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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.
- Source :
-
Biochemistry [Biochemistry] 1994 Nov 29; Vol. 33 (47), pp. 14126-35. - Publication Year :
- 1994
-
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.
- Subjects :
- Animals
Base Sequence
Binding Sites
Carbonic Anhydrases chemistry
Carbonic Anhydrases genetics
Cattle
Drug Stability
Escherichia coli
Histidine chemistry
Humans
Imino Acids metabolism
Molecular Sequence Data
Molecular Structure
Protein Engineering
Protein Folding
Protein Structure, Secondary
Recombinant Fusion Proteins isolation & purification
Retinol-Binding Proteins genetics
Retinol-Binding Proteins isolation & purification
Retinol-Binding Proteins metabolism
Structure-Activity Relationship
Thermodynamics
Retinol-Binding Proteins chemistry
Zinc metabolism
Subjects
Details
- Language :
- English
- ISSN :
- 0006-2960
- Volume :
- 33
- Issue :
- 47
- Database :
- MEDLINE
- Journal :
- Biochemistry
- Publication Type :
- Academic Journal
- Accession number :
- 7947824
- Full Text :
- https://doi.org/10.1021/bi00251a023