Hexahistidine-tag-specific optical probes for analyses of proteins and their interactions

The hexahistidine (His(6))/nickel(II)-nitrilotriacetic acid (Ni(2+)-NTA) system is widely used for affinity purification of recombinant proteins. The NTA group has many other applications, including the attachment of chromophores, fluorophores, or nanogold to His(6) proteins. Here we explore several applications of the NTA derivative, (Ni(2+)-NTA)(2)-Cy3. This molecule binds our two model His(6) proteins, N-ethylmaleimide sensitive factor (NSF) and O(6)-alklyguanine-DNA alkyltransferase (AGT), with moderate affinity (K approximately 1.5 x 10(6) M(-1)) and no effect on their activity. Its high specificity makes (Ni(2+)-NTA)(2)-Cy3 ideal for detecting His(6) proteins in complex mixtures of other proteins, allowing (Ni(2+)-NTA)(2)-Cy3 to be used as a probe in crude cell extracts and as a His(6)-specific gel stain. (Ni(2+)-NTA)(2)-Cy3 binding is reversible in 10mM ethylenediaminetetraacetic acid (EDTA) or 500 mM imidazole, but in their absence it exchanges slowly (k(exchange) approximately 5 x 10(-6) s(-1) with 0.2 microM labeled protein in the presence of 1 microM His(6) peptide). Labeling with (Ni(2+)-NTA)(2)-Cy3 allows characterization of hydrodynamic properties by fluorescence anisotropy or analytical ultracentrifugation under conditions that prevent direct detection of protein (e.g., high ADP absorbance). In addition, fluorescence resonance energy transfer (FRET) between (Ni(2+)-NTA)(2)-Cy3-labeled proteins and suitable donors/acceptors provides a convenient assay for binding interactions and for measurements of donor-acceptor distances.