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

• Applicant: Professorin Dr. Enrica Bordignon
• Subject Area: Analytical Chemistry
• Term: from 2015 to 2019
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 276452545

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.

• DFG Programme: Priority Programmes
• Subproject of: SPP 1601: New Frontiers in Sensitivity for EPR Spectroscopy: from Biological Cells to Nano Materials
• International Connection: USA
• Cooperation Partner: Professor Dr. Andrzej Rajca