Iodination of Escherichia coli lac repressor. Effect of tyrosine modification on repressor activity

Abstract

Treatment of Escherichia coli lac repressor with iodine (Kl3) at 4 degrees and pH 7.5 resulted in the rapid loss of repressor DNA binding activity. At a 30-fold molar excess of iodine to repressor, inactivation was complete within 15 sec. Inducer binding under the same conditions was only slightly affected. Iodinated repressor remained tetrameric, indicating that no gross structural alteration of the protein has taken place. Control experiments demonstrated that side products of the reaction did not contribute to the observed activity loss. Moreover, no restoration of binding activity was observed when iodinated repressor was assayed under a variety of assay conditions. Incubation of repressor with lac operator containing gamma plac DNA during the iodination reaction resulted in approximately 50% protection of binding activity. This protective effect was only partially operator specific, as gammaDNA lacking the operator binding site afforded roughly 25% protection under the same conditions. Incubation with either inducer and anti-inducer molecules during the iodination reaction did not protect repressor DNA binding activity. Iodination with K-131I-3 demonstrated that at complete inactivation virtually all of the bound iodine was recovered as iodotyrosine. A minor amount of cysteine oxidation to cysteinesulfonic acid was also detected. This oxidation encompassed no more than 30% of a single cysteine residue )tentatively identified as cysteine-107). Unstable intermediate oxidation products of cysteine did not appear to be involved in the loss of DNA binding activity. Modification of amino acids other than tyrosine and cysteine was not observed. Tryptic digestion of -131I-labeled repressor suggested that approximately 90% of the incorporated radioactivity was located in the repressor N-terminal tryptic peptide. Automated sequence analysis of iodinated repressor confirmed that at roughly 0.5 bound iodine atoms per repressor subunit (corresponding to approximately 5-10% activity loss) tyrosine residues 7, 12, and 17 were labeled in the ratios 1.0:0.5:0.8. Doubling the amount of bound iodine to 1.0 atom per subunit (corresponding to approximately 50-60% activity loss) did not significantly change the pattern of incorporation.