A ZN(II)-BINDING SITE ENGINEERED INTO RETINOL-BINDING PROTEIN EXHIBITS METAL-ION SPECIFICITY AND ALLOWS HIGHLY EFFICIENT AFFINITY PURIFICATION WITH A NEWLY DESIGNED METAL-LIGAND

Background: The Zn(II)-binding site from the active center of human ca rbonic anhydrase II, formed by three His side chains, can be grafted o nto the recombinant serum retinol-binding protein (REP). The artificia l binding site in the resulting variant RBP/H-3(A) has high affinity f or Zn(II) and stabilizes the protein against denaturation. Results: Th e metal-ion specificity of the grafted Zn(II) binding site in RBP/H-3( 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. Competit ion experiments with the chelate ligands iminodiacetic acid (IDA) and nitrilotriacetic acid (NTA) suggested that both Ni(II) and Cu(II) boun d to the protein in an octahedral manner with three vacant coordinatio n sites, as previously observed for Zn(II). A substituted pyrrolidine- dicarboxylic acid was designed as a structurally rigid IDA compound an d coupled to a matrix. Using this support in an immobilized metal affi nity chromatography (IMAC), RBP/H-3(A) was purified from the bacterial cell extract in one step with unprecedented efficiency. Conclusions: Although the His, metal-binding site used here had been removed from t he substrate pocket of an enzyme and exposed to solvent on a protein s urface, 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 matr ix with improved behavior was designed, allowing highly selective puri fication of RBP/H-3(A) and of His(3)-tagged REP as well. Such rational design of supramolecular recognition may be generally useful in the f ields of protein engineering and drug design.