Green Fluorescent Probe for Imaging His₆-Tagged Proteins Inside Living Cells

Small molecule-based fluorescent probes offer great opportunities for specifically tracking proteins in living systems with minimal perturbation on the protein function and localization. Herein, we report a small green fluorescent probe (Ni²⁺- NTA-AF) consisting of a Ni²⁺-NTA moiety, a fluorescein, and an arylazide group, that binds specifically to His₆-tagged proteins with fluorescence enhancement in vitro upon photoactivation of the arylazide group. Importantly, the probe can cross the cell membranes and stoichiometrically label His₆-tagged proteins rapidly (∼15 min) in living prokaryotic and eukaryotic cells exemplified by a DNA repair protein Xeroderma pigmentosum group A (XPA). Using the probe, we successfully visualized Sirtuin 5, which is localized to the mitochondria. This probe exhibits high quantum yields and improved solubility, offering a new opportunity for imaging intracellular His₆-tagged proteins inside living cells with better contrast.