Friday, May 19, 2017

T1 - Dynamic Nuclear Polarization Signal Enhancement with High-Affinity Biradical Tags #DNPNMR

Rivkah Rogawski, Ivan V. Sergeyev, Yongjun Li, M. Francesca Ottaviani, Virginia Cornish, and Ann E. McDermott The Journal of Physical Chemistry B 2017 121 (6), 1169-1175

Dynamic nuclear polarization is an emerging technique for sensitizing solid-state NMR experiments by transferring polarization from electrons to nuclei. Stable biradicals, the polarization source for the cross effect mechanism, are typically codissolved at millimolar concentrations with proteins of interest. Here we describe the high-affinity biradical tag TMP-T, created by covalently linking trimethoprim, a nanomolar affinity ligand of dihydrofolate reductase (DHFR), to the biradical polarizing agent TOTAPOL. With TMP-T bound to DHFR, large enhancements of the protein spectrum are observed, comparable to when TOTAPOL is codissolved with the protein. In contrast to TOTAPOL, the tight binding TMP-T can be added stoichiometrically at radical concentrations orders of magnitude lower than in previously described preparations. Benefits of the reduced radical concentration include reduced spectral bleaching, reduced chemical perturbation of the sample, and the ability to selectively enhance signals for the protein of interest.