Evaluation of spin labels for in-cell EPR by analysis of nitroxide reduction in cell extract of Xenopus laevis oocytes

Abstract

Spin-label electron paramagnetic resonance (SL-EPR) spectroscopy has become a powerful and useful tool for studying structure and dynamics of biomacromolecules. However, utilizing these methods at physiological temperatures for in-cell studies is hampered by reduction of the nitroxide spin labels and thus short half-lives in the cellular environment. Consequently, reduction kinetics of two structurally different nitroxides was investigated in cell extracts of Xenopus laevis oocytes using rapid-scan cw-experiments at X-band. The five member heterocyclic ring nitroxide PCA (3-carboxy-2,2,5,5-tetramethylpyrrolidinyl-1-oxy) under investigation features much higher stability against intracellular reduction than the six member ring analog TOAC (2,2,6,6-tetramethylpiperidine-N-oxyl-4-amino-4-carboxilic acid) and is therefore a suitable spin label type for in-cell EPR. The kinetic data can be described according to the Michaelis-Menten model and thus suggest an enzymatic or enzyme-mediated reduction process.

New NMR tools for protein structure and function: Spin tags for dynamic nuclear polarization solid state NMR

“…Similarly, in cells expressing proteins with the unnatural amino acid SLK-1, reduction of the EPR probe in cells and in lysate was reported, and prevented successful measurements in vivo [104,105,139]. It has been shown that 5-membered piperidine rings have improved stability in biological systems in comparison with the six membered pyrollidines typically used in DNP applications [140,141]. Substitution of the positions flanking the nitroxides radical with bulky moieties has been shown to increase resistance to reduction [142,143], and nitroxide spin tags based on these bulky tags may be promising for in cell DNP.…”

Towards characterization of DNA structure under physiological conditions in vivo at the single-molecule level using single-pair FRET

“…However, compared with in-cell PELDOR, the in-cell spFRET setup offers one particularly important advantage, namely extended measurement time. Whereas the in-cell spFRET measurement time is primarily limited by the life span of the cell (typically several hours for the ATTO680/ATTO740 donor–acceptor system used in this study), the in-cell PELDOR measurement time is typically limited to ∼70 min due to the rapid reduction of the spin labels by the cellular environment (3,4,42). …”

Intrazelluläre Elektronenspinresonanz‐Spektroskopie

“…7,8) Eine der größten Schwierigkeiten bei intrazellulären DEER-Experimenten in Kombination mit Nitroxid-Markierung liegt in der Reduktion des Nitroxids in der zellulären Umgebung und der daraus resultierenden kurzen Lebensdauer der Spinmarkierung. Da dieser Reduktionsprozess enzymatisch ist, 9) führen geringe Änderungen an der Struktur des Spinmarkers zu deutlichen Unterschieden in der Halbwertszeit in der Zelle. So weist der in Abbildung 1 gezeigte Spinmarker eine Halbwertszeit von etwa 30 Minuten auf, während es das Ana-logon mit einem Sechsring auf eine Halbwertszeit von lediglich einer Minute bringt.…”