In situ EPR spectroscopy of a bacterial membrane transporter using an expanded genetic code.

Anandi Kugele,Sophie Ketter,Benesh Joseph,Bjarne Silkenath,Malte Drescher,Valentin Wittmann

doi:10.1039/d1cc04612h

Anandi Kugele, Sophie Ketter + Show 4 more

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https://doi.org/10.1039/d1cc04612h

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Abstract

The membrane transporter BtuB is site-directedly spin labelled on the surface of living Escherichia coli via Diels–Alder click chemistry of the genetically encoded amino acid SCO-l-lysine. The previously introduced photoactivatable nitroxide PaNDA prevents off-target labelling, is used for distance measurements, and the temporally shifted activation of the nitroxide allows for advanced experimental setups. This study describes significant evolution of Diels–Alder-mediated spin labelling on cellular surfaces and opens up new vistas for the the study of membrane proteins.

Highlights

Anandi Kugele, a Sophie Ketter,b Bjarne Silkenath, a Valentin Wittmann, a Benesh Joseph *b and Malte Drescher *a
The membrane transporter BtuB is site-directedly spin labelled on the surface of living Escherichia coli via Diels–Alder click chemistry of the genetically encoded amino acid SCO-L-lysine
The previously introduced photoactivatable nitroxide photoactivatable nitroxide for Diels–Alder (PaNDA) prevents off-target labelling, is used for distance measurements, and the temporally shifted activation of the nitroxide allows for advanced experimental setups

Summary

Introduction

Anandi Kugele, a Sophie Ketter,b Bjarne Silkenath, a Valentin Wittmann, a Benesh Joseph *b and Malte Drescher *a. The membrane transporter BtuB is site-directedly spin labelled on the surface of living Escherichia coli via Diels–Alder click chemistry of the genetically encoded amino acid SCO-L-lysine. We presented the first approach applying inverse electron-demand Diels–Alder click chemistry[23,24,25,26,27] for SDSL of model proteins in vitro.[28] The photoactivatable nitroxide for Diels–Alder (PaNDA) spin label (Fig. 1A) distinguishes itself by an o-nitrobenzyl-based photoremovable protecting group (PPG) for the TEMPO-based nitroxide.[29,30,31,32] Upon UV irradiation at the 12980 | Chem.

Results

Conclusion