Characterization of His-X3-His sites in alpha-helices of synthetic metal-binding bovine somatotropin.
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 alpha-helix (His-X3-His variants). The affinities of these proteins for Cu(II) were characterized by measuring their partition 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-X3-His variants described here, these constants range from 2 x 10(4) to 1.6 x 10(6) M-1. The metal affinity of a His-X3-His site depends on the rigidity of the helix into which the site is engineered. The affinities of the His-X3-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.
Suh
S S
SS
Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena 91125.
Haymore
B L
BL
Arnold
F H
FH
D000818:Animals; D001665:Binding Sites; D002417:Cattle; D002614:Chelating Agents / Q000737:chemistry; D002846:Chromatography, Affinity; D003300:Copper / Q000737:chemistry; D003911:Dextrans; D013006:Growth Hormone / Q000737:chemistry* / Q000235:genetics; D006639:Histidine / Q000737:chemistry; D007098:Imino Acids; D007700:Kinetics; D008024:Ligands; D008667:Metalloproteins / Q000737:chemistry*; D011092:Polyethylene Glycols; D011487:Protein Conformation; D015202:Protein Engineering; D013816:Thermodynamics
D002614:Chelating Agents; D003911:Dextrans; D007098:Imino Acids; D008024:Ligands; D008667:Metalloproteins; D011092:Polyethylene Glycols; D006639:Histidine; D003300:Copper; D013006:Growth Hormone; C008109:iminodiacetic acid