Two surface coil resonators were tested at 1.0 GHz as alternatives to volume resonators that fully contain the sample; a 10 mm diameter coil with Q of 61 and efficiency of 0.044 mT/√W at ~2.5 mm above the coil and a 30 mm diameter coil with Q of 56 and efficiency of 0.0038 mT/√W at ~5 mm above the coil. The 10 mm diameter coil uses power more efficiently, but the 30 mm diameter surface coil detects signal from a larger volume and the B extends further into the sample. The signal intensity as a function of distance from the coil was measured for small samples of lithium phthalocyanine, a 1 mm thick plate of aqueous nitroxide solution, and a 7 mm internal diameter tube containing aqueous nitroxide solution. For a sample localized at a defined distance from the coil the signal intensity can be increased by increasing incident power to compensate for the decreases in B with distance from the coil. However, when the noise is dominated by the source, increasing power increases noise. If incident power is adjusted to compensate for the decrease in B with increasing distance from the coil the S/N for aqueous nitroxide in a 1 mm thick plate, was better for the 10 mm coil than for the 30 mm coil up to about a distance of about 6 mm but that advantage is lost at greater distances from the coil. Samples of nitroxide were used as a phantom to demonstrate 3D spatial imaging with the 30 mm coil.

The blue color that has made lazurite (lapis lazuli) a prized mineral is due to the same S radical that is in synthetic ultramarine blue (UMB), which has been proposed as a CW EPR standard. Continuous wave and pulsed EPR spectra and relaxation times of S are compared for three commercial sources of synthetic UMB, for samples of lapis lazuli from Afghanistan, Chile, Colorado USA, and Pakistan, and a solution in DMSO:dioxane. The spin concentrations in the UMB samples were high, in the range of 3×10 to 5×10 spins/g. The field-swept echo detected spectra of UMB samples have lineshapes at 4.2 K that depend on the field at which phase adjustment is performed, indicating strong spin-spin interaction. The spectra of the minerals included large spectral contributions from Mn, in addition to S for which the concentrations were 6×10 to 2.9×10 spins/g. Features in the spin-echo detected spectra attributed to forbidden Mn transitions were confirmed by comparison with Mn spectra in CaO powder. Large distributions in relaxation times caused derived results to depend strongly on the experimental acquisition windows for echo decay and inversion recovery curves. Short phase memory times are attributed to spin-spin interactions and to motion of the S in the lattices. Relatively weak temperature dependence of spin lattice relaxation rates below about 25 K is attributed to substantial spin-spin interaction and cross relaxation. The strong spin-spin interaction is not present for 0.4 mM S in DMSO:dioxane. The shorter T for S than for SO or SO is attributed to stronger spin orbit coupling.

We celebrate 80 years of EPR with a special issue of Applied Magnetic Resonance featuring both reviews and regular research articles. The focus is new opportunities for application of EPR and new directions for development of EPR. This introduction concisely surveys the scope of EPR and hints at future developments.

We examine the Electron Paramagnetic Resonance (EPR) and Electron-Nuclear Double Resonance (ENDOR) spectroscopy of three quite distinct high-spin Mn(II) systems and describe experimental techniques and methods of analysis that are useful in their study. We demonstrate that this S=5/2 metal center provides useful orientation-selection through the Zero-Field Splitting (ZFS) tensor that enables determination of a C hyperfine-coupling tensor with extremely small hyperfine interaction. We also demonstrate that Mims suppression effects can be used in concert with orientation-selection to edit complex H ENDOR patterns that can be produced by even a 'simple' center with a single Mn(II). We develop a perturbation-based approach to understanding second-order shifts in Mn(II) ENDOR responses that occur in systems with intermediate ZFS values, and show that these shifts can be used to estimate the values of the ZFS tensors.

Site directed spin labeling has enabled protein structure determination using electron spin resonance (ESR) pulsed dipolar spectroscopy (PDS). Small details in a distance distribution can be key to understanding important protein structure-function relationships. A major challenge has been to differentiate unimodal and overlapped multimodal distance distributions. They often yield similar distributions and dipolar signals. Current model-free distance reconstruction techniques such as Srivastava-Freed Singular Value Decomposition (SF-SVD) and Tikhonov regularization can suppress these small features in uncertainty and/or error bounds, despite being present. In this work, we demonstrate that continuous wavelet transform (CWT) can distinguish PDS signals from unimodal and multimodal distance distributions. We show that periodicity in CWT representation reflects unimodal distributions, which is masked for multimodal cases. This work is meant as a precursor to a cross-validation technique, which could indicate the modality of the distance distribution.

The majority of pathogenic Gram-negative bacteria benefit from intrinsic antibiotic resistance, attributed primarily to the lipopolysaccharide (LPS) coating of the bacterial envelope. To effectively coat the bacterial cell envelope, LPS is transported from the inner membrane by the LPS transport (Lpt) system, which comprises seven distinct Lpt proteins, LptA-G, that form a stable protein bridge spanning the periplasm to connect the inner and outer membranes. The driving force of this process, LptBFG, is an asymmetric ATP binding cassette (ABC) transporter with a novel architecture and function that ejects LPS from the inner membrane and facilitates transfer to the periplasmic bridge. Here, we utilize site-directed spin labeling (SDSL) electron paramagnetic resonance (EPR) spectroscopy to probe conformational differences between the periplasmic domains of LptF and LptG. We show that LptC solely interacts with the edge β-strand of LptF and does not directly interact with LptG. We also quantify the interaction of periplasmic LptC with LptF. Additionally, we show that LPS cannot enter the protein complex externally, supporting the unidirectional LPS transport model. Furthermore, we present our findings that the presence of LPS within the LptBFGC binding cavity and the membrane reconstitution environment affect the structural orientation of the periplasmic domains of LptF and LptG, but overall are relatively fixed with respect to one another. This study will provide insight into the structural asymmetry associated with the newly defined type VI ABC transporter class.

As new methods to interrogate glycan organization on cells develop, it is important to have a molecular level understanding of how chemical fixation can impact results and interpretations. Site-directed spin labeling technologies are well suited to study how the spin label mobility is impacted by local environmental conditions, such as those imposed by cross-linking effects of paraformaldehyde cell fixation methods. Here, we utilize three different azide-containing sugars for metabolic glycan engineering with HeLa cells to incorporate azido glycans that are modified with a DBCO-based nitroxide moiety via click reaction. Continuous wave X-band electron paramagnetic resonance spectroscopy is employed to characterize how the chronological sequence of chemical fixation and spin labeling impacts the local mobility and accessibility of the nitroxide-labeled glycans in the glycocalyx of HeLa cells. Results demonstrate that chemical fixation with paraformaldehyde can alter local glycan mobility and care should be taken in the analysis of data in any study where chemical fixation and cellular labeling occur.

Here we review applications of site-directed spin labeling (SDSL) with engineered cysteines in proteins, to study the structural dynamics of muscle and non-muscle proteins, using and developing the electron paramagnetic resonance (EPR) spectroscopic techniques of dipolar EPR, double electron electron resonance (DEER), saturation transfer EPR (STEPR), and orientation measured by EPR. The SDSL technology pioneered by Wayne Hubbell and collaborators has greatly expanded the use of EPR, including the measurement of distances between spin labels covalently attached to proteins and peptides. The Thomas lab and collaborators have applied these techniques to elucidate dynamic interactions in the myosin-actin complex, myosin-binding protein C, calmodulin, ryanodine receptor, phospholamban, utrophin, dystrophin, β-III-spectrin, and Aurora kinase. The ability to design and engineer cysteines in proteins for site-directed covalent labeling has enabled the use of these powerful EPR techniques to measure distances, while showing that they are complementary with optical spectroscopy measurements.

Site-directed spin labeling (SDSL) has been invaluable in the analysis of protein structure and dynamics, and has been particularly useful in the study of membrane proteins. ExoU, an important virulence factor in infections, is a bacterial phospholipase A2 that functions at the membrane - aqueous interface. Using SDSL methodology developed in the Hubbell lab, we find that the region surrounding the catalytic site of ExoU is buried within the tertiary structure of the protein in the soluble, apoenzyme state, but shows a significant increase in dynamics upon membrane binding and activation by ubiquitin. Continuous wave (CW) power saturation EPR studies show that the conserved serine hydrolase motif of ExoU localizes to the membrane surface in the active, holoenzyme state. SDSL studies on the C-terminal four-helix bundle (4HB) domain of ExoU similarly show a co-operative effect of ubiquitin binding and membrane association. CW power saturation studies of the 4HB domain indicate that two interhelical loops intercalate into the lipid bilayer upon formation of the holoenzyme state, anchoring ExoU at the membrane surface. Together these studies establish the orientation and localization of ExoU and the membrane surface, and illustrate the power of SDSL as applied to peripheral membrane proteins.

Electron spin relaxation times are reported for the SO radical in solid NaSO and KSO, and the SO radicals in NaSO, and KSO. Echo envelope modulation was observed for the radicals in the Na salts and characterized by HYSCORE. and were measured by spin echo methods from 40 to 293 K and by long-pulse saturation recovery at 293 K. At low temperature for the radicals in KSO (~14 μs) is longer than for the radicals in NaSO ( ~ 7 μs) or NaSO ( ~ 4 μs), which is attributed to the low magnetic moment of K. The shorter value of in NaSO is attributed in part to higher spin concentration. decreases with increasing temperature as approaches . In each lattice there is a distribution of spin lattice relaxation times that may be due to distributions in interspin distances. The long components in the distributions are longer for SO than for SO . In 0.5 M NaOH solution at 293 K SO has a relaxation-determined Lorentzian peak-to-peak linewidth of about 0.7 G, and ~ ~ 100 ns.

The viscosity measurements are of clinical significance for evaluation of the potential pathological conditions of biological lubricants such as synovial fluids of joints, and for formulation and characterization of peptide- and protein-based biotherapeutics. Due to inherent potential therapeutic activity, protein drugs have proven to be one of the most efficient therapeutic agents in treatment of several life-threatening disorders, such as diabetes and autoimmune diseases. However, home-use applications for treating chronic inflammatory diseases, such as diabetes and rheumatoid arthritis, necessitate the development of high-concentration insulin and monoclonal antibodies formulations for patient self-administration. High protein concentrations can affect viscosity of the corresponding drug solutions complicating their manufacture and administration. The measurements of the viscosity of new insulin analogs and monoclonal antibodies solutions under development is of practical importance to avoid unwanted highly viscous, and therefore, painful for injection drug formulations. Recently, we have demonstrated capability of the electron paramagnetic resonance (EPR) viscometry using viscosity-sensitive C-labeled trityl spin probe (C-dFT) to report the viscosity of human blood, and interstitial fluids measured in various organs in mice and in anesthetized mice, . In the present work, we demonstrate utility of the EPR viscometry using C-dFT to measure microviscosity of commercial insulin samples, antibodies solution, and human synovial fluids using small microliter volume samples (5-50 μL). This viscometry analysis approach provides useful tool to control formulations and administration of new biopharmaceuticals, and for evaluation of the state of synovial fluids of importance for clinical applications.

NMR spectroscopy is well known for its superb resolution, especially at high applied magnetic field. However, the sensitivity of this technique is very low. Liquid-state low-concentration photo-chemically-induced dynamic nuclear polarization (LC-photo-CIDNP) is a promising emerging methodology capable of enhancing NMR sensitivity in solution. LC-photo-CIDNP works well on solvent-exposed Trp and Tyr residues, either in isolation or within proteins. This study explores the magnetic-field dependence of the LC-photo-CIDNP experienced by two tryptophan isotopologs in solution upon LED-mediated optical irradiation. Out of the two uniformly C,N-labeled Trp (Trp-U-C,N) and Trp-α-C-β,β,2,4,5,6,7-d species employed here, only the latter bears a quasi-isolated H-C spin pair. Computer simulations of the predicted polarization due to geminate recombination of both species display a roughly bell-shaped field dependence. However, while Trp-U-C,N is predicted to show a maximum at ca. 500 MHz (11.7 T) and a fairly weak field dependence, Trp-α-C-β,β,2,4,5,6,7-d is expected to display a much sharper field dependence accompanied by a dramatic polarization increase at lower field (ca. 200 MHz, 4.7 T). Experimental LC-photo-CIDNP studies on both Trp isotopologs at 1μM concentration, performed at selected fields, are consistent with the theoretical predictions. In summary, this study highlights the prominent field-dependence of LC-photo-CIDNP enhancements () experienced by Trp isotopologs bearing a quasi-isolated spin pair.

[This corrects the article PMC10249954.].

The kinetics of the transfer of the chelate, ethylenediamine tetraacetate (EDTA), from Calcium(II) to Copper(II) in imidazole (Im) buffers near neutral pH, corresponding to the conversion, [Cu(II)Im]→ [Cu(II)EDTA], are characterized with stopped-flow absorption spectroscopy and implemented as a tool for calibrating the interval between mixing and freezing, the freeze-quench time ( ), of a rapid freeze-quench (RFQ) apparatus. The kinetics of this reaction are characterized by monitoring changes in UV-visible spectra (300 nm) due to changes in the charge-transfer band associated with the Cu ions upon EDTA binding. Stopped-flow measurements show that the rates of conversion of the Cu ions exhibit exponential kinetics on millisecond time scales at pH values less than 6.8. In parallel, we have developed a simple but precise method to quantitate the speciation of frozen solution mixtures of [Cu(II)(EDTA)] and tetraimidazole Cu(II) ([Cu(Im)]) in X-band EPR spectra. The results are implemented in a simple high-precision 'recipe' for determining . These procedures are more accurate and precise than the venerable reaction of aquometmyoglobin with azide for calibrating RFQ apparatus, with the benefit of avoiding high-concentrations of toxic azide solutions.

This review is motivated by the exciting new area of radiation therapy using a phenomenon termed FLASH in which oxygen is thought to have a central role. Well-established principles of radiation biology and physics suggest that if oxygen has a strong role, it should be the level at the DNA. The key aspect discussed is the rate of oxygen diffusion. If oxygen freely diffuses into cells and rapidly equilibrates, then measurements in the extracellular compartment would enable FLASH to be investigated using existing methodologies that can readily measure oxygen in the extracellular compartment. EPR spin-label oximetry allows evaluation of the oxygen permeability coefficient across lipid bilayer membranes. It is established that simple fluid phase lipid bilayers are not barriers to oxygen transport. However, further investigations indicate that many physical and chemical (compositional) factor can significantly decrease this permeation. In biological cell plasma membranes, the lipid bilayer forms the matrix in which integral membrane proteins are immersed, changing organization and properties of the lipid matrix. To evaluate oxygen permeability coefficients across these complex membranes, oxygen permeation across all membrane domains and components must be considered. In this review, we consider many of the factors that affect (decrease) oxygen permeation across cell plasma membranes. Finally, we address the question, can the plasma membrane of the cell form a barrier to the free diffusion of oxygen into the cell interior? If there is a barrier then this must be considered in the investigations of the role of oxygen in FLASH.

Multidimensional Magnetic Resonance Imaging (MRI) is a versatile tool for microstructure mapping. We use a diffusion weighted inversion recovery spin echo (DW-IR-SE) sequence with spiral readouts at ultra-strong gradients to acquire a rich diffusion-relaxation data set with sensitivity to myelin water. We reconstruct 1D and 2D spectra with a two-step convex optimization approach and investigate a variety of multidimensional MRI methods, including 1D multi-component relaxometry, 1D multi-component diffusometry, 2D relaxation correlation imaging, and 2D diffusion-relaxation correlation spectroscopic imaging (DR-CSI), in terms of their potential to quantify tissue microstructure, including the myelin water fraction (MWF). We observe a distinct spectral peak that we attribute to myelin water in multi-component T1 relaxometry, T1-T2 correlation, T1-D correlation, and T2-D correlation imaging. Due to lower achievable echo times compared to diffusometry, MWF maps from relaxometry have higher quality. Whilst 1D multi-component T1 data allows much faster myelin mapping, 2D approaches could offer unique insights into tissue microstructure and especially myelin diffusion.

Site-directed spin labeling electron paramagnetic resonance (SDSL-EPR) is an established tool for exploring protein structure and dynamics. Although nitroxide side chains attached to a single cysteine via a disulfide linkage are commonly employed in SDSL-EPR, their internal flexibility complicates applications to monitor slow internal motions in proteins and to structure determination by distance mapping. Moreover, the labile disulfide linkage prohibits the use of reducing agents often needed for protein stability. To enable the application of SDSL-EPR to the measurement of slow internal dynamics, new spin labels with hindered internal motion are desired. Here, we introduce a highly ordered nitroxide side chain, designated R9, attached at a single cysteine residue via a non-reducible thioether linkage. The reaction to introduce R9 is highly selective for solvent-exposed cysteine residues. Structures of R9 at two helical sites in T4 Lysozyme were determined by X-ray crystallography and the mobility in helical sequences was characterized by EPR spectral lineshape analysis, Saturation Transfer EPR, and Saturation Recovery EPR. In addition, interspin distance measurements between pairs of R9 residues are reported. Collectively, all data indicate that R9 will be useful for monitoring slow internal structural fluctuations, and applications to distance mapping via dipolar spectroscopy and relaxation enhancement methods are anticipated.

Biomolecular applications of pulse dipolar electron paramagnetic resonance spectroscopy (PDS) are becoming increasingly valuable in structural biology. Site-directed spin labelling of proteins is routinely performed using nitroxides, with paramagnetic metal ions and other organic radicals gaining popularity as alternative spin centres. Spectroscopically orthogonal spin labelling using different types of labels potentially increases the information content available from a single sample. When analysing experimental distance distributions between two nitroxide spin labels, the site-specific rotamer information has been projected into the distance and is not readily available, and the contributions of individual labelling sites to the width of the distance distribution are not obvious from the PDS data. Here, we exploit the exquisite precision of labelling double-histidine (dHis) motifs with Cu chelate complexes. The contribution of this label to the distance distribution widths in model protein GB1 has been shown to be negligible. By combining a dHis Cu labelling site with cysteine-specific nitroxide labelling, we gather insights on the label rotamers at two distinct sites, comparing their contributions to distance distributions based on different in silico modelling approaches and structural models. From this study, it seems advisable to consider discrepancies between different in silico modelling approaches when selecting labelling sites for PDS studies.

In this paper, we explore the robustness and sensitivity of Gd(III)-Gd(III) double electron-electron resonance (DEER) distance measurements in proteins for different spectrometer designs and three spin labels. To do this a protein was labeled at the same two positions with Gd(III) spin labels and measurements were performed on two home-built high-frequency (W-band, ~ 95 GHz) EPR spectrometers with different design approaches, and a commercial 150 W Q-band (34 GHz) spectrometer. The first W-band measurement approach uses a conventional, narrow band single mode cavity, while the second uses a broadband non-resonant induction mode sample holder. Both systems incorporate advanced arbitrary waveform generators (AWGs) that give flexibility over excitation bandwidth. We use three DOTA-like Gd(III) spin labels, Gd.C12, Gd.DO3A and Gd.L, conjugated to the calmodulin protein. We compare measurements taken by including or excluding the Gd(III) central transition excitation. The advantages and disadvantages of the EPR spectrometers for the measurement of Gd(III)-Gd(III) DEER are discussed in terms of the robustness of the resulting distance distribution width, absolute and concentration sensitivity, sample handling, ease of use, and flexibility of measurement.

The study of continuous wave (cw) electron paramagnetic resonance (EPR) spectra still poses a challenge for very broad signals, especially when the spectrum extends over a large part of the accessible field range. The difficulties derive from instrumental challenges, because of insufficient modulation depth and the need to apply measurement conditions that enhance cavity background. The biggest problem, however, is how to define a baseline such that spectral distortions are minimized. Conventional methods rely on a suitable choice of points outside the range of the signal of interest to perform a polynomial interpolation. These methods are effective in most cases where the signal of interest comprises only a narrow range of magnetic field (narrow features). In this study, a novel method of baseline correction for broad signals is proposed and compared to conventional methods. It takes into account that there are only few anchor points for the baseline. The method is applied to the signal of the iron-storage protein ferritin. The ferritin signal is a broad band that extends from zero to 0.8 T. An approach is developed by which this broad signal is analyzed reliably. The method is also extended to the case where the broad signal is superimposed on narrow signals and enables to extract the parameters of both types of signals in a fitting pipeline.

Temperature-dependent DEER effects are observed as a function of methyl rotation by either leucine- or nitroxide-specific protonated methyl groups in an otherwise deuterated background. Both species induce a site-specific enhancement in the apparent relaxation of the paramagnetic nitroxide label. The presence of a single protonated methyl group in close proximity (4-10 Å) to only one of the two nitroxide rotamer ensembles in AviTagged immunoglobulin-binding B domain of protein A results in a selective and substantial decrease in , manifested by differential decay of the peak intensities in the bimodal distance distribution as a function of the total dipolar evolution time, temperature, or both. The temperature-dependent differential decay of the individual distance components was globally analyzed by fitting the DEER dipolar time traces to a three-site jump model that is defined by the activation energy of leucine- or nitroxide-specific methyl rotation. Temperature-assisted T filtering will capture the DEER structural analysis of biomolecular systems heterogenic conformations, including complexes involving multimeric proteins.