Formation of mixed disulfide adducts at cysteine-281 of the lactose repressor protein affects operator and inducer binding parameters.
The lactose repressor protein has been modified with three sulfhydryl-specific reagents which form mixed disulfide adducts. Methyl methanethiosulfonate (MMTS) and 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) completely reacted with all three cysteine residues, whereas only partial reaction was observed with didansylcystine. Cysteines-107 and -140 reacted stoichiometrically with MMTS and DTNB, while Cys-281 was modified only at higher molar ratios. Didansylcystine reacted primarily with cysteines-107 and -140. Affinity of MMTS-modified repressor for 40 base pair operator DNA was decreased 30-fold compared to unmodified repressor, and this decrease correlated with modification of cysteine-281. DTNB-modified repressor bound operator DNA with a 50-fold weaker affinity than unmodified repressor. Modification of the lac repressor with didanylcystine decreased operator binding only 4-fold, and nonspecific DNA binding increased 3-fold compared to unmodified repressor. No change in the inducer equilibrium binding constant was observed following modification with any of these reagents. In contrast, inducer association and dissociation rate constants were decreased approximately 50-fold for repressor completely modified with MMTS or DTNB, while didansylcystine had minimal effect on inducer binding kinetics. Correlation between modification of Cys-281 and the observed decrease in rate constants indicates that this region of the protein regulates the accessibility of the sugar binding site. The parallel between the increase in the Kd for repressor binding to operator, the altered rate constant for inducer binding, and modification of cysteine-281 suggests that this region of the protein is crucially involved in the function of the repressor protein.
D003545:Cysteine; D003553:Cystine / Q000031:analogs & derivatives / Q000494:pharmacology; D003619:Dansyl Compounds / Q000494:pharmacology; D004220:Disulfides; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology; D007700:Kinetics; D008741:Methyl Methanesulfonate / Q000031:analogs & derivatives* / Q000494:pharmacology; D009579:Nitrobenzoates / Q000494:pharmacology*; D009875:Operator Regions, Genetic; D011485:Protein Binding; D012097:Repressor Proteins / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism*
D003619:Dansyl Compounds; D004220:Disulfides; D009579:Nitrobenzoates; D012097:Repressor Proteins; D014157:Transcription Factors; C038494:di(dansyl)cystine; C014674:methyl methanethiosulfonate; D007544:Isopropyl Thiogalactoside; D003553:Cystine; D004228:Dithionitrobenzoic Acid; D008741:Methyl Methanesulfonate; D003545:Cysteine