Exploiting the nitrilotriacetic acid moiety for biolabeling with ultrastable perylene dyes

Fluorescent probes are essential for the exploration of protein function, detection of molecular interactions, and conformational changes. The nitrilotriacetic acid derivatives of different chromophores were successfully used for site-selective noncovalent fluorescence labeling of histidine-tagged proteins. All of them, however, suffer from the same drawback--loss of the fluorescence upon binding of the nickel ions. Herein we present the solution and solid phase synthesis of water-soluble perylene(dicarboximide) functionalized with a nitrilotriacetic acid moiety (PDI-NTA). The photophysical properties of PDI-NTA revealed an exceptional photostability and fluorescence quantum yield that remained unchanged upon addition of nickel ions. The F1 complex of F0F1-ATP synthase from Escherichia coli, containing three hexahistidine tags, was labeled and the suitability for site-specific labeling of the new chromophore demonstrated using fluorescence correlation spectroscopy.