Background: The Zn(II)-binding site from the active center of human carbonic anhydrase II, formed by three His side chains, can be grafted onto the recombinant serum retinol-binding protein (RBP). The artificial binding site in the resulting variant RBP/H3(A) has high affinity for Zn(II) and stabilizes the protein against denaturation.
Results: The metal-ion specificity of the grafted Zn(II) binding site in RBP/H3(A) was investigated. Both Cu(II) and Ni(II) bound with high affinity, although the Kd values were not as low as for Zn(II) binding. Competition experiments with the chelate ligands iminodiacetic acid (IDA) and nitrilotriacetic acid (NTA) suggested that both Ni(II) and Cu(II) bound to the protein in an octahedral manner with three vacant coordination sites, as previously observed for Zn(II). A substituted pyrrolidine-dicarboxylic acid was designed as a structurally rigid IDA compound and coupled to a matrix. Using this support in an immobilized metal affinity chromatography (IMAC), RBP/H3(A) was purified from the bacterial cell extract in one step with unprecedented efficiency.
Conclusions: Although the His3 metal-binding site used here had been removed from the substrate pocket of an enzyme and exposed to solvent on a protein surface, it showed clear selectivity for Zn(II) compared to Cu(II) and Ni(II). Thus the properties of this structurally defined metal-binding site (which are not shared by isolated His residues or flexible oligo-His tags) can be preserved when it is added to proteins. An IMAC matrix with improved behaviour was designed, allowing highly selective purification of RBP/H3(A) and of His6-tagged RBP as well. Such rational design of supramolecular recognition may be generally useful in the fields of protein engineering and drug design.