The field of dynamic nuclear polarization has undergone tremendous developments and diversification since its inception more than 6 decades ago. In this review we provide an in-depth overview of the relevant topics involved in DNP-enhanced MAS NMR spectroscopy. This includes the theoretical description of DNP mechanisms as well as of the polarization transfer pathways that can lead to a uniform or selective spreading of polarization between nuclear spins. Furthermore, we cover historical and state-of-the art aspects of dedicated instrumentation, polarizing agents, and optimization techniques for efficient MAS DNP. Finally, we present an extensive overview on applications in the fields of structural biology and materials science, which underlines that MAS DNP has moved far beyond the proof-of-concept stage and has become an important tool for research in these fields.

We report magic angle spinning, dynamic nuclear polarization (DNP) experiments at magnetic fields of 9.4 T, 14.1 T, and 18.8 T using the narrow line polarizing agents 1,3-bisdiphenylene-2-phenylallyl (BDPA) dispersed in polystyrene, and sulfonated-BDPA (SA-BDPA) and trityl OX063 in glassy glycerol/water matrices. The 1 H DNP enhancement field profiles of the BDPA radicals exhibit a significant DNP Overhauser effect (OE) as well as a solid effect (SE) despite the fact that these samples are insulating solids. In contrast, trityl exhibits only a SE enhancement. Data suggest that the appearance of the OE is due to rather strong electron-nuclear hyperfine couplings present in BDPA and SA-BDPA, which are absent in trityl and perdeuterated BDPA (d 21 -BDPA). In addition, and in contrast to other DNP mechanisms such as the solid effect or cross effect, the experimental data suggest that the OE in non-conducting solids scales favorably with magnetic field, increasing in magnitude in going from 5 T, to 9.4 T, to 14.1 T, and to 18.8 T. Simulations using a model two spin system consisting of an electron hyperfine coupled to a 1 H reproduce the essential features of the field profiles and indicate that the OE in these samples originates from the zero and double quantum cross relaxation induced by fluctuating hyperfine interactions between the intramolecular delocalized unpaired electrons and their neighboring nuclei, and that the size of these hyperfine couplings is crucial to the magnitude of the enhancements. Microwave power dependent studies show that the OE saturates at considerably lower power levels than the solid effect in the same samples. Our results provide new insights into the mechanism of the Overhauser effect, and also provide a new approach to perform DNP experiments in chemical, biophysical, and physical systems at high magnetic fields. INTRODUCTIONThe last decade has witnessed a renaissance in the use of high frequency dynamic nuclear polarization (DNP) to enhance sensitivity in nuclear magnetic resonance (NMR) experiments. In particular, the development of gyrotron and other high frequency microwave sources permits DNP to be performed at magnetic fields used in contemporary NMR experiments (5-20 T). 1-8 To date these experiments, which have focused mostly on insulating solids formed from glassy, frozen solutions of proteins and other nonconducting materials, have relied primarily on narrow line monoradicals and the solid effect (SE) 1, 9-11 or nitroxide biradicals and the cross effect (CE) [12][13][14][15][16][17][18] to mediate the polarization process. These approaches have resulted in large signal enhancements and have enabled many experiments that would otherwise be impossible. [19][20][21][22][23] by Overhauser 25 and confirmed by Carver and Slichter, 26 has not been identified or utilized during the course of this renaissance. Although the possibility of an OE in insulator was discussed by Abragam, 27 the conventional wisdom is that Overhauser DNP is important only in systems with mobile electrons ...

Escherichia coli ribonucleotide reductase is an α2β2 complex that catalyzes the conversion of nucleotides to deoxynucleotides using a diferric-tyrosyl radical (Y122•) cofactor in β2 to initiate catalysis in α2. Each turnover requires reversible long-range proton-coupled electron transfer (PCET) over 35 Å between the two subunits by a specific pathway (Y122• ⇆ [W48?] ⇆ Y356 within β to Y731 ⇆ Y730 ⇆ C439 within α). Previously, we reported that a β2 mutant with 3-nitrotyrosyl radical (NO2Y•, 1.2 radicals/β2) in place of Y122• in the presence of α2, CDP and ATP catalyzes formation of 0.6 equiv dCDP and accumulates 0.6 equiv of a new Y• proposed to be located on Y356 in β2. We now report three independent methods that establish that Y356 is the predominant location (85 – 90%) of the radical with the remaining 10 – 15% delocalized onto Y731 and Y730 in α2. Pulsed electron-electron double resonance spectroscopy on samples prepared by rapid freeze quench (RFQ) methods identified three distances: 30 ± 0.4 Å (88 ± 3%), 33 ± 0.4 Å and 38 ± 0.5 Å (12 ± 3%) indicative of NO2Y122•-Y356•, NO2Y122•- NO2Y122•, and NO2Y122•-Y731(730)•, respectively. Radical distribution in α2 was supported by RFQ EPR studies using Y731(3,5-F2Y) or Y730(3,5-F2Y)-α2 which revealed F2Y•, and by studies using globally incorporated [β-2H2]Y-α2 and analysis using parameters obtained from 140 GHz EPR spectroscopy. The amount of Y• delocalized in α2 from these two studies varied from 6 to 15%. The studies together give the first insight into the relative redox potentials of the three transient Y•s in the PCET pathway and their conformations.

Biological processes occur in complex environments containing a myriad of potential interactors. Unfortunately, limitations on the sensitivity of biophysical techniques normally restrict structural investigations to purified systems, at concentrations that are orders of magnitude above endogenous levels. Dynamic nuclear polarization (DNP) can dramatically enhance the sensitivity of NMR spectroscopy and enable structural studies in biologically complex environments. Here we applied DNP NMR to investigate the structure of a protein containing both an environmentally sensitive folding pathway and an instrinsically disordered region, the yeast prion protein Sup35. We added an exogenously-prepared isotopically-labeled protein to deuterated lysates, rendering the biological environment “invisible” and enabling highly efficient polarization transfer for DNP. In this environment, structural changes occurred in a region known to influence biological activity but intrinsically disordered in purified samples. Thus, DNP makes structural studies of proteins at endogenous levels in biological contexts possible and such contexts can influence protein structure.

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.

The effects of nuclear signal quenching induced by the presence of a paramagnetic polarizing agent are documented for conditions used in magic angle spinning (MAS)-dynamic nuclear polarization (DNP) experiments on homogeneous solutions. In particular, we present a detailed analysis of three time constants: (1) the longitudinal build-up time constant TB for 1H; (2) the rotating frame relaxation time constant T1ρ for 1H and 13C and (3) T2 of 13C, the transverse relaxation time constant in the laboratory frame. These relaxation times were measured during microwave irradiation at a magnetic field of 5 T (140 GHz) as a function of the concentration of four polarizing agents: TOTAPOL, 4-amino-TEMPO, trityl (OX063), and Gd-DOTA and are compared to those obtained for a sample lacking paramagnetic doping. We also report the EPR relaxation time constants T1S and T2S, the DNP enhancements, ε, and the parameter E, defined below, which measures the sensitivity enhancement for the four polarizing agents as a function of the electron concentration. We observe substantial intensity losses (paramagnetic quenching) with all of the polarizing agents due to broadening mechanisms and cross relaxation during MAS. In particular, the monoradical trityl and biradical TOTAPOL induce ~40 and 50% loss of signal intensity. In contrast there is little suppression of signal intensity in static samples containing these paramagnetic species. Despite the losses due to quenching, we find that all of the polarizing agents provide substantial gains in signal intensity, and in particular that the net enhancement is optimal for biradicals that operate with the cross effect. We discuss the possibility that much of this polarization loss can be regained with the development of instrumentation and methods to perform electron decoupling.

We report the dynamic nuclear polarization of 1H spins in magic angle spinning spectra recorded at 5 T and 84K via the solid effect using Mn2+ and Gd3+ complexes as polarizing agents. We show that the magnitude of the enhancements can be directly related to the effective linewidth of the central (MS = −1/2 →+1/2) EPR transition. Using a Gd3+ complex with a narrow central transition EPR linewidth of 29 MHz, a maximum enhancement of ~13 is observed, which is comparable to previous results on the narrow linewidth trityl radical.

The synthesis of air-stable highly water-soluble organic radicals containing a 1,3-bisdiphenylene-2-phenylallyl (BDPA) core is reported. A sulfonated derivative, SA-BDPA, retains the narrow EPR linewidth (<30 MHz at 5 T) of the parent BDPA in highly concentrated glycerol/water solutions (40 mM), which enables its use as polarizing agent for solid effect dynamic nuclear polarization (SE DNP). Sensitivity enhancement of 110 was obtained in high field magic-angle-spinning nuclear magnetic resonance (MAS NMR) experiments. The ease of synthesis and high maximum enhancements obtained with the BDPA-based radicals constitute a major advance over the trityl-type narrow line polarization agents.