In-organello and in-cell EPR on orthogonally spin-labeled proteins

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Within the Schwerpunktprogramm New Frontiers in Sensitivity for EPR Spectroscopy: From Biological Cells to Nano Materials, this research focuses on the optimization of orthogonal spin labeling methodologies and state-of-the-art setups for pulsed dipolar EPR spectroscopy to extract structural information on proteins in organelles and in cells. We will perform pulsed dipolar EPR spectroscopy on multi-frequency commercially available spectrometers equipped with high power amplifiers and arbitrary waveform generators (AWG) to detect via interspin distances the conformational changes of apoptotic Bcl-2 proteins at the onset of apoptosis as well as the networking of protein-protein interaction in isolated mitochondria and in cells. For in-cell EPR, the method of choice is the internalization of spin-labeled purified proteins into mammalian cells. Relevant for the project is the development of these labeling strategies: i) a three-spin orthogonal labeling scheme for simultaneous detection of three proteins on the same sample; ii) lanthanide chelators to be attached to proteins to get both structural information by EPR and protein localization in cells via imaging techniques based on luminescence; ii) a trisNTA-based Gd-label for His-tagged proteins in living cells. This work will help establishing EPR as a valuable technique to retrieve high resolution site-specific structural information under physiological conditions.