¹H Dynamic Nuclear Polarization Based on an Endogenous Radical

Abstract

We demonstrate a 15-fold enhancement of solid-state NMR signals via dynamic nuclear polarization (DNP) based on a stable, naturally occurring radical in a protein: the flavin mononucleotide (FMN) semiquinone of flavodoxin. The linewidth of flavodoxin’s EPR signal suggests that the dominant DNP mechanism is the solid effect, consistent with the field-dependent DNP enhancement profile. The magnitude of the enhancement as well as the bulk-polarization build-up time constant (τB) with which it develops are dependent on the isotopic composition of the protein. Deuteration of the protein to 85 % increased the nuclear longitudinal relaxation time T1n and τB by factors of five and seven, respectively. Slowed dissipation of polarization can explain the two-fold higher maximal enhancement than that obtained in proteated protein, based on the endogenous semiquinone. In contrast, the long τB of TOTAPOL-based DNP in non-glassy samples was not accompanied by a similarly important long T1n, and in this case the enhancement was greatly reduced. The low concentrations of radicals occurring naturally in biological systems limit the magnitude of DNP enhancement that is attainable by this means. However, our enhancement factors of up to 15 can nonetheless make an important difference to the feasibility of applying solid-state NMR to biochemical systems. We speculate that DNP based on endogenous radicals may facilitate MAS NMR characterization of biochemical complexes and even organelles, and could also serve as a source of additional structural and physiological information.

Is solid-state NMR enhanced by dynamic nuclear polarization?

“…This is also true with Matrix-Free samples in mind, where polarizing agents with common functional groups will be designed so as best to interact with a large range of samples. Polarizing agents may also move past the current trend of exogenous nitroxide-based radicals, to endogenous radicals [85,86], metal ions [87], and thin-line (with respect to EPR linewidths) radicals [6], so that a greater assortment of samples and nuclei can be hyperpolarized directly and analyzed efficiently. However, further technological advancements will be required for this to be realized.…”

Bacterial expansins and related proteins from the world of microbes

“…One technical challenge with this approach is that the concentration of expansin binding sites in the wall is so low that ssNMR signals would be too low to detect by standard methods. This limitation was overcome by use of a technique called dynamic nuclear polarization to enhance the sensitivity of ssNMR detection by ~25-fold (Maly et al 2012; Takahashi et al 2013). The results showed that BsEXLX1 bound to a rather specific site within the complex wall that contains cellulose, but with a slightly different structure from bulk cellulose.…”

Cysteine-Specific Labeling of Proteins with a Nitroxide Biradical for Dynamic Nuclear Polarization NMR

“…In the first demonstration of DNP from covalently attached radicals, Miller, Griffin, and co-workers have employed an endogenously present stable flavin mononucleotide radical, semiquinone, to enhance the NMR signal of flavodoxin. 37 Jannin, Bodenhausen et al have covalently attached TOTAPOL to the C-terminal amino acid of a decapeptide through the ester bond. 38 McDermott et al reported on DNP enhancement from a "pseudo-biradical" formed upon the dimerization of gramicidin labeled by monoradicals at the dimer interface.…”