thèse Annalisa PIERRO

16/11/2021 - 13:30 - Amphi. Desnuelles
Annalisa PIERRO, BIP 07

Protein structural dynamics in bacteria via nitroxide-based SDSL-EPR spectroscopy: from method improvements to in-cell studies

The study of biomolecules in their native environment has been one of the main goals of structural biology in the last decade. As a result, we are assisting to a remarkable increase of new « in-cell » approaches, like Cryo-Electron Tomography, FRET microscopy and NMR spectroscopy. Among these approaches, Site-Directed Spin Labeling (SDSL) coupled to Electron Paramagnetic Resonance (EPR) spectroscopy shows competitive and advantageous features to capture protein dynamics inside cells. In particular, nitroxide-based SDSL-EPR combines the advantages of high sensitivity and the lack of size constraints on the biomolecule of interest with the ability to capture protein structural transitions and interactions at physiological temperature. Despite the methodological advancements of the technique that have allowed the community to obtain increasingly relevant results, progresses still need to be done.
In this work, the main limitation of nitroxide-based SDSL-EPR has been addressed. In the first time, we focused on the development of delivery methods to introduce the labeled protein in bacterial cells. Next, the stability of nitroxide labels in reducing environments and in-cell has been assessed, monitoring in parallel the viability of the cells during the EPR measurements.
Thanks to the results achieved in this methodological part, we were able to study the structural dynamics of two flexible chaperone proteins directly in bacterial cells: NarJ from Escherichia coli and UreG from Sporosarcina pasteurii.
Finally, to go further in understanding the impact of the cellular environment on the protein dynamics, the data obtained in cellular context were compared with those obtained in vitro or in a cell-mimicking environment.

Keywords: SDSL-EPR, in-cell EPR, nitroxide labels, chaperone proteins, structural dynamics, bacterial cells



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