Abstract
Photoactivatable fluorophores are emerging optical probes for biological applications. Most photoactivatable fluorophores are relatively large in size and need to be activated by ultraviolet light; this dramatically limits their applications. To introduce photoactivatable fluorophores into proteins, recent investigations have explored several protein-labeling technologies, including fluorescein arsenical hairpin (FlAsH) Tag, HaloTag labeling, SNAPTag labeling, and other bioorthogonal chemistry-based methods. However, these technologies require a multistep labeling process. Here, by using genetic code expansion and a single sulfur-for-oxygen atom replacement within an existing fluorescent amino acid, we have site-specifically incorporated the photoactivatable fluorescent amino acid thioacridonylalanine (SAcd) into proteins in a single step. Moreover, upon exposure to visible light, SAcd can be efficiently desulfurized to its oxo derivatives, thus restoring the strong fluorescence of labeled proteins.