Probing the Structural Dynamics of a Bacterial Chaperone in Its Native Environment by Nitroxide-Based EPR
Annalisa Pierro1,2, Alessio Bonucci1, Davide Normanno3,4
1Aix Marseille Univ, CNRS, BIP Bioénérgetique et Ingénierie des Protéines, IMM, 13009, Marseille, France.
Chemistry (Weinheim an Der Bergstrasse, Germany)
|October 6, 2022
Summary
Site-directed spin labeling electron paramagnetic resonance (SDSL-EPR) enables studying protein dynamics within living cells. This method confirmed NarJ chaperone activity and revealed cellular environment effects on its structural dynamics.
Area of Science:
- Structural biology
- Biophysics
- Molecular dynamics
Background:
- Studying protein dynamics in complex cellular environments remains a significant challenge in structural biology.
- Electron paramagnetic resonance spectroscopy coupled to site-directed spin labeling (SDSL-EPR) is a powerful technique for investigating protein local dynamics and conformational ensembles.
Purpose of the Study:
- To apply SDSL-EPR to investigate the structural dynamics of the NarJ chaperone within its native host, Escherichia coli.
- To demonstrate the feasibility of performing SDSL-EPR studies under conditions that preserve cellular integrity and protein activity.
Main Methods:
- Utilized nitroxide labels sensitive to local protein dynamics at room temperature.
- Employed site-directed spin labeling electron paramagnetic resonance (SDSL-EPR) spectroscopy.
- Performed experiments within intact Escherichia coli cells.
Main Results:
- Demonstrated that spin-labeled NarJ retains its biological activity within the cellular environment.
- Showed that the cellular medium influences NarJ structural dynamics in a site-specific manner.
- Confirmed that the overall structural flexibility of NarJ is maintained in vivo.
Conclusions:
- SDSL-EPR is a viable method for studying protein dynamics in living cells without compromising protein function or cell viability.
- The cellular environment modulates the structural dynamics of NarJ, highlighting the importance of in-cell studies.
- Presented time-resolved data on NarJ dynamics in its native cellular context.
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