Title: Dynamic Nuclear Polarization with Biradical Affinity Reagents
Description: Dynamic nuclear polarization (DNP) is an emerging method for increasing signal-to-noise in solid-state NMR experiments by transferring polarization from electrons to nuclei. Stable nitroxide biradicals, the polarization source for the cross effect mechanism, are typically co-dissolved at millimolar concentrations with biomolecules of interest. There has been considerable recent interest, however, in co-localizing the paramagnetic polarizing agent with the analyte of interest through covalent or noncovalent interactions. In this thesis, we bound the biradical to the protein of interest through conjugation to a high affinity, non-covalent inhibitor using E.coli dihydrofolate reductase (DHFR) and its nanomolar inhibitor trimethoprim (TMP). Two different biradical affinity reagents (TMP-T and TMP-V-T) were created by covalently linking trimethoprim to the biradical polarizing agent TOTAPOL. These TMP-TOTAPOL compounds provided excellent enhancements of the DHFR NMR spectrum, comparable to when TOTAPOL is co-dissolved with the protein.

In contrast to TOTAPOL, the specific interactions of these compounds enabled them to be added stoichiometrically to samples of DHFR at micromolar concentrations while retaining excellent enhancements. Benefits of lowered biradical concentration include reduced chemical perturbation of the sample and the ability to selectively enhance signals for the protein of interest in a perdeuterated solvent matrix. The binding of TMP-V-T to DHFR confines the paramagnetic TOTAPOL moiety to the protein binding pocket. We capitalized on this fact to site-specifically study paramagnet induced signal quenching in DNP samples. Using crystallography and solution NMR spectroscopy, we demonstrated that although the TMP fragment is well ordered the TOTAPOL moiety is disordered when bound to DHFR. We prepared site-specific isotpically enriched samples of DHFR and measured signal quenching in DNP samples. Intriguingly, the bleaching patterns seen in DNP samples are similar to those observed in room temperature studies.

We argue that static disorder plays a role in this observation, among other contributions. Our results provide design principles for DNP experiments with affinity biradicals, and demonstrate the utility of affinity biradicals for DNP studies given the confined signal quenching. Affinity biradicals hold great promise for enabling selective DNP investigations of dilute proteins in whole cells or cellular lysates. We explored how to use TMP-T and TMP-V-T to study DHFR in whole cells and cellular lysates. We developed strategies to overcome fast chemical reduction of the nitroxide moiety in the cellular environment, and sample preparation protocols to obtain overexpressed DHFR with isotopic enrichment patterns that enable selective DNP enhancements. We demonstrate excellent enhancements of whole cell pellets by TOTAPOL, and also provide evidence for selective DHFR enhancement by TMP-V-T in cellular lysates. Our results form guidelines for future selective studies of DHFR in cellular contexts.

These results collectively demonstrate the potential of the TMP-T(V-T) compounds and similar affinity biradical reagents both for mechanistic DNP studies and selective NMR studies in cellular contexts.