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gem-Diethyl Pyrroline Nitroxide Spin Labels: Synthesis,EPR Characterization,Rotamer Libraries and Biocompatibility |
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| Authors: | Dr. Stephanie Bleicken Tufa E. Assafa Dr. Hui Zhang Christina Elsner Irina Ritsch Dr. Maren Pink Dr. Suchada Rajca Prof. Dr. Gunnar Jeschke Prof. Dr. Andrzej Rajca Prof. Dr. Enrica Bordignon |
| Affiliation: | 1. Faculty of Chemistry and Biochemistry, Ruhr University Bochum, Universitaetsstrasse 150, 44801 Bochum, Germany ZEMOS, Ruhr University Bochum, Universitaetsstrasse 150, 44801, Bochum, Germany;2. Faculty of Chemistry and Biochemistry, Ruhr University Bochum, Universitaetsstrasse 150, 44801 Bochum, Germany These authors contributed equally to this work.;3. Department of Chemistry, University of Nebraska, Lincoln, Nebraska, 68588-0304 USA These authors contributed equally to this work.;4. Faculty of Chemistry and Biochemistry, Ruhr University Bochum, Universitaetsstrasse 150, 44801 Bochum, Germany;5. ETH Zurich, Laboratory of Physical Chemistry, Vladimir-Prelog-Weg 2, CH-8093 Zurich, Switzerland.;6. IUMSC, Department of Chemistry, Indiana University, Bloomington, Indiana, 47405-7102 USA;7. Department of Chemistry, University of Nebraska, Lincoln, Nebraska, 68588-0304 USA |
| Abstract: | The availability of bioresistant spin labels is crucial for the optimization of site-directed spin labeling protocols for EPR structural studies of biomolecules in a cellular context. As labeling can affect proteins’ fold and/or function, having the possibility to choose between different spin labels will increase the probability to produce spin-labeled functional proteins. Here, we report the synthesis and characterization of iodoacetamide- and maleimide-functionalized spin labels based on the gem-diethyl pyrroline structure. The two nitroxide labels are compared to conventional gem-dimethyl analogs by site-directed spin labeling (SDSL) electron paramagnetic resonance (EPR) spectroscopy, using two water soluble proteins: T4 lysozyme and Bid. To foster their use for structural studies, we also present rotamer libraries for these labels, compatible with the MMM software. Finally, we investigate the “true” biocompatibility of the gem-diethyl probes comparing the resistance towards chemical reduction of the NO group in ascorbate solutions and E. coli cytosol at different spin concentrations. |
| Keywords: | biocompatibility nitroxide EPR pyrrolines spin labeling |