The introduction of specific sites for heavy metal binding in a crystalline protein

Heavy metal derivatives of the galactose binding protein of Salmonella typhimurium were obtained by the treatment of crystals with carbon disulfide under anaerobic conditions, followed by exposure to mercury-containing reagents. Carbon disulfide reacts with protein amino groups to give a metastable dithiocarbamate, which is susceptible to covalent derivatization by mercurials. The number of amino groups which react for any particular crystalline protein will depend on the pH, the composition of the crystal mother liquor, and the steric accessibility limitations imposed by crystal packing. Direct reaction with protein crystals, rather than solution derivatization followed by purification and subsequent crystallization, is used to promote isomorphism of the derivative crystal with the native and to limit the number of available sites. For the S. typhimurium galactose binding protein, carbon disulfide treatment, followed by reaction with 2-chloromercuri-4-nitrophenol, resulted in binding at two sites at pH 8.0. Similar treatment with dimercury acetate gave one binding site for the dimercurial at the same pH. Both derivatives were isomorphous with the native crystal to a resolution of at least 3.5 A. These heavy atom derivatives have been used to produce an interpretable electron density map of the protein at 3-A resolution.