Overhauser enhanced liquid state nuclear magnetic resonance spectroscopy in one and two dimensions.

Nuclear magnetic resonance (NMR) is fundamental in the natural sciences, from chemical analysis and structural biology, to medicine and physics. Despite its enormous achievements, one of its most severe limitations is the low sensitivity, which arises from the small population difference of nuclear spin states. Methods such as dissolution dynamic nuclear polarization and parahydrogen induced hyperpolarization can enhance the NMR signal by several orders of magnitude, however, their intrinsic limitations render multidimensional hyperpolarized liquid-state NMR a challenge. Here, we report an instrumental design for 9.4 Tesla liquid-state dynamic nuclear polarization that enabled enhanced high-resolution NMR spectra in one and two-dimensions for small molecules, including drugs and metabolites. Achieved enhancements of up to two orders of magnitude translate to signal acquisition gains up to a factor of 10,000. We show that hyperpolarization can be transferred between nuclei, allowing DNP-enhanced two-dimensional 13C-13C correlation experiments at 13C natural abundance. The enhanced sensitivity opens up perspectives for structural determination of natural products or characterization of drugs, available in small quantities. The results provide a starting point for a broader implementation of DNP in liquid-state NMR.

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.

Statistical analysis of ENDOR spectra.

Electron-nuclear double resonance (ENDOR) measures the hyperfine interaction of magnetic nuclei with paramagnetic centers and is hence a powerful tool for spectroscopic investigations extending from biophysics to material science. Progress in microwave technology and the recent availability of commercial electron paramagnetic resonance (EPR) spectrometers up to an electron Larmor frequency of 263 GHz now open the opportunity for a more quantitative spectral analysis. Using representative spectra of a prototype amino acid radical in a biologically relevant enzyme, the [Formula: see text] in Escherichia coli ribonucleotide reductase, we developed a statistical model for ENDOR data and conducted statistical inference on the spectra including uncertainty estimation and hypothesis testing. Our approach in conjunction with 1H/2H isotopic labeling of [Formula: see text] in the protein unambiguously established new unexpected spectral contributions. Density functional theory (DFT) calculations and ENDOR spectral simulations indicated that these features result from the beta-methylene hyperfine coupling and are caused by a distribution of molecular conformations, likely important for the biological function of this essential radical. The results demonstrate that model-based statistical analysis in combination with state-of-the-art spectroscopy accesses information hitherto beyond standard approaches.

Studies of transmembrane peptides by pulse dipolar spectroscopy with semi-rigid TOPP spin labels.

Electron paramagnetic resonance (EPR)-based pulsed dipolar spectroscopy measures the dipolar interaction between paramagnetic centers that are separated by distances in the range of about 1.5-10 nm. Its application to transmembrane (TM) peptides in combination with modern spin labelling techniques provides a valuable tool to study peptide-to-lipid interactions at a molecular level, which permits access to key parameters characterizing the structural adaptation of model peptides incorporated in natural membranes. In this mini-review, we summarize our approach for distance and orientation measurements in lipid environment using novel semi-rigid TOPP [4-(3,3,5,5-tetramethyl-2,6-dioxo-4-oxylpiperazin-1-yl)-L-phenylglycine] labels specifically designed for incorporation in TM peptides. TOPP labels can report single peak distance distributions with sub-angstrom resolution, thus offering new capabilities for a variety of TM peptide investigations, such as monitoring of various helix conformations or measuring of tilt angles in membranes.

Spin density localization and accessibility of organic radicals affect liquid-state DNP efficiency.

We report a large variation in liquid DNP performance of up to a factor of about five in coupling factor among organic radicals commonly used as polarizing agents. A comparative study of 1H and 13C DNP in model systems shows the impact of the spin density distribution and accessibility of the radical site by the target molecule.

1H high field electron-nuclear double resonance spectroscopy at 263 GHz/9.4 T.

We present and discuss the performance of 1H electron-nuclear double resonance (ENDOR) at 263 GHz/9.4 T by employing a prototype, commercial quasi optical spectrometer. Basic instrumental features of the setup are described alongside a comprehensive characterization of the new ENDOR probe head design. The performance of three different ENDOR pulse sequences (Davies, Mims and CP-ENDOR) is evaluated using the 1H BDPA radical. A key feature of 263 GHz spectroscopy - the increase in orientation selectivity in comparison with 94 GHz experiments - is discussed in detail. For this purpose, the resolution of 1H ENDOR spectra at 263 GHz is verified using a representative protein sample containing approximately 15 picomoles of a tyrosyl radical. Davies ENDOR spectra recorded at 5 K reveal previously obscured spectral features, which are interpreted by spectral simulations aided by DFT calculations. Our analysis shows that seven internal proton couplings are detectable for this specific radical if sufficient orientation selectivity is achieved. The results prove the fidelity of 263 GHz experiments in reporting orientation-selected 1H ENDOR spectra and demonstrate that new significant information can be uncovered in complex molecular systems, owing to the enhanced resolution combined with high absolute sensitivity and no compromise in acquisition time.

Dynamic Nuclear Polarization of 13 C Nuclei in the Liquid State over a 10†Tesla Field Range.

Nuclear magnetic resonance (NMR) techniques play an essential role in natural science and medicine. In spite of the tremendous utility associated with the small energies detected, the most severe limitation is the low signal-to-noise ratio. Dynamic nuclear polarization (DNP), a technique based on transfer of polarization from electron to nuclear spins, has emerged as a tool to enhance sensitivity of NMR. However, the approach in liquids still faces several challenges. Herein we report the observation of room-temperature, liquid DNP 13 C signal enhancements in organic small molecules as high as 600 at 9.4 Tesla and 800 at 1.2 Tesla. A mechanistic investigation of the 13 C-DNP field dependence shows that DNP efficiency is raised by proper choice of the polarizing agent (paramagnetic center) and by halogen atoms as mediators of scalar hyperfine interaction. Observation of sizable DNP of 13 CH2 and 13 CH3 groups in organic molecules at 9.4 T opens perspective for a broader application of this method.

Photo-induced radical polarization and liquid-state dynamic nuclear polarization using fullerene nitroxide derivatives.

We report on radical polarization and optically-driven liquid DNP using nitroxide radicals functionalized by photoexcitable fullerene derivatives. Pulse laser excitation of the fullerene moiety leads to transient nitroxide radical polarization that is one order of magnitude larger than that at the Boltzmann equilibrium. The life time of the radical polarization increases with the size of the fullerene derivative and is correlated with the electronic spin-lattice relaxation time T1e. Overhauser NMR signal enhancements of toluene solvent protons were observed under steady-state illumination, which replaced microwave irradiation.

High-resolution measurement of long-range distances in RNA: pulse EPR spectroscopy with TEMPO-labeled nucleotides.

Structural information at atomic resolution of biomolecular assemblies, such as RNA and RNA protein complexes, is fundamental to comprehend biological function. Modern spectroscopic methods offer exceptional opportunities in this direction. Here we present the capability of pulse EPR to report high-resolution long-range distances in RNAs by means of a recently developed spin labeled nucleotide, which carries the TEMPO group directly attached to the nucleobase and preserves Watson-Crick base-pairing. In a representative RNA duplex with spin-label separations up to 28 base pairs (≈8 nm) we demonstrate that the label allows for a model-free conversion of inter-spin distances into base-pair separation (Δbp) if broad-band pulse excitation at Q band frequencies (34 GHz) is applied. The observed distance distribution increases from ±0.2 nm for Δbp = 10 to only ±0.5 nm for Δbp = 28, consistent with only small deviations from the "ideal" A-form RNA structure. Molecular dynamics (MD) simulations conducted at 20 °C show restricted conformational freedom of the label. MD-generated structural deviations from an "ideal" A-RNA geometry help disentangle the contributions of local flexibility of the label and its neighboring nucleobases and global deformations of the RNA double helix to the experimental distance distributions. The study demonstrates that our simple but strategic spin labeling procedure can access detailed structural information on RNAs at atomic resolution over distances that match the size of macromolecular RNA complexes.

A high saturation factor in Overhauser DNP with nitroxide derivatives: the role of (14)N nuclear spin relaxation.

Overhauser DNP enhancements of toluene were measured at a magnetic field of 0.35 Tesla in a series of chemically functionalized nitroxide radicals. We observe that the enhancements increase systematically with polarizer size and rotational correlation time. Examination of the saturation factor of (14)N nitroxides by pulsed ELDOR spectroscopy led to a quantitative interpretation of the enhancements, for which the saturation factor increases up to almost unity due to enhanced nuclear ((14)N) relaxation in the nitroxide radical. The observation has a direct impact on the choice of optimum DNP polarizers in liquids.

High DNP efficiency of TEMPONE radicals in liquid toluene at low concentrations.

We show that at low concentrations (≤5 mM) TEMPONE radicals in liquid toluene exhibit higher DNP efficiency than in water. In spite of reduced coupling factors, the improved DNP performance in toluene results from favourable saturation and leakage factors, as determined by pulse electron-electron double resonance (ELDOR) and NMR relaxation, respectively. The extracted coupling factors at 0.35 Tesla support theoretical predictions of the Overhauser mechanism.

Orientation selection in distance measurements between nitroxide spin labels at 94 GHz EPR with variable dual frequency irradiation.

Pulsed electron-electron double resonance (PELDOR, also known as DEER) has become a method of choice to measure distances in biomolecules. In this work we show how the performance of the method can be improved at high EPR frequencies (94 GHz) using variable dual frequency irradiation in a dual mode cavity in order to obtain enhanced resolution toward orientation selection. Dipolar evolution traces of a representative RNA duplex and an α-helical peptide were analysed in terms of possible bi-radical structures by considering the inherent ambiguity of symmetry-related solutions.

W-band orientation selective DEER measurements on a Gd3+/nitroxide mixed-labeled protein dimer with a dual mode cavity.

Double electron-electron resonance (DEER) at W-band (95 GHz) was applied to measure the distance between a pair of nitroxide and Gd(3+) chelate spin labels, about 6 nm apart, in a homodimer of the protein ERp29. While high-field DEER measurements on systems with such mixed labels can be highly attractive in terms of sensitivity and the potential to access long distances, a major difficulty arises from the large frequency spacing (about 700 MHz) between the narrow, intense signal of the Gd(3+) central transition and the nitroxide signal. This is particularly problematic when using standard single-mode cavities. Here we show that a novel dual-mode cavity that matches this large frequency separation dramatically increases the sensitivity of DEER measurements, allowing evolution times as long as 12 µs in a protein. This opens the possibility of accessing distances of 8 nm and longer. In addition, orientation selection can be resolved and analyzed, thus providing additional structural information. In the case of W-band DEER on a Gd(3+)-nitroxide pair, only two angles and their distributions have to be determined, which is a much simpler problem to solve than the five angles and their distributions associated with two nitroxide spin labels.

A dual-mode microwave resonator for double electron-electron spin resonance spectroscopy at W-band microwave frequencies.

We present a dual-mode resonator operating at/near 94 GHz (W-band) microwave frequencies and supporting two microwave modes with the same field polarization at the sample position. Numerical analysis shows that the frequencies of both modes as well as their frequency separation can be tuned in a broad range up to GHz. The resonator was constructed to perform pulsed ELDOR experiments with a variable separation of "pump" and "detection" frequencies up to Δν=350 MHz. To examine its performance, test ESE/PELDOR experiments were performed on a representative biradical system.

Optimization of dynamic nuclear polarization experiments in aqueous solution at 15 MHz/9.7 GHz: a comparative study with DNP at 140 MHz/94 GHz.

Dynamic nuclear polarization is emerging as a potential tool to increase the sensitivity of NMR aiming at the detection of macromolecules in liquid solution. One possibility for such an experimental design is to perform the polarization step between electrons and nuclei at low magnetic fields and then transfer the sample to a higher field for NMR detection. In this case, an independent optimization of the polarizer and detection set ups is required. In the present paper we describe the optimization of a polarizer set up at 15 MHz (1)H NMR/9.7 GHz EPR frequencies based on commercial hardware. The sample consists of the nitroxide radical TEMPONE-D,(15)N in water, for which the dimensions were systematically decreased to fit the homogeneous B(1) region of a dielectric ENDOR resonator. With an available B(1) microwave field up to 13 G we observe a maximum DNP enhancement of -170 at room temperature by irradiating on either one of the EPR lines. The DNP enhancement was saturated at all polarizer concentrations. Pulsed ELDOR experiments revealed that the saturation level of the two hyperfine lines is such that the DNP enhancements are well consistent with the coupling factors derived from NMRD data. By raising the polarizing field and frequencies 10-fold, i.e. to 140 MHz (1)H/94 GHz EPR, we reach an enhancement of -43 at microwave field strengths (B(1) approximately 5 G). The results are discussed in view of an application for a DNP spectrometer.

(1)H and (13)C dynamic nuclear polarization in aqueous solution with a two-field (0.35 T/14 T) shuttle DNP spectrometer.

Dynamic nuclear polarization (DNP) permits increasing the NMR signal of nuclei by pumping the electronic spin transitions of paramagnetic centers nearby. This method is emerging as a powerful tool to increase the inherent sensitivity of NMR in structural biology aiming at detection of macromolecules. In aqueous solution, additional technical issues associated with the penetration of microwaves in water and heating effects aggravate the performance of the experiment. To examine the feasibility of low-field (9.7 GHz/0.35 T) DNP in high resolution NMR, we have constructed the prototype of a two-field shuttle DNP spectrometer that polarizes nuclei at 9.7 GHz/0.35 T and detects the NMR spectrum at 14 T. We report our first (1)H and (13)C DNP enhancements with this spectrometer. Effective enhancements up to 15 were observed for small molecules at (1)H 600 MHz/14 T as compared to the Boltzmann signal. The results provide a proof of principle for the feasibility of a shuttle DNP experiment and open up perspectives for the application potential of this method in solution NMR.