title: Grafting of a high-affinity Zn(II)-binding site on the beta-barrel of retinol-binding protein results in enhanced folding stability and enables simplified purification.
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.
authors.affiliation: Max-Planck-Institut für Biophysik, Abteilung Molekulare Membranbiologie, Frankfurt am Main, Germany.
authors.forename: H N
authors.initials: HN
authors.lastname: Müller
authors.forename: A
authors.initials: A
authors.lastname: Skerra
mesh_terms: D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002256:Carbonic Anhydrases / Q000737:chemistry / Q000235:genetics; D002417:Cattle; D004355:Drug Stability; D004926:Escherichia coli; D006639:Histidine / Q000737:chemistry; D006801:Humans; D007098:Imino Acids / Q000378:metabolism; D008969:Molecular Sequence Data; D015394:Molecular Structure; D015202:Protein Engineering; D017510:Protein Folding; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification; D012177:Retinol-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013329:Structure-Activity Relationship; D013816:Thermodynamics; D015032:Zinc / Q000378:metabolism*
chemical_list: D007098:Imino Acids; D011993:Recombinant Fusion Proteins; D012177:Retinol-Binding Proteins; D006639:Histidine; D002256:Carbonic Anhydrases; D015032:Zinc; C008109:iminodiacetic acid