Site-Specific Dynamic Nuclear Polarization (DNP) NMR Agents

Dynamic Nuclear Polarization (DNP) enhances the signal in solid-state NMR of proteins by transferring polarization from electronic spins to the nuclear spins of interest.  Typically, both the protein and an exogenous source of electronic spins, such as a biradical, are co-dissolved or suspended and then frozen in a glycerol/water glassy matrix to achieve a homogeneous distribution. The use of such a matrix reduces the available sample volume (by ca. a factor of 4 in our experiments) and causes proportional NMR signal loss.

Recently we have demonstrated an alternative approach, which completely eliminates the need for a glassy matrix. Specifically, Prof. Maxim Voynov in Smirnov group synthesizes a new biradical, ToSMTSL, which is based on the known DNP agent TOTAPOL, and in addition contains a thiol-specific methanethiosulfonate group to allow for incorporating this biradical into a protein in a site-directed manner.

ToSMTSL was characterized by EPR and tested for DNP of a heptahelical transmembrane protein, Anabaena Sensory Rhodopsin (ASR), by covalent modification of solvent-exposed cysteine residues in two 15N-labeled ASR mutants. DNP enhancements were measured at 400 MHz/263 GHz NMR/EPR frequencies for a series of samples prepared in deuterated and protonated buffers and with varied biradical/protein ratios.  The maximum DNP enhancement of 15 obtained in these samples is comparable to that observed for an ASR sample co-suspended with ~17 mM TOTAPOL in a d8-glycerol/D2O/H2O matrix, but the resulting signal is fourfold greater due to the gain in the filling factor.

We anticipate that the DNP enhancements could be further improved by optimizing the biradical structure. The use of covalently attached biradicals would broaden the applicability of DNP NMR to studies of proteins (1, 2) .

  1. Voinov MA, Good DB, Ward ME, Milikisiyants S, Marek A, Caporini MA, et al. Cysteine-Specific Labeling of Proteins with a Nitroxide Biradical for Dynamic Nuclear Polarization NMR. Journal of Physical Chemistry B. 2015;119(32):10180-90.
  2. Smirnov A, MA V. Site-specific dynamic nuclear polarization NMR agents. US Patent App 15/222,428. 2017.