposed by the oxalate ion. The bathochromic shift of the visible p π–d π * CT band for iron(III)–BTf and the hypsochromic shift of the p π–d σ * CT band for copper(II)–BTf, on replacing carbonate by oxalate as the associated anion, are consistent with the relative positions of these anionic ligands in the spectrochemical series and the nature of the d-type acceptor orbitals involved in the CT transitions. The binding and spectroscopic properties of bovine serum transferrin – a serum transferrin – very nearly mirror those of human serum transferrin, but differ significantly from those of human lactoferrin.