- 11:30 - Visio
Annalisa PIERRO, BIP 07
In-cell EPR spectroscopy to study protein dynamics in their natural environment
In the past decade, understanding how the intracellular medium influences protein structural dynamics and protein-protein interactions is an intriguing topic that structural biology is trying to address. As the cellular environment is seldom reproducible in vitro, the scientific community is adapting the existing techniques to study proteins directly inside the cells. Site-Directed Spin Labeling (SDSL) coupled to Electron Paramagnetic Resonance (EPR) spectroscopy is one of the techniques available which exhibits competitive and advantageous features to capture protein dynamics inside cells. In particular, nitroxides-based SDSL-EPR combines the high sensitivity, no limit in the biomolecule size and the ability to study structural transitions and interactions.
In this work, we set up an efficient protocol to overcome the current limits of the use of nitroxides labels in SDSL-EPR in-cell studies. We will discuss the results achieved by the investigation of local and global structural dynamics of NarJ, a flexible chaperone protein involved in the biogenesis of nitrate reductase A, directly inside its endogenous host cells (E. coli). We were able to prove that NarJ was active inside the cells and to follow local structural dynamics changes in a physiologically relevant environment. The data obtained will be discussed in light of the ones obtained in vitro, highlighting how the cellular environment can impact the behavior of this protein.
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