Terahertz EPR spectroscopy using a 36-tesla high-homogeneity series-connected hybrid magnet.

Electron Paramagnetic Resonance (EPR) is a powerful technique to study materials and biological samples on an atomic scale. High-field EPR in particular enables extracting very small g-anisotropies in organic radicals and half-filled 3d and 4f metal ions such as MnII (3d5) or GdIII (4f7), and resolving EPR signals from unpaired spins with very close g-values, both of which provide high-resolution details of the local atomic environment. Before the recent commissioning of the high-homogeneity Series Connected Hybrid magnet (SCH, superconducting + resistive) at the National High Magnetic Field Laboratory (NHMFL), the highest-field, high-resolution EPR spectrometer available was limited to 25 T using a purely resistive "Keck" magnet at the NHMFL. Herein, we report the first EPR experiments performed using the SCH magnet capable of reaching the field of 36 T, corresponding to an EPR frequency of 1 THz for g = 2. The magnet's intrinsic homogeneity (25 ppm, that is 0.9 mT at 36 T over 1 cm diameter, 1 cm length cylinder) was previously established by NMR. We characterized the magnet's temporal stability (5 ppm, which is 0.2 mT at 36 T over one-minute, the typical acquisition time) using 2,2-diphenyl-1-picrylhydrazyl (DPPH). This high resolution enables resolving the weak g-anisotropy of 1,3-bis(diphenylene)-2-phenylallyl (BDPA), Δg = 2.5 × 10-4 obtained from measurements at 932 GHz and 33 T. Subsequently, we recorded EPR spectra at multiple frequencies for two GdIII complexes with potential applications as spin labels. We demonstrated a significant reduction in line broadening in Gd[DTPA], attributed to second order zero field splitting, and a resolution enhancement of g-tensor anisotropy for Gd[sTPATCN]-SL.

A Complete Triad of Zero-Valent 17-Electron Monoradicals of Group 7 Elements Stabilized by m-Terphenyl Isocyanides.

The first consistent series of mononuclear 17-electron complexes of three Group 7 elements has been isolated in crystalline form and studied by X-ray diffraction and spectroscopic methods. The paramagnetic compounds have a composition of [M0 (CO)(CNp-F-ArDArF2 )4 ] (M=Mn, Tc, Re; ArDArF2 =2,6-(3,5-(CF3 )2 C6 H3 )2 C6 H2 F) and are stabilized by four sterically encumbering isocyanides, which prevent the metalloradicals from dimerization. They have a square pyramidal structure with the carbonyl ligands as apexes. The frozen-solution EPR spectra of the rhenium and technetium compounds are clearly anisotropic with large 99 Tc and 185,187 Re hyperfine interactions for one component. High-field EPR (Q band and W band) has been applied for the elucidation of the EPR parameters of the manganese(0) complex.

Resolution of chemical shift anisotropy in 19F ENDOR spectroscopy at 263 GHz/9.4 T.

Pulsed 19F ENDOR spectroscopy provides a selective method for measuring angstrom to nanometer distances in structural biology. Here, the performance of 19F ENDOR at fields of 3.4 T and 9.4 T is compared using model compounds containing one to three 19F atoms. CF3 groups are included in two compounds, for which the possible occurrence of uniaxial rotation might affect the distance distribution. At 9.4 T, pronounced asymmetric features are observed in many of the presented 19F ENDOR spectra. Data analysis by spectral simulations shows that these features arise from the chemical shift anisotropy (CSA) of the 19F nuclei. This asymmetry is also observed at 3.4 T, albeit to a much smaller extent, confirming the physical origin of the effect. The CSA parameters are well consistent with DFT predicted values and can be extracted from simulation of the experimental data in favourable cases, thereby providing additional information about the geometrical and electronic structure of the spin system. The feasibility of resolving the CSA at 9.4 T provides important information for the interpretation of line broadening in ENDOR spectra also at lower fields, which is relevant for developing methods to extract distance distributions from 19F ENDOR spectra.

Application of spherical harmonics for DEER data analysis in systems with a conformational distribution.

Double electron-electron resonance (DEER) and other pulse electron paramagnetic resonance (EPR) techniques are valuable tools for determining distances between paramagnetic centres. DEER theory is well developed for a scenario where relative orientations of paramagnetic centres do not affect the DEER data. In particular, such theory enables a number of approaches for extracting distance distributions. However, in a more general case, when orientation selection effects become substantial, the analytical theory of DEER is less well developed, therefore quite commonly researchers rely on a comparison of some model-based simulations with experimental data. This work elaborates the theory of DEER with orientation selection effects, focusing on a scenario of a moderate conformational disorder, leading to an orientation distribution in a pair of paramagnetic centres. The analytical treatment based on expansions into spherical harmonics, provides important insights into the structure of DEER data. As follows from this treatment, DEER spectra with orientation selection can be represented as a linear combination of modified Pake pattern (MPP) components. The conformational disorder has a filtering effect on the weights of MPP components, specifically by significantly suppressing MPP components of higher degrees. The developed theory provides a pathway for model-based simulations of DEER data where orientation distribution is defined by analytical functions with parameters. The theory based on spherical harmonics expansions was also applied to develop an iterative processing algorithm based on Tikhonov regularization, which disentangles the distance and orientation information in a model-free manner. As an input, this procedure takes several DEER datasets measured at various positions of an EPR line, and outputs a distance distribution and orientation distribution information encoded in a set of coefficients related to the weights of MPP components. The model-based and model-free approaches based on the developed theory were validated for a nitroxide biradical and a spin-labelled protein.

The Transient Complex of Cytochrome c and Cytochrome c Peroxidase: Insights into the Encounter Complex from Multifrequency EPR and NMR Spectroscopy.

We present a novel approach to study transient protein-protein complexes with standard, 9 GHz, and high-field, 95 GHz, electron paramagnetic resonance (EPR) and paramagnetic NMR at ambient temperatures and in solution. We apply it to the complex of yeast mitochondrial iso-1-cytochrome c (Cc) with cytochrome c peroxidase (CcP) with the spin label [1-oxyl-2,2,5,5-tetramethyl-Δ3-pyrroline-3-methyl)-methanethiosulfonate] attached at position 81 of Cc (SL-Cc). A dissociation constant KD of 20±4×10-6  M (EPR and NMR) and an equal amount of stereo-specific and encounter complex (NMR) are found. The EPR spectrum of the fully bound complex reveals that the encounter complex has a significant population (60 %) that shares important features, such as the Cc-interaction surface, with the stereo-specific complex.

Photonic band-gap resonators for high-field/high-frequency EPR of microliter-volume liquid aqueous samples.

High-field EPR provides significant advantages for studying structure and dynamics of molecular systems possessing an unpaired electronic spin. However, routine use of high-field EPR in biophysical research, especially for aqueous biological samples, is still facing substantial technical difficulties stemming from high dielectric millimeter wave (mmW) losses associated with non-resonant absorption by water and other polar molecules. The strong absorbance of mmW's by water also limits the penetration depth to just fractions of mm or even less, thus making fabrication of suitable sample containers rather challenging. Here we describe a radically new line of high Q-factor mmW resonators that are based on forming lattice defects in one-dimensional photonic band-gap (PBG) structures composed of low-loss ceramic discs of λ/4 in thickness and having alternating dielectric constants. A sample (either liquid or solid) is placed within the E = 0 node of the standing mm wave confined within the defect. A resonator prototype has been built and tested at 94.3 GHz. The resonator performance is enhanced by employing ceramic nanoporous membranes as flat sample holders of controllable thickness and tunable effective dielectric constant. The experimental Q-factor of an empty resonator was  ≈ 420. The Q-factor decreased slightly to  ≈ 370 when loaded with a water-containing nanoporous disc of 50 µm in thickness. The resonator has been tested with a number of liquid biological samples and demonstrated about tenfold gain in concentration sensitivity vs. a high-Q cylindrical TE012-type cavity. Detailed HFSS Ansys simulations have shown that the resonator structure could be further optimized by properly choosing the thickness of the aqueous sample and employing metallized surfaces. The PBG resonator design is readily scalable to higher mmW frequencies and is capable of accommodating significantly larger sample volumes than previously achieved with either Fabry-Perot or cylindrical resonators.

Triple resonance EPR spectroscopy determines the Mn2+ coordination to ATP.

Mn2+ often serves as a paramagnetic substitute to Mg2+, providing means for exploring the close environment of Mg2+ in many biological systems where it serves as an essential co-factor. This applies to proteins with ATPase activity, where the ATP hydrolysis requires the binding of Mg2+-ATP to the ATPase active site. In this context, it is important to distinguish between the Mn2+ coordination mode with free ATP in solution as compared to the protein bound case. In this work, we explore the Mn2+ complexes with ATP, the non-hydrolysable ATP analog, AMPPNP, and ADP free in solution. Using W-band 31P electron-nuclear double resonance (ENDOR) we obtained information about the coordination to the phosphates, whereas from electron-electron double resonance (ELDOR) - detected NMR (EDNMR) we determined the coordination to an adenosine nitrogen. The coordination to these ligands has been reported earlier, but whether the nitrogen and phosphate coordination is within the same nucleotide molecules or different ones is still under debate. By applying the correlation technique, THYCOS (triple hyperfine correlation spectroscopy), and measuring 15N-31P correlations we establish that in Mn-ATP in solution both phosphates and a nitrogen are coordinated to the Mn2+ ion. We also carried out DFT calculations to substantiate this finding. In addition, we expanded the understanding of the THYCOS experiment by comparing it to 2D-EDNMR for 55Mn-31P correlation experiments and through simulations of THYCOS and 2D-EDNMR spectra with 15N-31P correlations.

Double resonance calibration of g factor standards: Carbon fibers as a high precision standard.

The g factor of paramagnetic defects in commercial high performance carbon fibers was determined by a double resonance experiment based on the Overhauser shift due to hyperfine coupled protons. Our carbon fibers exhibit a single, narrow and perfectly Lorentzian shaped ESR line and a g factor slightly higher than gfree with g=2.002644=gfree·(1+162ppm) with a relative uncertainty of 15ppm. This precisely known g factor and their inertness qualify them as a high precision g factor standard for general purposes. The double resonance experiment for calibration is applicable to other potential standards with a hyperfine interaction averaged by a process with very short correlation time.

Arbitrary waveform modulated pulse EPR at 200GHz.

We report here on the implementation of arbitrary waveform generation (AWG) capabilities at ∼200GHz into an Electron Paramagnetic Resonance (EPR) and Dynamic Nuclear Polarization (DNP) instrument platform operating at 7T. This is achieved with the integration of a 1GHz, 2 channel, digital to analog converter (DAC) board that enables the generation of coherent arbitrary waveforms at Ku-band frequencies with 1ns resolution into an existing architecture of a solid state amplifier multiplier chain (AMC). This allows for the generation of arbitrary phase- and amplitude-modulated waveforms at 200GHz with >150mW power. We find that the non-linearity of the AMC poses significant difficulties in generating amplitude-modulated pulses at 200GHz. We demonstrate that in the power-limited regime of ω1<1MHz phase-modulated pulses were sufficient to achieve significant improvements in broadband (>10MHz) spin manipulation in incoherent (inversion), as well as coherent (echo formation) experiments. Highlights include the improvement by one order of magnitude in inversion bandwidth compared to that of conventional rectangular pulses, as well as a factor of two in improvement in the refocused echo intensity at 200GHz.

Spin Probe Multi-Frequency EPR Study of Unprocessed Cotton Fibers.

Known since the ancient times, cotton continues to be one of the essential materials for the human civilization. Cotton fibers are almost pure cellulose and contain both crystalline and amorphous nanodomains with different physicochemical properties. While understanding of interactions between the individual cellulose chains within the crystalline phase is important from a perspective of mechanical properties, studies of the amorphous phase lead to characterization of the essential transport parameters, such as solvent diffusion, dyeing, drug release, and toxin absorption, as well as more complex processes of enzymatic degradation. Here, we describe the use of spin probe electron paramagnetic resonance methods to study local polarity and heterogeneous viscosity of two types of unprocessed cotton fibers, G. hirsutum and G. barbadense, harvested in the State of North Carolina, USA. These fibers were loaded with two small molecule nitroxide probes that differ in polarity-Tempo and its more hydrophilic derivative Tempol-using a series of polar and non-polar solvents. The electron paramagnetic resonance spectra of the nitroxide-loaded cotton fibers were analyzed both semi-empirically and by least-squares simulations using a rigorous stochastic theory of electron paramagnetic resonance spectra developed by Freed and coworkers. A software package and least-squares fitting protocols were developed to carry out automatic simulations of multi-component electron paramagnetic resonance spectra in both first-derivative and the absorption forms at multiple resonance frequencies such as X-band (9.5 GHz) and W-band (94.3 GHz). The results are compared with the preceding electron paramagnetic resonance spin probe studies of a commercial bleached cotton sheeting carried out by Batchelor and coworkers. One of the results of this study is a demonstration of a co-existence of cellulose nanodomains with different physicochemical properties such as polarity and microviscosity that are affected by solvents and temperature. Spin labeling studies also revealed a macroscopic heterogeneity in the domain distribution along the cotton fibers and a critical role the cuticular layer is playing as a barrier for spin probe penetration. Finally but not lastly, the simultaneous multi-component least-squares simulation method of electron paramagnetic resonance spectra acquired at different resonant frequencies and the display forms (e.g., absorption and first-derivative displays) and the strategy of spectral parameter sharing could be potentially applicable to other heterogeneous biological systems in addition to the cotton fibers studies here.

Gd³âº Spin Labeling for Measuring Distances in Biomacromolecules: Why and How?

Applications of distance measurements by pulse dipolar electron-paramagnetic resonance (PD-EPR) spectroscopy to structural biology are based on introducing spin labels (SLs) at well-defined locations in the biomacromolecule. The most commonly used SLs are nitroxyl radicals, but recently SLs based on high-spin Gd(3+) (S=7/2) complexes have been shown to be an attractive alternative for PD-EPR, particularly double electron-electron resonance (DEER), at spectrometer frequencies higher than 30 GHz. In this chapter, we describe the advantage of using this new family of SLs in terms of sensitivity, stability, and chemical diversity. We present current labeling strategies for proteins, discuss the approximations under which DEER data analysis of a pair of Gd(3+) SLs (GdSLs) is equivalent to that of a pair of S=1/2 SLs, and discuss the reduction in multispin effects in a cluster of GdSLs, as opposed to a cluster of nitroxide labels, which can be found in oligomeric systems. In addition, we provide a brief overview of the current, rather limited, knowledge of Gd(3+) phase relaxation behavior and describe experimental strategies in terms of optimizing sensitivity. The possibility of using several types of SLs in a system allows one to isolate effects due to the chemical nature of the SL itself; several such examples are presented, focusing on comparing nitroxide and GdSLs. Finally, we will discuss the initial results on in-cell DEER with GdSLs.

Continuous wave W- and D-band EPR spectroscopy offer "sweet-spots" for characterizing conformational changes and dynamics in intrinsically disordered proteins.

Site-directed spin labeling (SDSL) electron paramagnetic resonance (EPR) spectroscopy is a powerful tool for characterizing conformational sampling and dynamics in biological macromolecules. Here we demonstrate that nitroxide spectra collected at frequencies higher than X-band (∼9.5 GHz) have sensitivity to the timescale of motion sampled by highly dynamic intrinsically disordered proteins (IDPs). The 68 amino acid protein IA3, was spin-labeled at two distinct sites and a comparison of X-band, Q-band (35 GHz) and W-band (95 GHz) spectra are shown for this protein as it undergoes the helical transition chemically induced by tri-fluoroethanol. Experimental spectra at W-band showed pronounced line shape dispersion corresponding to a change in correlation time from ∼0.3 ns (unstructured) to ∼0.6 ns (α-helical) as indicated by comparison with simulations. Experimental and simulated spectra at X- and Q-bands showed minimal dispersion over this range, illustrating the utility of SDSL EPR at higher frequencies for characterizing structural transitions and dynamics in IDPs.

High-field ELDOR-detected NMR study of a nitroxide radical in disordered solids: towards characterization of heterogeneity of microenvironments in spin-labeled systems.

The combination of high-field EPR with site-directed spin-labeling (SDSL) techniques employing nitroxide radicals has turned out to be particularly powerful in probing the polarity and proticity characteristics of protein/matrix systems. This information is concluded from the principal components of the nitroxide Zeeman (g), nitrogen hyperfine (A) and quadrupole (P) tensors of the spin labels attached to specific sites. Recent multi-frequency high-field EPR studies underlined the complexity of the problem to treat the nitroxide microenvironment in proteins adequately due to inherent heterogeneities which result in several principal x-components of the nitroxide g-tensor. Concomitant, but distinctly different nitrogen hyperfine components could, however, not be determined from high-field cw EPR experiments owing to the large intrinsic EPR linewidth in fully protonated guest/host systems. It is shown in this work that, using the W-band (95GHz) ELDOR- (electron-electron double resonance) detected NMR (EDNMR) method, different principal nitrogen hyperfine, Azz, and quadrupole, Pzz, tensor values of a nitroxide radical in glassy 2-propanol matrix can be measured with high accuracy. They belong to nitroxides with different hydrogen-bond situations. The satisfactory resolution and superior sensitivity of EDNMR as compared to the standard ENDOR (electron-nuclear double resonance) method are demonstrated.

Optimization of an absolute sensitivity in a glassy matrix during DNP-enhanced multidimensional solid-state NMR experiments.

Thanks to instrumental and theoretical development, notably the access to high-power and high-frequency microwave sources, high-field dynamic nuclear polarization (DNP) on solid-state NMR currently appears as a promising solution to enhance nuclear magnetization in many different types of systems. In magic-angle-spinning DNP experiments, systems of interest are usually dissolved or suspended in glass-forming matrices doped with polarizing agents and measured at low temperature (down to ∼100K). In this work, we discuss the influence of sample conditions (radical concentration, sample temperature, etc.) on DNP enhancements and various nuclear relaxation times which affect the absolute sensitivity of DNP spectra, especially in multidimensional experiments. Furthermore, DNP-enhanced solid-state NMR experiments performed at 9.4 T are complemented by high-field CW EPR measurements performed at the same magnetic field. Microwave absorption by the DNP glassy matrix is observed even below the glass transition temperature caused by softening of the glass. Shortening of electron relaxation times due to glass softening and its impact in terms of DNP sensitivity is discussed.

X-ray detected magnetic resonance: a unique probe of the precession dynamics of orbital magnetization components.

X-ray Detected Magnetic Resonance (XDMR) is a novel spectroscopy in which X-ray Magnetic Circular Dichroism (XMCD) is used to probe the resonant precession of local magnetization components in a strong microwave pump field. We review the conceptual bases of XDMR and recast them in the general framework of the linear and nonlinear theories of ferromagnetic resonance (FMR). Emphasis is laid on the information content of XDMR spectra which offer a unique opportunity to disentangle the precession dynamics of spin and orbital magnetization components at given absorbing sites. For the sake of illustration, we focus on selected examples in which marked differences were found between FMR and XDMR spectra simultaneously recorded on ferrimagnetically ordered iron garnets. With pumping capabilities extended up to sub-THz frequencies, high-field XDMR should allow us to probe the precession of orbital magnetization components in paramagnetic organometallic complexes with large zero-field splitting. Even more challenging, we suggest that XDMR spectra might be recorded on selected antiferromagnetic crystals for which orbital magnetism is most often ignored in the absence of any supporting experimental evidence.