Background

Electron paramagnetic resonance (EPR) spectroscopy is the method of choice for characterization of paramagnetic systems (Atherton, 1993; Gerson et al., 1994; Jeschke and Schweiger, 2001). Diamagnetic bio-macromolecules can be made accessible for EPR spectroscopy by site-directed spin labeling (SDSL), commonly using nitroxides as spin labels (Hubbell and Altenbach, 1994; Feix and Klug, 2002; Likhtenshtein et al., 2008; Berliner and Reuben, 2012). The combination of SDSL with in-cell EPR spectroscopy is a powerful tool to gain information about structure and dynamics of bio-macromolecules such as proteins or nucleotides in their natural environment (Azarkh et al., 2013; Martorana et al., 2014; Qi et al., 2014; Cattani et al., 2017). The most common experimental procedure for the fledging technique of in-cell EPR is based on the microinjection of the target molecules into oocytes from the African frog Xenopus laevis, which are a widely used eukaryotic cell model (Kay, 1991; Barnard et al., 1982; Mishina et al., 1984; Dawid and Sargent, 1988; Richter, 1999).

The advantages of Xenopus laevis oocytes for in-cell EPR are the large size of approximately 1 mm in diameter (approximately 1 µl cell volume), the resulting easy handling and the fact that only three or five of them are required for an in-cell EPR sample (Qi et al., 2014; Cattani et al., 2017). Consequently, bio-macromolecules can be introduced relatively easily in the quantity required for EPR measurements into the Xenopus laevis oocyte by microinjection. Hence, there have been numerous intracellular distance measurements of spin labelled DNA and proteins performed by double electron-electron resonance (DEER) measurements after microinjection into Xenopus laevis oocytes (Igarashi et al., 2010; Azarkh et al., 2011; Krstic et al., 2011; Azarkh et al., 2013; Martorana et al., 2014; Wojciechowski et al., 2015; Cattani et al., 2017).