Site-Specific Incorporation of a Photoactivatable Fluorescent Amino Acid

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.

Keywords: genetic code expansion; noncanonical amino acids; optical probes; photoactivatable fluorophores; protein labeling.

Figure 1.

Synthesis of SAcd.

Figure 2.

(A) Overlay of ¹H NMR spectra (7.2 – 8.9 ppm) of Acd taken at the indicated light irradiation times (470/60 nm, 0.4 μW cm⁻²). (B) Normalized absorbance (left) and fluorescence (right) spectra of SAcd after photoactivation.

Figure 3.

(A) Incorporation of SACD into proteins expressed in E. coli, followed by the photo-activation. (B) SDS-PAGE analysis of GFP, scFv, and FB mutants isolated from E. coli. (C) ESI-MS analysis of GFP, scFv, and FB mutants with SACD. (D) Fluorescence spectra of FB-H19SACD after light irradiation for different time (470/60 nm, 0.4 μW cm⁻²).