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 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.

Automated summary

Photoactivatable fluorophores are limited in their applications due to their size and the need for ultraviolet light activation. Research has focused on more efficient protein labeling technologies, including single-step labeling processes. By using genetic code expansion and replacing oxygen with sulfur within existing fluorescent amino acids, the photoactivatable fluorescent amino acid thioacridonylalanine (SAcd) has been successfully incorporated into proteins in a single step.