Site-Specific Incorporation of a Photoactivatable Fluorescent Amino Acid.

Photoactivatable fluorophores are emerging optical probes for biological applications. Most photoactivatable fluorophores are relatively large in size and need to be activated using ultraviolet light, dramatically limiting 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 bioorthogonal conjugation. However, these techniques require a multi-step 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.