Diaminoterephthalate Turn‐On Fluorescence Probes for Thiols—Tagging of Recoverin and Tracking of its Conformational Change

Diaminoterephthalates with a maleimide moiety were synthesized and used as fluorescence dyes for sensing thiols. Whereas these “NiWa Blue” dyes showed no emission, the conjugate addition of a thiol to the maleimide group turned on a fluorescence at about 400 nm when irradiating the dye at 338 nm. The neuronal‐calcium sensor protein recoverin possesses a single cysteine residue at position 39, which reacts with NiWa Blue, and is therefore labeled by a fluorophore with an emission at about 440 nm. In the absence of Ca 2+ , irradiation at 280 nm of a tryptophan residue in close proximity to Cys‐bound NiWa Blue lead to strong FRET, which was detected by emission of the dye at 440 nm. In the presence of Ca 2+ , the protein holds a conformation with distal Trp and Cys residues, thus FRET of irradiated Trp to Cys‐bound NiWa Blue was significantly weakened.