Strategies for 1H‐Detected Dynamic Nuclear Polarization Magic‐Angle Spinning NMR Spectroscopy

Abstract Combining dynamic nuclear polarization with proton detection significantly enhances the sensitivity of magic‐angle spinning NMR spectroscopy. Herein, the feasibility of proton‐detected experiments with slow (10 kHz) magic angle spinning was demonstrated. The improvement in sensitivity permits the acquisition of indirectly detected 14N NMR spectra allowing biomolecular structures to be characterized without recourse to isotope labelling. This provides a new tool for the structural characterization of environmental and medical samples, in which isotope labelling is frequently intractable.


Sample Preparation
b2-microglobulin was expressed and purified from inclusion bodies (IBs) in E.coli. Briefly, E.coli BL21 (DE3) were transformed with a pET11 a vector containing the coding sequence for the b 2-microglobulin gene. For unlabeled samples the E.coli were grown on Luria Broth (LB) (Sigma-Aldrich) supplemented with 100 µg/ml ampicillin at 37°C with 200 rpm shaking until an OD600 of 0.6-0.8 was reached. Expression was induced through the addition of IPTG to a final concentration of 1 mM and grown for a further 4 hours prior to harvesting by centrifugation at 3,000 rpm. For samples enriched in 15 N the overnight culture was used to inoculate 500 mL of M9 media supplemented with 100 µg/ml ampicillin and grown to a OD600 of between 0.6 and 0.8. The bacteria were harvested by centrifugation at 4,000 x g and resuspended in 500 mL of minimal media containing 1 g L -1 15 N ammonium chloride and 2 g L -1 unlabeled glucose. After 8 hours the cells were harvested by centrifugation at 6900 x g for 20 minutes at 4°C and frozen until required for purification.
The bacterial pellet was resuspended in 30 mL HEPES (20 mM, pH= 7.4), and the cells broken by sonication using a stud sonicator. The IBs were resuspended in 25 mL of wash buffer (10 mM Tris-HCl pH 7.5, 2.5 mM MgCl2, 0.5 mM CaCl2) containing DNaseI and lysozyme and incubated for 1 hour before pelleting by centrifugation (20 min, 16000 x g). The IBs were washed a further 4 times by resuspending the them in Triton-buffer (50 mM Tris-HCl pH 8.0, 100 mM NaCl, 0.5% Triton X-100) and pelleting between washes (20 min, 16000 x g). Finally the IBs were dissolved in solubilization buffer (8M urea, 50 mM MES, 0.1 mM EDTA 0.2 mM DTT), incubating overnight at 4 °C. The solubilized material was then clarified by centrifugation (20 min, 16000 x g).
The solubilised b2m was refolded by dilution at 4°C. The solubilised b2m was first diluted 1:1 with refold buffer (100 mM Tris-HCl pH 8.0, 400 mM L-Arginine-HCl, 2 mM EDTA, 5 mM Glutathione-reduced, 0.5 mM Glutathione-oxidised, 0.1 mM PMSF), and then added drop wise at 0.1 ml/min using a peristaltic pump to ice cold refold buffer such that the final concentration of b2m would be less than or equal to 5 µM. After refold, the b2m was concentrated using a 5000 MWCO Kvick filtration system (GE Healthcare) to a concentration of ~2 mg/ml as determined by nanodrop.
Monomeric b2m was isolated by size exclusion chromatography using a Sephadex 75 Hiload 16/60 column (GE Healthcare) equilibrated with gel filtration buffer (10 mM HEPES pH 7.4, 50 mM KCl, 0.1 % Sodium azide buffer). The column was run at a flow rate of 1 mL min -1 with the sample loaded as a 2 mL aliquot. Fractions corresponding to monomeric b2m were pooled and then concentrated to 1 mg/ml by centrifugal filtration. To form fibrils, b2m was mixed 1:1 with a low pH sodium citrate buffer to lower its pH to 2.5. The solutions were then incubated at 37 °C with 200rpm orbital shaking for 3 days.
To facilitate proton detection, residual protonated buffer components were removed prior to resuspending the b2m fibrils in a deuterated DNP matrix. The extent to which the protons were removed and its influence on the overall 15 N spectra were monitored by 1 H MAS-NMR and 15 N CP-MAS studies. Washing of the samples four times with D2O significantly reduced the strength of the water resonance ( Figure S1B). However to completely abolish the water signal the sample was lyophilized overnight resulting in spectra dominated by non-labile protons attached to the protein.

NMR Experiments.
All DNP experiments were performed on a Bruker Avance III spectrometer operating at 14.1 T (600 MHz of 1 H Larmor frequency) equipped with a gyrotron oscillator at 395 GHz and a low temperature, triple-resonance 3.2 mm MAS probe tuned to 1 H/ 13 C/ 14 N or 1 H/ 13 C/ 15 N. Spectra were recorded at a temperature of 100 K with a MAS frequency of 9.8 kHz. Data were processed in matNMR 3 .
Carbon-13 MAS spectra were acquired with cross-polarization from protons to 13 C using a linear ramp from 90-100% proton amplitude and a 50 kHz 13 C spin-lock field. Optimal transfer was obtained at the n=+1 Hartmann-Hahn condition with a 1.75 ms contact time.
During acquisition protons were decoupled with 100 kHz SWf-TPPM 4 decoupling with a 5.1 µs pulse and ± 15° phase flip.  1 H/ 14 N 2D correlation spectra were recorded with the pulse sequence shown in Figure S3. The excitation and reconversion pulses applied to 14 N were rotor synchronized and at an RF amplitude of 30 kHz. Two-dimensional data were acquired using States-TPPI 5 . Unless stated data were acquired with a 5 second recycle, with 32 rotor synchronized t1 increments. PMLG decoupling was applied during 14 N excitation, evolution and reconversion, with similar windowed PMLG applied during observation. Typically 15 PMLG cycles were applied per rotor period. Spectra were zerofilled to 2048 points in either dimension, and exponential linebroadening of 50 Hz was applied in the F1 dimension before 2D Fourier transform.

CQ estimation
The 14 N second order isotropic quadrupolar shift (SOIQS) is given by 6 : where n0 is the Larmor frequency and wQ is the quadrupolar product: where CQ is the quadrupolar coupling constant and h the asymmetry parameter.
By using equantion [1] and [2] we can estimate the distribution of quadrupolar couplings present.
Subtracting from the centre of the amide region of the 14 N spectrum of b2m, 450 ppm, the centre of the amide region of the 15 N spectrum, 120 ppm, one can determine an ''average'' amide SOIQS in b2m of 380 ppm at 14.1 T. From eqn [1] and [2], one can determine this to be consistent with CQ values of 2.49-2.88 MHz, without any knowledge of the asymmetry parameter. The is dependent upon the size of the quadrupolar interaction as described above, a parameter that can vary by 100's of kHz even for nitrogen in exhibiting subtle differences in hydrogen bonding such as those observed between amide nitrogens in protein backbones adopting a-helical as opposed to b-sheeted conformations. In addition to providing valuable structural information, this also provides addition resolution in the 14N dimension. In contrast, the resolution in the 15N dimension of a 1 H/ 15 N correlation spectrum is determined by the isotropic chemical shift which in proteins shows relatively small dispersion (~10-15 ppm as opposed to the 100's ppm observed in the 14 N spectrum). Both spectra acquired under the conditions described in the Materials and Methods.