Studying ion channel conformation dynamics by encoding coumarin as unnatural amino acid.

Monitoring the conformational changes of proteins is critical to understand their function. Ion channels are membrane-bound minute machines controlling the passage of ions across biological membranes. The precise labeling of ion channels with fluorescent probes allows studying their dynamics and facilitates their characterization by high-resolution optical techniques. Here we describe a protocol for the use of a small fluorescent reporter, incorporated by expansion of the genetic code in the host cell. An important advantage of using small probes is that they are less likely to perturb protein structure, function, and trafficking. In our hands, Tyr-coumarin proved to be useful to measure the conformational changes occurring in the narrow space of the permeation pathway in single capsaicin receptors. The method described here could be directly translated to the study of membrane receptors, non-electrogenic transporters, or membrane-bound enzymes.