Genetically Encoding Single-Molecule Fluorophores into Ion Channels in Living Cells

Abstract

We have developed a new approach to track protein dynamics in living cells on a single-molecule level with genetically encoded fluorescent non-canonical amino acids (ncAAs). The probes are based on amino acids with free primary amines that are chemically coupled to commercially available fluorophores, such as Cy3 and Cy5. Specific incorporation of fluorescent ncAAs was achieved by nonsense suppression in Xenopus laevis oocytes and confirmed by two-electrode voltage clamp of rescued ionic currents as well as by total internal reflection fluorescence microscopy detecting single fluorophores in the plasma membrane. Overall, the data show that these fluorescent ncAA were accepted as substrates for the X. laevis translation machinery. To date, we have encoded five fluorescent ncAAs into intracellular, extracellular and transmembrane regions of a CLC chloride channel or a voltage-gated sodium channel. Moreover, the variation in the linker length between the alpha carbon atom and the fluorescent moiety of the ncAA was explored by coupling to para-amino-phenylalanine, lysine and diaminopropionic acid and shows that linker length does not significantly affect the incorporation efficiency. This approach overcomes limitations of existing methods to study protein dynamics in living cells and is applicable to virtually any membrane protein. New applications include the detection of stoichiometry of macromolecular complexes by photo-bleaching of diffraction-limited puncta or the acquisition of relative distance measurements resulting from dynamic conformational changes within a membrane protein by Forster resonance energy transfer, both on a single-molecule level.