ENGINEERING METAL COORDINATION SITES INTO THE ANTIBODY LIGHT-CHAIN

A three-histidine Zn2+ binding site based on the carbonic anhydrase B site has been engineered into the four available sites on the light chain of the fluorescein binding antibody 4-4-20. All mutant antibodies bind fluorescein. Transition metal binding was assayed by tryptophan fluorescence quenching, Two of the four sites exhibit metal affinities consistent with complexation by three ligands. The specificity of the highest affinity site was probed by mutagenesis. For various combinations of histidine, aspartate, and glutamate residues, affinities range from -5 to -10 kcal/mol for Cu2+, -3 to -6.5 kcal/mol for Zn2+, and -3 and -5.5 kcal/mol for Cd2+. Binding is also observed between at least one mutant and Co2+ or Ni2+. The second highest affinity site shows a metal-dependent increase in fluorescein binding, indicating a ternary complex. Several ligand combinations give affinities in a potentially useful range for antibody catalysis with only four amino acid changes.