High-Field Dynamic Nuclear Polarization for Solid and Solution Biological NMR

Abstract

Dynamic nuclear polarization (DNP) results in a substantial nuclear polarization enhancement through a transfer of the magnetization from electrons to nuclei. Recent years have seen considerable progress in the development of DNP experiments directed towards enhancing sensitivity in biological nuclear magnetic resonance (NMR). This review covers the applications, hardware, polarizing agents, and theoretical descriptions that were developed at the Francis Bitter Magnet Laboratory at Massachusetts Institute of Technology for high-field DNP experiments. In frozen dielectrics, the enhanced nuclear polarization developed in the vicinity of the polarizing agent can be efficiently dispersed to the bulk of the sample via (1)H spin diffusion. This strategy has been proven effective in polarizing biologically interesting systems, such as nanocrystalline peptides and membrane proteins, without leading to paramagnetic broadening of the NMR signals. Gyrotrons have been used as a source of high-power (5-10 W) microwaves up to 460 GHz as required for the DNP experiments. Other hardware has also been developed allowing in situ microwave irradiation integrated with cryogenic magic-angle-spinning solid-state NMR. Advances in the quantum mechanical treatment are successful in describing the mechanism by which new biradical polarizing agents yield larger enhancements at higher magnetic fields. Finally, pulsed methods and solution experiments should play a prominent role in the future of DNP.

Fig. 1

Spin diffusion spectrum of U-¹³C,¹⁵N-proline in 40 mM TEMPO and glycerol/water. The DNP enhancement is 9 at 5 T and a temperature of 90 K. Reproduced with permission from ref. . (Copyright 2002 American Chemical Society).

Fig. 2

DNP experiments on [20% U-¹³C,¹⁵N-GNNQ]QNY nanocrystals in d₈-glycerol/D₂O/H₂O (60/30/10) with 10 mM TOTAPOL. a Illustration of heterogeneously mixed DNP sample of crystalline peptide and the DNP solvent matrix, based on the transmission electron microscopy data on GNNQQNY nanocrystals. Arrows illustrate the diffusion of the enhanced polarization from the matrix into the crystals, b Differential polarization enhancement buildup of peptide and matrix carbon signals. The signal intensities are normalized to the equilibrium off signal, c DNP-enhanced 2-D ¹³C-¹³C dipolar-assisted rotary resonance or radiofrequency-assisted diffusion correlation spectrum of the same sample. Adapted from and reproduced with permission ref. . (Copyright 2006 American Chemical Society).

Fig. 3

a ¹⁵N MAS spectra of light-adapted ζ-¹⁵N-Lys-bR. Top: Spectrum acquired on a 317 MHz spectrometer using a 5 mm ZrO₂ rotor with a 160 ml sample volume, 10000 scans, 3.5 days (about 5000 min) of data acquisition, T = 200 K. Bottom: Spectrum acquired with DNP-250 GHz mw irradiation using a 4 mm sapphire rotor, 40 ml sample volume, T = 90 K, 384 scans, 30 min of data acquisition. The resonances from left to right are: protonated Schiff base ¹⁵N at 165 ppm, natural-abundance amide backbone at 130 ppm, natural-abundance guanidine-HCl at 80 ppm (only in the 380 MHz spectrum), six free ζ-¹⁵N-Lys signals at 50 ppm. ω_r/2π = 7 kHz. b 2-D nitrogen carbon correlation spectrum of dark-adapted bR recorded with 250 GHz DNP, showing heterogeneity which cannot be resolved in the 1-D nitrogen spectrum. Reprinted from ref. . (Copyright 2007, with permission of Elsevier).

Fig. 4

Drawings of a cryogenic SSNMR DNP probe, a 1, probehead; 2, cut-out of the vacuum dewar; 3, tuning elements of the RF circuit located in the box; 4, corrugated waveguide from gyrotron; 5, concave and flat mirrors to direct microwaves into the vertical waveguide; 6, vacuum-jacketed transfer lines for the bearing and drive cryogens. b Probehead for rotors with a diameter of 4 mm. 1, stator housing; 2, sample rotor within RF coil (at the magic angle); 3, metal mirror miter; 4, the inner conductor of the coaxial RF transmission line is corrugated on the inside and serves as an over-moded waveguide; 5, outer conductor of the RF coaxial line is stainless steel for thermal isolation but is coated with silver and gold for good electrical performance.

Fig. 5

a Cross-sectional schematic of the cylindrically symmetric 460 GHz gyrotron tube, not shown to scale, indicating key components. Adapted from ref. . (Reproduced by permission of IEEE), b 250 GHz line layout for DNP experiments. Adapted from ref. . (Reproduced by permission of IEEE).

Fig. 6

Quantum mechanical diagrams of the electron-nuclear transitions (dashed arrows) in the SE (a), CE (b) and TM mechanisms (c), which involve single, paired and multiple electron spins, respectively. Note that the probabilities of electron-nuclear transitions are always small in the SE but could be large in the CE and TM, especially when there is degeneracy between the states with alternating nuclear spin quantum numbers. Adapted from ref. .

Fig. 7

a Illustration of the EPR spectrum of monomeric TEMPO nitroxide at 5 T. Note that the breadth of the spectrum is about 600 MHz and is large compared to that of the ¹H Larmor frequency (211 MHz). Arrows indicate the approximate frequencies of two electron spins e₁ and e₂, separated by ωI/2π, expected to participate in the CE/TM DNP enhancement process, b Illustration of the mw-driven three-spin process associated with TM or CE DNP, where two coupled electrons undergo an energy-conserving flip-flop process that leads to the enhanced nuclear spin polarization of nucleus n. c The molecular structure of the BTnE biradicals, where m is the number of ethylene glycol units that tether two nitroxide radicals (TEMPO). The dots represent the two unpaired electrons, whose displacement is approximated as the oxygen-oxygen distance, R_O-O. Reproduced with permission from ref. . (Copyright 2004 American Chemical Society).

Fig. 8

Histogram of DNP enhancements (with error bars) in 4 mm (white) and 2.5 mm (black) rotors with TOTAPOL biradical, a series of BTnE biradicals, and monomeric TEMPO. The data illustrate that TOTAPOL yields the largest enhancement, especially when the mw penetration depth is optimal for 2.5 mm rotors. Reproduced with permission from ref. . (Copyright 2006 American Chemical Society).

Fig. 9

a Illustration of the growth of the nuclear polarization as a result of mw irradiation using the biradical TOTAPOL (b) as a polarizing agent. Integration of the spectral intensities with and without irradiation yields a ¹H enhancement of ε ~ 290 measured indirectly through the ¹³C CP signal using the pulse sequence shown in panel c. The measurements were performed on a sample of 3 mM TOTAPOL and 2 M ¹³C-urea in d₆-DMSO/D₂O/H₂O (60:34:6 w/w/w) at 90 K, 5 T, and ω_r/2π = 7 kHz MAS. The time constant associated with the growth is about 9 s, approximately the nuclear T₁ of the sample. Reproduced with permission from ref. . (Copyright 2006 American Chemical Society).

Fig. 10

a Pulse sequence used in the TJ-DNP experiment. The samples are irradiated with 140 GHz microwaves at 90 K, polarizing the ¹H spins in the sample. Enhanced ¹H polarization is then transferred to ¹³C via CP. During the heating period using a 10.6 mm CO₂ laser, the ¹³C magnetization is stored along the z-axis of the rotating frame. The ¹³C spectrum is detected following a 90° pulse in the presence of WALTZ ¹H decoupling, b Experimental spectra obtained for U-¹³C-urea. Left, solid-state MAS spectrum produced after 40 s DNP time at 90 K (ε ~ 290); right, liquid-state spectrum with an enhancement ε^† ~ 400, after a 1.2 s melting period. Note that the DNP spectrum was acquired in a single scan, whereas the room-temperature spectrum required 256 scans, c ¹³C TJ-DNP NMR spectra of Na[1,2-¹³C₂,²H₃]-acetate in 60% ²H₈-glycerol and 40% water (80% ²H₂O/20% H₂O) after 40 s polarization and 1 s melting, d ¹³C TJ-DNP NMR spectra of [U-¹³C₆,²H₇]-glucose in H₂O after 30 s polarization time and 1.5 s melting period. Samples contained 3–5 mM TOTAPOL biradical polarizing agent, corresponding to 6–10 mM electrons. The TJ-DNP spectra (the top traces in each panel) were recorded with a single scan, while the room-temperature spectra were recorded with 128 (c) and 512 scans (d), respectively. Reproduced with permission from ref. . (Copyright 2006 American Chemical Society).

Fig. 11

Energy level diagram comparing various polarization transfer schemes, a CP, b laboratory frame solid effect (LFSE), c NRF-SE, d DSSE. The polarization transfer is achieved when either allowed or forbidden transitions are irradiated (dashed lines). The highlighted terms represent interactions that mix eigenstates and allow RF/mw irradiations to drive the polarization transfer.

Fig. 12

a NRF-DNP pulse sequence. The NRF-DNP enhancements are determined from a comparison with the laboratory frame signal obtained with a solid-echo pulse sequence and no mw irradiation or nuclear spin-lock, b Pulse sequence for the indirect detection of electron-nuclear CP. The electron spin echo intensity is monitored after an electron spin-lock and a refocusing π-pulse. The echo intensities of two sequences with and without RF pulses are subtracted and recorded as a function of the RF frequency, ω_RF. Reprinted from refs. and . (Copyright 2000 and 2006, with permission of Elsevier).

Fig. 13

DSSE/e-NCP experiment on perdeuterated BDPA for various settings of the mw (ω_1S/2π ~ 1.75, 0.9 and 0.5 MHz) and RF field strengths (ω_1I/2π ~ 100 kHz at 350 W). The CP contact time was set to 3 µs. Reprinted from refs. and . (Copyright 2006, with permission of Elsevier).