Mechanisms of dynamic nuclear polarization in insulating solids

Dynamic nuclear polarization (DNP) is a technique used to enhance signal intensities in NMR experiments by transferring the high polarization of electrons to their surrounding nuclei. The past decade has witnessed a renaissance in the development of DNP, especially at high magnetic fields, and its application in several areas including biophysics, chemistry, structural biology and materials science. Recent technical and theoretical advances have expanded our understanding of established experiments: for example, the cross effect DNP in samples spinning at the magic angle. Furthermore, new experiments suggest that our understanding of the Overhauser effect and its applicability to insulating solids needs to be re-examined. In this article, we summarize important results of the past few years and provide quantum mechanical explanations underlying these results. We also discuss future directions of DNP and current limitations, including the problem of resolution in protein spectra recorded at 80–100 K.National Institute for Biomedical Imaging and Bioengineering (U.S.) (EB-002804)National Institute for Biomedical Imaging and Bioengineering (U.S.) (EB-001960)National Institute for Biomedical Imaging and Bioengineering (U.S.) (EB-003151)National Institute for Biomedical Imaging and Bioengineering (U.S.) (EB-002026

Time domain DNP with the NOVEL sequence

We present results of a pulsed dynamic nuclear polarization (DNP) study at 0.35 T (9.7 GHz/14.7 MHz for electron/1H Larmor frequency) using a lab frame-rotating frame cross polarization experiment that employs electron spin locking fields that match the 1H nuclear Larmor frequency, the so called NOVEL (nuclear orientation via electron spin locking) condition. We apply the method to a series of DNP samples including a single crystal of diphenyl nitroxide (DPNO) doped benzophenone (BzP), 1,3-bisdiphenylene-2-phenylallyl (BDPA) doped polystyrene (PS), and sulfonated-BDPA (SA-BDPA) doped glycerol/water glassy matrices. The optimal Hartman-Hahn matching condition is achieved when the nutation frequency of the electron matches the Larmor frequency of the proton, ω[subscript 1S] = ω[subscript 0I], together with possible higher order matching conditions at lower efficiencies. The magnetization transfer from electron to protons occurs on the time scale of ∼100 ns, consistent with the electron-proton couplings on the order of 1-10 MHz in these samples. In a fully protonated single crystal DPNO/BzP, at 270 K, we obtained a maximum signal enhancement of ε = 165 and the corresponding gain in sensitivity of ε(T[subscript 1]/T[subscript B])[superscript 1/2] = 230 due to the reduction in the buildup time under DNP. In a sample of partially deuterated PS doped with BDPA, we obtained an enhancement of 323 which is a factor of ∼3.2 higher compared to the protonated version of the same sample and accounts for 49% of the theoretical limit. For the SA-BDPA doped glycerol/water glassy matrix at 80 K, the sample condition used in most applications of DNP in nuclear magnetic resonance, we also observed a significant enhancement. Our findings demonstrate that pulsed DNP via the NOVEL sequence is highly efficient and can potentially surpass continuous wave DNP mechanisms such as the solid effect and cross effect which scale unfavorably with increasing magnetic field. Furthermore, pulsed DNP is also a promising avenue for DNP at high temperature.National Institute for Biomedical Imaging and Bioengineering (U.S.) (Grant No. EB-002804)National Institute for Biomedical Imaging and Bioengineering (U.S.) (Grant No. EB-002026)National Institute of General Medical Sciences (U.S.) (Grant No. GM095843

Frequency-Swept Integrated Solid Effect

The efficiency of continuous wave dynamic nuclear polarization (DNP) experiments decreases at the high magnetic fields used in contemporary high-resolution NMR applications. To recover the expected signal enhancements from DNP, we explored time domain experiments such as NOVEL which matches the electron Rabi frequency to the nuclear Larmor frequency to mediate polarization transfer. However, satisfying this matching condition at high frequencies is technically demanding. As an alternative we report here frequency-swept integrated solid effect (FS-ISE) experiments that allow low power sweeps of the exciting microwave frequencies to constructively integrate the negative and positive polarizations of the solid effect, thereby producing a polarization efficiency comparable to (±10 % difference) NOVEL. Finally, the microwave frequency modulation results in field profiles that exhibit new features that we coin the “stretched” solid effect.National Institute of Biomedical Imaging and Bioengineering (U.S.) (Grant EB-002804)National Institute of Biomedical Imaging and Bioengineering (U.S.) (Grant EB-002026)National Institute of General Medical Sciences (U.S.) (Grant GM095843

ベトナム国南部における稲遺伝資源の探索収集

Collaborative exploration between Japan and Vietnam was conducted in the Mekong Delta area of Southern Vietnam from November 17 to December 16, 1998. A total of 124 landraces and 12 wild relatives, including Oryza rufipogon and O. officinalis were collected. Land races which are adapted to deep water conditions, acid sulfurate soils, aluminum soil, or salinity were found in various locations. Some land races were grown for their high quality as aromatic rice and glutinous rice. Twelve accessions of wild relatives of rice, which include Oryza rufipogon and O. officinalis, were also collected near former fields. Oryza officinalis was collected in Ca Mau province, which is the first accession of that species from that province

New methods for dynamic nuclear polarization in insulating solids : the Overhauser effect and time domain techniques

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Chemistry, 2017.Cataloged from PDF version of thesis.Includes bibliographical references.Dynamic nuclear polarization (DNP) is now established as a powerful technique for improving the sensitivity of NMR signals by several orders of magnitude, enabling otherwise impossible experiments. Unfortunately, the enhancements obtained at high magnetic fields (> 9 T) are only a small fraction of the theoretical limit due to the fact that current DNP mechanisms, including the cross effect and solid effect, utilize continuous wave (CW) microwave irradiation, and scale unfavorably with B0. This has motivated us to develop new DNP methods that do not suffer from the same field dependences. Our first attempt resulted in the observation of the Overhauser effect in insulating solids doped with 1,3-bisdiphenylene-2-phenylallyl (BDPA) or sulfonated-BDPA (SA-BDPA) radical. As opposed to all other CW DNP mechanisms, the enhancement of the OE in insulating solids scales favorably with B0, increasing in magnitude in going from 5 T, to 9.4 T, to 14.1 T, and to 18.8 T. This finding sheds a new light on the seemingly well-understood Overhauser effect. Our second approach is to perform time domain or pulsed DNP, which differs fundamentally from CW DNP, and like CP and INEPT transfers, is in principle independent of B0. In particular, we have investigated the performance of two related pulse sequences including the nuclear orientation via electron spin locking (NOVEL) and integrated solid effect (ISE) at magnetic fields ranging from 0.35 T to 3.35 T. The NOVEL pulse sequence relies on a matching condition between the nuclear Larmor frequency and the electron Rabi frequency, resulting in a fast polarization transfer from electron to protons (hundreds of ns time scale). Furthermore, we showed that adding amplitude modulation to the microwave field, analogous to a ramped CP experiment, led to longer mixing time (ps time scale) but improved the enhancement by a factor of 1.4 to 2. Finally, we implemented a new version of the integrated solid effect (ISE) by modulating the microwave frequency instead of sweeping the B0 which is technically challenging in high field superconducting magnets. In comparison to NOVEL, ISE gives similar DNP enhancement even far below the NOVEL condition. Our study sets the foundation for further development of time domain DNP at high fields.by Thach V. Can.Ph. D

Adiabatic Solid Effect

The solid effect (SE) is a two spin dynamic nuclear polarization (DNP) mechanism that enhances the sensitivity in NMR experiments by irradiation of the electron-nuclear spin transitions with continuous wave (CW) microwaves at ω0S ± ω0I, where ω0S and ω0I are electron and nuclear Larmor frequencies, respectively. Using trityl (OX063), dispersed in a 60/40 glycerol/water mixture at 80 K, as a polarizing agent, we show here that application of a chirped microwave pulse, with a bandwidth comparable to the EPR line width applied at the SE matching condition, improves the enhancement by a factor of 2.4 over the CW method. Furthermore, the chirped pulse yields an enhancement that is ∼20% larger than obtained with the ramped-amplitude NOVEL (RA-NOVEL), which to date has achieved the largest enhancements in time domain DNP experiments. Numerical simulations suggest that the spins follow an adiabatic trajectory during the polarization transfer; hence, we denote this sequence as an adiabatic solid effect (ASE). We foresee that ASE will be a practical pulsed DNP experiment to be implemented at higher static magnetic fields due to the moderate power requirement. In particular, the ASE uses only 13% of the maximum microwave power required for RA-NOVEL

Ramped-amplitude NOVEL

We present a pulsed dynamic nuclear polarization (DNP) study using a ramped-amplitude nuclear orientation via electron spin locking (RA-NOVEL) sequence that utilizes a fast arbitrary waveform generator (AWG) to modulate the microwave pulses together with samples doped with narrow-line radicals such as 1,3-bisdiphenylene-2-phenylallyl (BDPA), sulfonated-BDPA (SA-BDPA), and trityl-OX063. Similar to ramped-amplitude cross polarization in solid-state nuclear magnetic resonance, RA-NOVEL improves the DNP efficiency by a factor of up to 1.6 compared to constant-amplitude NOVEL (CA-NOVEL) but requires a longer mixing time. For example, at τ mix = 8 μs, the DNP efficiency reaches a plateau at a ramp amplitude of ∼20 MHz for both SA-BDPA and trityl-OX063, regardless of the ramp profile (linear vs. tangent). At shorter mixing times (τ mix = 0.8 μs), we found that the tangent ramp is superior to its linear counterpart and in both cases there exists an optimum ramp size and therefore ramp rate. Our results suggest that RA-NOVEL should be used instead of CA-NOVEL as long as the electronic spin latti ce relaxation T 1e is sufficiently long and/or the duty cycle of the microwave amplifier is not exceeded. To the best of our knowledge, this is the first example of a time domain DNP experiment that utilizes modulated microwave pulses. Our results also suggest that a precise modulation of the microwave pulses can play an important role in optimizing the efficiency of pulsed DNP experiments and an AWG is an elegant instrumental solution for this purpose.National Institute of Biomedical Imaging and Bioengineering (U.S.) (Grant EB-002804)National Institute of Biomedical Imaging and Bioengineering (U.S.) (Grant EB-002026)National Institute of General Medical Sciences (U.S.) (Grant GM-095843

Reverse Micelles As a Platform for Dynamic Nuclear Polarization in Solution NMR of Proteins

Despite tremendous advances in recent years, solution NMR remains fundamentally restricted due to its inherent insensitivity. Dynamic nuclear polarization (DNP) potentially offers significant improvements in this respect. The basic DNP strategy is to irradiate the EPR transitions of a stable radical and transfer this nonequilibrium polarization to the hydrogen spins of water, which will in turn transfer polarization to the hydrogens of the macromolecule. Unfortunately, these EPR transitions lie in the microwave range of the electromagnetic spectrum where bulk water absorbs strongly, often resulting in catastrophic heating. Furthermore, the residence times of water on the surface of the protein in bulk solution are generally too short for efficient transfer of polarization. Here we take advantage of the properties of solutions of encapsulated proteins dissolved in low viscosity solvents to implement DNP in liquids. Such samples are largely transparent to the microwave frequencies required and thereby avoid significant heating. Nitroxide radicals are introduced into the reverse micelle system in three ways: attached to the protein, embedded in the reverse micelle shell, and free in the aqueous core. Significant enhancements of the water resonance ranging up to ∼−93 at 0.35 T were observed. We also find that the hydration properties of encapsulated proteins allow for efficient polarization transfer from water to the protein. These and other observations suggest that merging reverse micelle encapsulation technology with DNP offers a route to a significant increase in the sensitivity of solution NMR spectroscopy of proteins and other biomolecules.National Institutes of Health (U.S.) (Grant P41 EB002026)National Institutes of Health (U.S.) (Grant P41 EB002804)Netherlands Organization for Scientific Research (Rubicon Fellowship

Primary Transfer Step in the Light-Driven Ion Pump Bacteriorhodopsin: An Irreversible U‑Turn Revealed by Dynamic Nuclear Polarization-Enhanced Magic Angle Spinning NMR

Despite much attention, the path of the highly consequential primary proton transfer in the light-driven ion pump bacteriorhodopsin (bR) remains mysterious. Here we use DNP-enhanced magic angle spinning (MAS) NMR to study critical elements of the active site just before the Schiff base (SB) deprotonates (in the L intermediate), immediately after the SB has deprotonated and Asp85 has become protonated (in the M_o intermediate), and just after the SB has reprotonated and Asp96 has deprotonated (in the N intermediate). An essential feature that made these experiments possible is the 75-fold signal enhancement through DNP. ¹⁵N­(SB)–¹H correlations reveal that the newly deprotonated SB is accepting a hydrogen bond from an alcohol and ¹³C–¹³C correlations show that Asp85 draws close to Thr89 before the primary proton transfer. Concurrently, ¹⁵N–¹³C correlations between the SB and Asp85 show that helices C and G draw closer together just prior to the proton transfer and relax thereafter. Together, these results indicate that Thr89 serves to relay the SB proton to Asp85 and that creating this pathway involves rapprochement between the C and G helices as well as chromophore torsion

Gd(III) and Mn(II) complexes for dynamic nuclear polarization : small molecular chelate polarizing agents and applications with site-directed spin labeling of proteins

We investigate complexes of two paramagnetic metal ions Gd3+ and Mn2+ to serve as polarizing agents for solid-state dynamic nuclear polarization (DNP) of 1H, 13C, and 15N at magnetic fields of 5, 9.4, and 14.1 T. Both ions are half-integer high-spin systems with a zero-field splitting and therefore exhibit a broadening of the mS = −1/2 ↔ +1/2 central transition which scales inversely with the external field strength. We investigate experimentally the influence of the chelator molecule, strong hyperfine coupling to the metal nucleus, and deuteration of the bulk matrix on DNP properties. At small Gd-DOTA concentrations the narrow central transition allows us to polarize nuclei with small gyromagnetic ratio such as 13C and even 15N via the solid effect. We demonstrate that enhancements observed are limited by the available microwave power and that large enhancement factors of >100 (for 1H) and on the order of 1000 (for 13C) can be achieved in the saturation limit even at 80 K. At larger Gd(III) concentrations (≥10 mM) where dipolar couplings between two neighboring Gd3+ complexes become substantial a transition towards cross effect as dominating DNP mechanism is observed. Furthermore, the slow spin-diffusion between 13C and 15N, respectively, allows for temporally resolved observation of enhanced polarization spreading from nuclei close to the paramagnetic ion towards nuclei further removed. Subsequently, we present preliminary DNP experiments on ubiquitin by site-directed spin-labeling with Gd3+ chelator tags. The results hold promise towards applications of such paramagnetically labeled proteins for DNP applications in biophysical chemistry and/or structural biology