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

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.