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, binds specifically to His6-tagged proteins, leading to over 15-fold 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 mins) 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 visual contrast.