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.
Keywords: His-tagged protein; live cell imaging; membrane permeability; photoactivation; small fluorescent probe.