The inherently low sensitivity of NMR spectroscopy, in particular in the solid-state, can be overcome with a methodology called dynamic nuclear polarization (DNP). DNP relies on the transfer of polarization from electron spins to nuclei and involves the addition of polarization agents (stable radical species that contain unpaired electron spins) to the sample of interest. While this technique can achieve experimental signal enhancements in the range 10 – 200 for <i>in vitro</i> biological samples, there is a great interest in its potential for structural biology applications in the cellular environment. To adapt DNP for in-cell NMR applications, we have designed a new sensitivity-enhancement strategy where the polarization agent is directly attached to the protein of interest. In this approach, we take advantage of chemical biology approaches that allow the incorporation of unnatural amino acids into proteins by genetic means (amber suppression) and tetrazine-based bio-orthogonal conjugation chemistry suitable for the cellular environment. Using this strategy, we have generated ubiquitin with a site-specifically attached polarization agent and have measured preliminary DNP signal enhancements of ~ 15. More importantly, however, this approach has allowed us to record NMR structural data from just 5 μg of isotopically labeled protein material. In the future, we expect that this will be a powerful tool to illuminate the structure and function of proteins inside bacterial and mammalian cells at endogenous concentrations.<br/>

New tools for sensitivity-enhanced in-cell NMR spectroscopy

2019 Fall National Meeting

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