Histidine-Tag-Specific Optical Probes for Analytical Ultracentrifugation Analysis

Abstract

The hexahistidine (His6)/nickel(II)-nitrilotriacetic acid (Ni2+-NTA) system is a universal tool for the affinity purification of recombinant proteins. Additionally, the NTA group can be exploited for the attachment of fluorophores and chromophores to His6 proteins at unique user-defined locations. The applications of one such derivative, (Ni2+-NTA)2-Cy3 is characterized. The derivative binds two model His6 proteins, N-ethylmaleimide sensitive factor (NSF) and O6-alklyguanine-DNA alkyltransferase (AGT) with moderate affinity (K∼1.5x106M−1). The activity of these proteins remains unaffected by the attachment of the derivative. The (Ni2+-NTA)2-Cy3 has high specificity which makes it suitable for the detection of His6 proteins within complex mixtures containing other proteins. This property allows the derivative to be used as a probe of crude cell extracts and as a His6-specfic gel stain. The binding of the (Ni2+-NTA)2-Cy3 derivative is reversible in the presence of 10 mM ethylenediaminetetraacetic acid (EDTA) or 500 mM imidazole, and in the absence of such agents the exchange occurs slowly (kexchange∼5x10−6S−1 with 0.2 M labeled protein in the presence of 1 M His6 peptide). The hydrodynamic properties can be explored with the attachment of the (Ni2+-NTA)2-Cy3 derivative utilizing fluorescent anisotropy or analytical ultracentrifugation within environments which prevent direct detection of the protein. Additionally, the (Ni2+-NTA)2-Cy3 derivative can be utilized during fluorescence resonance energy transfer (FRET) between labeled proteins and suitable donors/acceptors creating a practical assay for binding interactions and measurements of donor-acceptor distances.