期刊
JOURNAL OF MAGNETIC RESONANCE
卷 303, 期 -, 页码 115-120出版社
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.jmr.2019.04.013
关键词
Dynamic nuclear polarization (DNP); In-cell NMR; Nitroxide reduction; TOTAPOL
资金
- National Science Foundation (NSF) [MCB1412253]
- National Institute of Health [P41GM118302]
- NYSTAR
- ORIP/NIH facility improvement grant [CO6RR015495]
- NIH [S10RR029249]
- National Institute of Health Training Program in Molecular Biophysics [T32GM008281]
- NSF Graduate Fellowship [DGE 16-44869]
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 N-ethylmaleimide 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. (C) 2019 Published by Elsevier Inc.
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