Stability of Nitroxide Biradical TOTAPOL in Biological Samples

Abstract

We characterize chemical reduction of a nitroxide biradical, TOTAPOL, used in dynamic nuclear polarization (DNP) experiments, specifically probing the stability in whole-cell pellets and lysates, and present a few strategies to stabilize the biradicals for DNP studies. DNP solid-state NMR experiments use paramagnetic species such as nitroxide biradicals to dramatically increase NMR signals. Although there is considerable excitement about using nitroxide-based DNP for detecting the NMR spectra of proteins in whole cells, nitroxide radicals are reduced in minutes in bacterial cell pellets, which we confirm and quantify here. We show that addition of the covalent cysteine blocker Nethylmaleimide to whole cells significantly slows the rate of reduction, suggesting that cysteine thiol radicals are important to in vivo radical reduction. The use of cell lysates rather than whole cells also slows TOTAPOL reduction, which suggests a possible role for the periplasm and oxidative phosphorylation metabolites in radical degradation. Reduced TOTAPOL in lysates can also be efficiently reoxidized with potassium ferricyanide. These results point to a practical and robust set of strategies for DNP of cellular preparations.

Targeted DNP for biomolecular solid-state NMR

“…108 DNP experiments need to be performed relatively rapidly after addition of the PA in order to limit radical reduction. 109 In this approach, the challenge is to detect the labeled protein at M concentration, which can be overcome using multiple-quantum filtered pulse sequences that only select NMR-active nuclei that are close in space. This works well for the study of soluble proteins (e.g.…”