Stability of nitroxide biradical TOTAPOL in biological samples
Stability of nitroxide biradical TOTAPOL in biological samples
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DOI：
10.1016/j.jmr.2019.04.013
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发表时间：
2019-06-01
影响因子：
2.2
通讯作者：
McDermott, Ann E.
中科院分区：
文献类型：
--
作者：
McCoy, Kelsey M.;Rogawski, Rivkah;McDermott, Ann E.
我们的特点是化学还原的氮氧双自由基，TOTAPOL，用于动态核极化（DNP）实验，特别是探测在全细胞颗粒和裂解物的稳定性，并提出了一些策略，以稳定的双自由基的DNP研究。DNP固态NMR实验使用顺磁性物质，如氮氧双自由基，以显着增加NMR信号。虽然使用基于氮氧自由基的DNP来检测整个细胞中蛋白质的NMR光谱是相当令人兴奋的，但在细菌细胞团中，氮氧自由基在几分钟内就被减少了，我们在这里证实并量化了这一点。我们表明，除了共价半胱氨酸阻断剂N-乙基马来酰亚胺的全细胞显着降低的速度，表明半胱氨酸巯基自由基是重要的，在体内自由基还原。使用细胞裂解物而不是全细胞也减缓了TOTAPOL的还原，这表明周质和氧化磷酸化代谢物在自由基降解中可能起作用。裂解物中还原的TOTAPOL也可以用铁氰化钾有效地再氧化。这些结果指出了一套实用而稳健的细胞制剂DNP策略。(C)2019由Elsevier Inc.出版
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.