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Super-Resolution Imaging of Bacterial Secreted Proteins Using Genetic Code Expansion
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In situ EPR spectroscopy of a bacterial membrane transporter using an expanded genetic code
Anandi Kugele1, Sophie Ketter2, Bjarne Silkenath1
1Department of Chemistry and Konstanz Research School Chemical Biology (KoRS-CB), University of Konstanz, Universitätsstraße 10, 78457 Konstanz, Germany. malte.drescher@uni-konstanz.de.
Chemical Communications (Cambridge, England)
|November 18, 2021
Summary
This study advances spin labeling on living bacterial cell surfaces using Diels-Alder click chemistry. This technique enables precise studies of membrane proteins like BtuB in Escherichia coli.
Area of Science:
- Biochemistry
- Molecular Biology
- Biophysics
Background:
- Membrane proteins are crucial cellular components.
- Studying membrane proteins in their native environment is challenging.
- Site-directed spin labeling (SDSL) is a powerful technique for structural and dynamic studies.
Purpose of the Study:
- To evolve Diels-Alder click chemistry for site-directed spin labeling on the surface of living bacterial cells.
- To investigate the membrane transporter BtuB in *Escherichia coli*.
- To explore advanced experimental setups using photoactivatable nitroxides.
Main Methods:
- Genetically encoding SCO-L-lysine in *Escherichia coli* for site-specific labeling.
- Utilizing Diels-Alder click chemistry for covalent attachment of spin labels.
- Employing the photoactivatable nitroxide PaNDA for controlled labeling and distance measurements.
Main Results:
- Successfully achieved site-directed spin labeling of the BtuB transporter on living *E. coli*.
- Demonstrated the utility of PaNDA for preventing off-target labeling and enabling distance measurements.
- Showcased temporally shifted activation for advanced experimental designs.
Conclusions:
- This work represents a significant advancement in Diels-Alder-mediated spin labeling on cellular surfaces.
- The developed methodology opens new possibilities for studying membrane protein structure and dynamics in situ.
- This technique provides a powerful tool for future investigations in membrane biophysics.
