Genetically Incorporable, Tryptophan-Based Cyan Fluorescent Amino Acid for Protein Spectroscopy and Microscopy

Abstract

6-acetyl-L-tryptophan (6Ac-Trp) was synthesized as a tryptophan (Trp) analog with an absorption spectrum extending to ca. 400 nm, an emission spectrum spanning the blue-to-green visible region. Its fluorescence quantum yield is environment-sensitive (i.e., ca. 0.54 in water and <0.01 in hydrophobic solvents). A highly active synthetase was evolved for site-specific incorporation of 6Ac-Trp into proteins in both Escherichia coli and mammalian cells. Moreover, the utility of 6Ac-Trp was demonstrated by two-photon excitation fluorescence microscopy imaging of cell-penetrating peptide distributions in live HeLa cells and nuclear localization of histone H3 in HEK293T cells. Given its structural similarity to tryptophan, compatibility with chemical peptide synthesis, and genetic encoding, 6Ac-Trp provides a useful minimal fluorophore for site-specific studies of protein dynamics, interactions, and localization in complex biological systems.