ABSTRACT
Technological advances in gene sequencing, mutagenesis, and X-ray structure deter mination should continue to lead to more information on possible zinc binding sites and crystal structures of biological metal binding sites. Thus, the search for putative zinc sites can be increasingly guided by the growing number of zinc sites available for reference. Since a family of proteins that have this site will likely conserve the zinc binding ligands, this should be the first level of comparison. In addition, there may be a catalytic group or a possible secondary ligand orienter that is important to the stability of the zinc that is conserved in the vicinity of the proposed zinc ligands. This can also narrow the search for prospective family members. After such a group of proteins has been compiled, the expression and purification of a member of this group can be carried out. Analytical measurement of zinc by an established analytical means that provides information on the metal to protein stoichiometry will give the first indication of the validity of the postulate. Methods such as zinc blotting should be avoided since they are not quantitative and are artifact-prone. Thus, the original studies indicated that several known zinc enzymes did not exchange with 65Zn and several nonmetalloproteins did bind 65Zn [285-287]. XAFS can give infor mation on the type and number of zinc ligands. Mutagenesis of putative zinc ligands in conjunction with zinc binding and enzyme activity measurements will provide further evidence for the predicted zinc binding site. In these experiments some form of affinity chromatography or affinity labeling of the expressed protein should be made to avoid ambiguous answers due to a mixture of active and inactive proteins, some of which bind zinc. The ultimate verification of the zinc binding site will need structural determination by X-ray crystallography or NMR.
