Rigid orthogonal bis-TEMPO biradicals with improved solubility for dynamic nuclear polarization.

The synthesis and characterization of oxidized bis-thioketal-trispiro dinitroxide biradicals that orient the nitroxides in a rigid, approximately orthogonal geometry are reported. The biradicals show better performance as polarizing agents in dynamic nuclear polarization (DNP) NMR experiments as compared to biradicals lacking the constrained geometry. In addition, the biradicals display improved solubility in aqueous media due to the presence of polar sulfoxides. The results suggest that the orientation of the radicals is not dramatically affected by the oxidation state of the sulfur atoms in the biradical, and we conclude that a biradical polarizing agent containing a mixture of oxidation states can be used for improved solubility without a loss in performance.

Solid effect dynamic nuclear polarization and polarization pathways.

Using dynamic nuclear polarization (DNP)/nuclear magnetic resonance instrumentation that utilizes a microwave cavity and a balanced rf circuit, we observe a solid effect DNP enhancement of 94 at 5 T and 80 K using trityl radical as the polarizing agent. Because the buildup rate of the solid effect increases with microwave field strength, we obtain a sensitivity gain of 128. The data suggest that higher microwave field strengths would lead to further improvements in sensitivity. In addition, the observation of microwave field dependent enhancements permits us to draw conclusions about the path that polarization takes during the DNP process. By measuring the time constant for the polarization buildup and enhancement as a function of the microwave field strength, we are able to compare models of polarization transfer, and show that the major contribution to the bulk polarization arises via direct transfer from electrons, rather than transferring first to nearby nuclei and then transferring to bulk nuclei in a slow diffusion step. In addition, the model predicts that nuclei near the electron receive polarization that can relax, decrease the electron polarization, and attenuate the DNP enhancement. The magnitude of this effect depends on the number of near nuclei participating in the polarization transfer, hence the size of the diffusion barrier, their T(1), and the transfer rate. Approaches to optimizing the DNP enhancement are discussed.

Simplified THz Instrumentation for High-Field DNP-NMR Spectroscopy.

We present an alternate simplified concept to irradiate a nuclear magnetic resonance sample with terahertz (THz) radiation for dynamic nuclear polarization (DNP) experiments using the TE(01) circular waveguide mode for transmission of the THz power and the illumination of the DNP sample by either the TE(01) or TE(11) mode. Using finite element method and 3D electromagnetic simulations we demonstrate that the average value of the transverse magnetic field induced by the THz radiation and responsible for the DNP effect using the TE(11) or the TE(01) mode are comparable to that generated by the HE(11) mode and a corrugated waveguide. The choice of the TE(11)/TE(01) mode allows the use of a smooth-walled, oversized waveguide that is easier to fabricate and less expensive than a corrugated waveguide required for transmission of the HE(11) mode. Also, the choice of the TE(01) mode can lead to a simplification of gyrotron oscillators that operate in the TE(0n) mode, by employing an on-axis rippled-wall mode converter to convert the TE(0n) mode into the TE(01) mode either inside or outside of the gyrotron tube. These novel concepts will lead to a significant simplification of the gyrotron, the transmission line and the THz coupler, which are the three main components of a DNP system.

Compact, tunable polarization transforming reflector for quasi-optical devices used in terahertz science.

We describe the design, fabrication, and characterization of a compact polarization transforming reflector (PTR). The device can be easily tuned over a broad frequency range, has very little insertion losses, and can easily be integrated into quasi-optical systems that are based on a half-cube design. By varying the distance between the wire grid and a flat mirror, the polarization state of an arbitrary polarized Gaussian incident beam can be set to an output Gaussian beam in either linear or circular polarization. In addition, by varying the orientation of the wire grid, the PTR can be used as a universal polarizer, a property that has not been discussed or demonstrated in the literature. The ability to control the electric field polarization at Terahertz (THz) frequencies is essential for many applications, such as THz spectroscopy and high-field electron paramagnetic resonance spectroscopy.

Recent advances in microresonators and supporting instrumentation for electron paramagnetic resonance spectroscopy.

Electron paramagnetic resonance (EPR) spectroscopy characterizes the magnetic properties of paramagnetic materials at the atomic and molecular levels. Resonators are an enabling technology of EPR spectroscopy. Microresonators, which are miniaturized versions of resonators, have advanced inductive-detection EPR spectroscopy of mass-limited samples. Here, we provide our perspective of the benefits and challenges associated with microresonator use for EPR spectroscopy. To begin, we classify the application space for microresonators and present the conceptual foundation for analysis of resonator sensitivity. We summarize previous work and provide insight into the design and fabrication of microresonators as well as detail the requirements and challenges that arise in incorporating microresonators into EPR spectrometer systems. Finally, we provide our perspective on current challenges and prospective fruitful directions.

Multifrequency pulsed electron paramagnetic resonance on metalloproteins.

Metalloproteins often contain metal centers that are paramagnetic in some functional state of the protein; hence electron paramagnetic resonance (EPR) spectroscopy can be a powerful tool for studying protein structure and function. Dipolar spectroscopy allows the determination of the dipole-dipole interactions between metal centers in protein complexes, revealing the structural arrangement of different paramagnetic centers at distances of up to 8 nm. Hyperfine spectroscopy can be used to measure the interaction between an unpaired electron spin and nuclear spins within a distance of 0.8 nm; it therefore permits the characterization of the local structure of the paramagnetic center's ligand sphere with very high precision. In this Account, we review our laboratory's recent applications of both dipolar and hyperfine pulsed EPR methods to metalloproteins. We used pulsed dipolar relaxation methods to investigate the complex of cytochrome c and cytochrome c oxidase, a noncovalent protein-protein complex involved in mitochondrial electron-transfer reactions. Hyperfine sublevel correlation spectroscopy (HYSCORE) was used to study the ligand sphere of iron-sulfur clusters in complex I of the mitochondrial respiratory chain and substrate binding to the molybdenum enzyme polysulfide reductase. These examples demonstrate the potential of the two techniques; however, they also highlight the difficulties of data interpretation when several paramagnetic species with overlapping spectra are present in the protein. In such cases, further approaches and data are very useful to enhance the information content. Relaxation filtered hyperfine spectroscopy (REFINE) can be used to separate the individual components of overlapping paramagnetic species on the basis of differences in their longitudinal relaxation rates; it is applicable to any kind of pulsed hyperfine or dipolar spectroscopy. Here, we show that the spectra of the iron-sulfur clusters in complex I can be separated by this method, allowing us to obtain hyperfine (and dipolar) information from the individual species. Furthermore, performing pulsed EPR experiments at different magnetic fields is another important tool to disentangle the spectral components in such complex systems. Despite the fact that high magnetic fields do not usually lead to better spectral separation for metal centers, they provide additional information about the relative orientation of different paramagnetic centers. Our high-field EPR studies on cytochrome c oxidase reveal essential information regarding the structural arrangement of the binuclear Cu(A) center with respect to both the manganese ion within the enzyme and the cytochrome in the protein-protein complex with cytochrome c.

Overhauser dynamic nuclear polarization (ODNP)-enhanced two-dimensional proton NMR spectroscopy at low magnetic fields.

The majority of low-field Overhauser dynamic nuclear polarization (ODNP) experiments reported so far have been 1D NMR experiments to study molecular dynamics and in particular hydration dynamics. In this work, we demonstrate the application of ODNP-enhanced 2D J-resolved (JRES) spectroscopy to improve spectral resolution beyond the limit imposed by the line broadening introduced by the paramagnetic polarizing agent. Using this approach, we are able to separate the overlapping multiplets of ethyl crotonate into a second dimension and clearly identify each chemical site individually. Crucial to these experiments is interleaved spectral referencing, a method introduced to compensate for temperature-induced field drifts over the course of the NMR acquisition. This method does not require additional hardware such as a field-frequency lock, which is especially challenging when designing compact systems.

High-resolution MAS NMR analysis of PI3-SH3 amyloid fibrils: backbone conformation and implications for protofilament assembly and structure .

The SH3 domain of the PI3 kinase (PI3-SH3 or PI3K-SH3) readily aggregates into fibrils in vitro and has served as an important model system in the investigation of the molecular properties and mechanism of formation of amyloid fibrils. We describe the molecular conformation of PI3-SH3 in amyloid fibril form as revealed by magic-angle spinning (MAS) solid-state nuclear magnetic resonance (NMR) spectroscopy. The MAS NMR spectra of these fibrils display excellent resolution, with narrow (13)C and (15)N line widths, representing a high degree of structural order and the absence of extensive molecular motion for the majority of the polypeptide chain. We have identified the spin systems of 82 of the 86 residues in the protein and obtained sequential resonance assignments for 75 of them. Chemical shift analysis indicates that the protein subunits making up the fibril adopt a compact conformation consisting of four well-defined beta-sheet regions and four random-coil elements with varying degrees of local dynamics or disorder. The backbone conformation of PI3-SH3 in fibril form differs significantly from that of the native state of the protein, both in secondary structure and in the location of dynamic or disordered segments. The site-specific MAS NMR analysis of PI3-SH3 fibrils we report here is compared with previously published mechanistic and structural data, resulting in a detailed interpretation of the factors that mediate fibril formation by PI3-SH3 and allowing us to propose a possible model of the core structure of the fibrils. Our results confirm the structural similarities between PI3-SH3 fibrils and amyloid assemblies directly related to degenerative and infectious diseases.

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

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

2H-DNP-enhanced 2H-13C solid-state NMR correlation spectroscopy.

Perdeuteration of biological macromolecules for magic angle spinning solid-state NMR spectroscopy can yield high-resolution (2)H-(13)C correlation spectra and the method is therefore of great interest for the structural biology community. Here we demonstrate that the combination of sample deuteration and dynamic nuclear polarization yields resolved (2)H-(13)C correlation spectra with a signal enhancement of epsilon > or = 700 compared to a spectrum recorded with microwaves off and otherwise identical conditions. To our knowledge, this is the first time that (2)H-DNP has been employed to enhance MAS-NMR spectra of a biologically relevant system. The DNP process is studied using several polarizing agents and the technique is applied to obtain (2)H-(13)C correlation spectra of U-[(2)H, (13)C] proline.

High-resolution Overhauser dynamic nuclear polarization enhanced proton NMR spectroscopy at low magnetic fields.

Dynamic nuclear polarization (DNP) has gained large interest due to its ability to increase signal intensities in nuclear magnetic resonance (NMR) experiments by several orders of magnitude. Currently, DNP is typically used to enhance high-field, solid-state NMR experiments. However, the method is also capable of dramatically increasing the observed signal intensities in solution-state NMR spectroscopy. In this work, we demonstrate the application of Overhauser dynamic nuclear polarization (ODNP) spectroscopy at an NMR frequency of 14.5 MHz (0.35 T) to observe DNP-enhanced high-resolution NMR spectra of small molecules in solutions. Using a compact hybrid magnet with integrated shim coils to improve the magnetic field homogeneity we are able to routinely obtain proton linewidths of less than 4 Hz and enhancement factors >30. The excellent field resolution allows us to perform chemical-shift resolved ODNP experiments on ethyl crotonate to observe proton J-coupling. Furthermore, recording high-resolution ODNP-enhanced NMR spectra of ethylene glycol allows us to characterize the microwave induced sample heating in-situ, by measuring the separation of the OH and CH2 proton peaks.

Dynamic nuclear polarization with a rigid biradical.

A new polarizing agent with superior performance in dynamic nuclear polarization experiments is introduced, and utilizes two TEMPO (2,2,6,6-tetramethylpiperidine-1-oxyl) moieties connected through a rigid spiro tether (see structure). The observed NMR signal intensities were enhanced by a factor of 1.4 compared to those of TOTAPOL, a previously described TEMPO-based biradical with a flexible tether.

Thermo-mechanical analysis of a probe for electron paramagnetic resonance spectroscopy operating at cryogenic temperatures.

In this article, we present the thermo-mechanical analysis of an electron paramagnetic resonance (EPR) probe operating at cryogenic temperatures using finite element analysis. Thermo-mechanical analysis plays a key role in the mechanical design evaluation process as EPR probes are often subjected to large stresses under such extreme conditions. For simplification, we assume thermal conduction to be the dominant mode of heat transfer over convection and radiation. The simulation model consists of a cryostat-probe assembly with appropriate thermal and structural boundary conditions. The predicted temperature distributions from the steady-state thermal analysis is then used for the stress analysis of the EPR probe. The stress analysis indicated that stresses in the EPR probe are below the ultimate strengths of each component, and thus safe for running EPR experiments. Furthermore, the simulation results were confirmed experimentally, and we found that the predicted heat losses for the EPR probe assembly and the sample holder are in excellent agreement with the experimental measurements.

2D-REFINE spectroscopy: separation of overlapping hyperfine spectra.

We show on a mixture of three spectrally overlapping paramagnetic compounds TEMPO, BDPA and CuHis that it is possible to separate their field-swept and hyperfine spectra based on the difference in their longitudinal relaxation times T1. This was achieved in a two-dimensional experiment, where one dimension corresponds to the spectral domain and the second dimension encodes the relaxation behavior of the individual compound. Inverse Laplace Transform with respect to this domain separates the field-swept and hyperfine spectra of the individual compounds in the relaxation rate domain. This extends our formerly proposed Relaxation Filtered Hyperfine (REFINE) method to be applicable to more than two spectrally overlapping spectra by adding a further dimension to the chosen EPR experiment.

A ferromagnetic shim insert for NMR magnets - Towards an integrated gyrotron for DNP-NMR spectroscopy.

In recent years high-field Dynamic Nuclear Polarization (DNP) enhanced NMR spectroscopy has gained significant interest. In high-field DNP-NMR experiments (⩾400MHz 1H NMR, ⩾9.4T) often a stand-alone gyrotron is used to generate high microwave/THz power to produce sufficiently high microwave induced B1e fields at the position of the NMR sample. These devices typically require a second, stand-alone superconducting magnet to operate. Here we present the design and realization of a ferroshim insert, to create two iso-centers inside a commercially available wide-bore NMR magnet. This work is part of a larger project to integrate a gyrotron into NMR magnets, effectively eliminating the need for a second, stand-alone superconducting magnet.

A field-sweep/field-lock system for superconducting magnets--Application to high-field EPR.

We describe a field-lock/field-sweep system for the use in superconducting magnets. The system is based on a commercially available field mapping unit and a custom designed broad-band 1H NMR probe. The NMR signal of a small water sample is used in a feedback loop to set and control the magnetic field to high accuracy. The current instrumental configuration allows field sweeps of +/-0.4 T and a resolution of up to 10(-5) T (0.1 G) and the performance of the system is demonstrated in a high-field electron paramagnetic resonance (EPR) application. The system should also be of utility in other experiments requiring precise and reproducible sweeps of the magnetic field such as DNP, ENDOR or PELDOR.

1H dynamic nuclear polarization based on an endogenous radical.

We demonstrate a 15-fold enhancement of solid-state NMR signals via dynamic nuclear polarization (DNP) based on a stable, naturally occurring radical in a protein: the flavin mononucleotide (FMN) semiquinone of flavodoxin. The line width of flavodoxin's EPR signal suggests that the dominant DNP mechanism is the solid effect, consistent with the field-dependent DNP enhancement profile. The magnitude of the enhancement as well as the bulk-polarization build-up time constant (τ(B)) with which it develops are dependent on the isotopic composition of the protein. Deuteration of the protein to 85% increased the nuclear longitudinal relaxation time T(1n) and τ(B) by factors of five and seven, respectively. Slowed dissipation of polarization can explain the 2-fold higher maximal enhancement than that obtained in proteated protein, based on the endogenous semiquinone. In contrast, the long τ(B) of TOTAPOL-based DNP in nonglassy samples was not accompanied by a similarly important long T(1n), and in this case the enhancement was greatly reduced. The low concentrations of radicals occurring naturally in biological systems limit the magnitude of DNP enhancement that is attainable by this means. However, our enhancement factors of up to 15 can nonetheless make an important difference to the feasibility of applying solid-state NMR to biochemical systems. We speculate that DNP based on endogenous radicals may facilitate MAS NMR characterization of biochemical complexes and even organelles, and could also serve as a source of additional structural and physiological information.

Synthesis of a BDPA-TEMPO biradical.

The synthesis and characterization of a biradical containing a 1,3-bisdiphenylene-2-phenylallyl (BDPA) free radical covalently attached to a 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) free radical are described. The synthesis of the biradical is a step toward improved polarizing agents for dynamic nuclear polarization (DNP).