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Abstract

In a rational protein design approach, the His₃ Zn(II)-binding site from the active center of human carbonic anhydrase II was transplanted on the beta-barrel of mammalian serum retinol-binding protein (RBP) in a solvent-accessible location on the protein's outer surface. Several mutants of RBP were generated and produced in Escherichia coli, and their Zn(II)-binding properties were investigated in equilibrium dialysis experiments. One mutant, RBP/H₃(A), with His residues introduced at the positions 46, 54, and 56 in the polypeptide sequence was shown to bind Zn(II) specifically with a stoichiometry of 1 and a corresponding dissociation constant equal to 36 +/- 10 nM. Binding of Zn(II) had no influence on the binding of retinoic acid, a natural ligand of RBP. In guanidinium chloride-induced unfolding experiments the mutant was found to be significantly stabilized in the presence of small concentrations of ZnSO₄. This effect could be quantitatively explained using thermodynamic theory. Furthermore, it was demonstrated that the protein-bound Zn(II) is accessible to iminodiacetic acid as an additional chelating ligand without competition for the metal ion. Thus it was possible to use the grafted metal-binding site for the efficient purification of the engineered, bifunctional RBP via immobilized metal affinity chromatography from the bacterial protein extract.