Sterically shielded spin labels for in-cell EPR spectroscopy: Analysis of stability in reducing environment

Post-print (lokagerð höfundar) Electron paramagnetic resonance (EPR) spectroscopy is a powerful and widely used technique for studying structure and dynamics of biomolecules under bio-orthogonal conditions. In-cell EPR is an emerging area in this field; however, it is hampered by the reducing enviro...

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Bibliographic Details
Published in:Free Radical Research
Main Authors: Jagtap, A. P., Krstic, I., Kunjir, N. C., Hänsel, R., Prisner, Thomas F., Sigurdsson, Snorri
Other Authors: Raunvísindadeild (HÍ), Faculty of Physical Sciences (UI), Raunvísindastofnun (HÍ), Science Institute (UI), Verkfræði- og náttúruvísindasvið (HÍ), School of Engineering and Natural Sciences (UI), Háskóli Íslands, University of Iceland
Format: Article in Journal/Newspaper
Language:English
Published: Informa UK Limited 2014
Subjects:
Nitroxide reduction
Aminoxyl radical
Radical stability
Spin labeling
Trityl radical
Litrófsgreining
Efnagreining
Iceland
Online Access:https://hdl.handle.net/20.500.11815/1954
https://doi.org/10.3109/10715762.2014.979409
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Summary:Post-print (lokagerð höfundar) Electron paramagnetic resonance (EPR) spectroscopy is a powerful and widely used technique for studying structure and dynamics of biomolecules under bio-orthogonal conditions. In-cell EPR is an emerging area in this field; however, it is hampered by the reducing environment present in cells, which reduces most nitroxide spin labels to their corresponding diamagnetic N-hydroxyl derivatives. To determine which radicals are best suited for in-cell EPR studies, we systematically studied the effects of substitution on radical stability using five different classes of radicals, specifically piperidine-, imidazolidine-, pyrrolidine-, and isoindoline-based nitroxides as well as the Finland trityl radical. Thermodynamic parameters of nitroxide reduction were determined by cyclic voltammetry; the rate of reduction in the presence of ascorbate, cellular extracts, and after injection into oocytes was measured by continuous-wave EPR spectroscopy. Our study revealed that tetraethyl-substituted nitroxides are good candidates for in-cell EPR studies, in particular pyrrolidine derivatives, which are slightly more stable than the trityl radical. This work was supported by the Icelandic Research Fund (120001021), the Deutsche Forschungsgemeinschaft (SFB 902, Molecular principles of RNA-based regulation) and by a doctoral fellowship to A. P. Jagtap from the University of Iceland Research Fund. Peer reviewed

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