Solid state nuclear magnetic resonance with magic-angle spinning and dynamic nuclear polarization below 25K

Abstract

We describe an apparatus for solid state nuclear magnetic resonance (NMR) with dynamic nuclear polarization (DNP) and magic-angle spinning (MAS) at 20–25 K and 9.4 Tesla. The MAS NMR probe uses helium to cool the sample space and nitrogen gas for MAS drive and bearings, as described earlier (Thurber et al., J. Magn. Reson. 2008) [1], but also includes a corrugated waveguide for transmission of microwaves from below the probe to the sample. With a 30 mW circularly polarized microwave source at 264 GHz, MAS at 6.8 kHz, and 21 K sample temperature, greater than 25-fold enhancements of cross-polarized 13C NMR signals are observed in spectra of frozen glycerol/water solutions containing the triradical dopant DOTOPA-TEMPO when microwaves are applied. As demonstrations, we present DNP-enhanced one-dimensional and two-dimensional 13C MAS NMR spectra of frozen solutions of uniformly 13C-labeled L-alanine and melittin, a 26-residue helical peptide that we have synthesized with four uniformly 13C-labeled amino acids.

Cellular solid-state NMR investigation of a membrane protein using dynamic nuclear polarization

“…For example, the average distance of observed correlation in a 2D 13 C/ 13 C PDSD experiment could be about 5.5 Å for the transmembrane α-helical region. This experiment allowed us to observe the reasonably well-resolved cross peaks for [3- 13 C] Ala/[2- 13 C] Leu, [2- 13 C] Leu/Trp (indole ring-2- 13 C), [3- 13 C] Ala/[1- 13 C] Val, and [2- 13 C] Leu/[1- 13 C] Val sites of cytochrome b5 even at 99.5 K. It should be noted that previous studies have shown that solid-state NMR experiments at low-temperatures result in line-broadening mainly due to the restriction of the molecular motions [47,80-82]. Thus, our preliminary DNP results at cryogenic temperature demonstrate that it is feasible to acquire reasonably well-resolved MAS spectra if the isotopically labeled sites in the embedded protein of interest are carefully chosen.…”

Perturbation of nuclear spin polarizations in solid state NMR of nitroxide-doped samples by magic-angle spinning without microwaves

“…6 All samples contained 50 mM 15 N, 13 C 3 -L-alanine in partially protonated glycerol/water. Nitroxidedoped samples contained 30 mM of nitroxide radicals (10 mM of the triradicals DOTOPA-4OH or DOTOPA-Ethanol 2,7,8 or 15 mM of the biradical TOTAPOL 9 ), while samples without nitroxides contained 0.2 mM DyEDTA to reduce the lowtemperature nuclear spin-lattice relaxation times (T 1n ). Sample identities and compositions are given in Table I.…”

“…In addition, many solid-state NMR experiments rely on magic-angle spinning (MAS) to average out anisotropic nuclear spin interactions and thereby produce sharp solid-state NMR lines. As a result, the combination of DNP with MAS has recently become prevalent in applications of solid-state NMR to a variety of chemical and biochemical systems, 1,2 making it important to understand how DNP mechanisms are affected by MAS. 3,4 This paper reports experiments and simulations showing that MAS alone can perturb nuclear spin polarizations in frozen solutions that are paramagnetically doped with nitroxide-based compounds, even without microwave irradiation.…”

Theoretical aspects of Magic Angle Spinning - Dynamic Nuclear Polarization

“…In that case, the KBr powder was put at the bottom of the NMR rotor and isolated from the buffer by a silicon rubber seal. The proline was the analyte to be detected via DNP enhanced MAS-NMR experiments, and KBr was used for internal (or in situ) temperature-calibration [38,28,26]. The concentrations of the radicals were set by adding appropriate amounts of 4-amino TEMPO, protonated and deuterated TOTAPOL and AMUPOL [23,26].…”