Characterization of His-X_3-His sites in α-helices of synthetic metal-binding bovine somatotropin

Abstract

Variants of bovine somatotropin have been engineered to contain synthetic metal-binding sites consisting of two solvent-exposed histidines separated by a single turn of an α-helix (His-X_3-His variants). The affinities of these proteins for Cu(II) were characterized by measuring their partitioning coefficients in an aqueous two-phase polymer system. The partition coefficients were used to generate binding constants for formation of a complex between the engineered metal-binding site and Cu(II) chelated to an iminodiacetic acid derivative of polyethylene glycol. For three His-X_3-His variants described here, these constants range from 2×104 to 1.66×610^6 M^(-1). The metal affinity of a His-X_3-His site depends on the rigidity of the helix into which the site is engineered. The affinities of the His-X_3-His sites for Cu(II) are large enough to dramatically increase not only the partitioning of these proteins in aqueous two-phase systems, but also their retention times on a metal-affinity chromatography column. Both these features can greatly facilitate the purification of engineered proteins. Criteria for choosing positions for incorporating metal-binding sites are discussed.