Characterization and modification of a monomeric mutant of the lactose repressor protein.
A monomeric mutant lactose repressor protein (T-41), containing serine at position 282 in place of tyrosine [Schmitz, A., Schmeissner, U., Miller, J. H., & Lu, P. (1976) J. Biol. Chem. 251, 3359-3366], has been purified by a series of chromatographic and precipitation methods. The molecular weight of the mutant as determined by gel filtration was approximately 40,000. The inducer equilibrium binding constant for the mutant was comparable to that of the tetrameric wild-type repressor at pH 7.5, whereas operator DNA binding was not detectable. In contrast to wild-type repressor, equilibrium and kinetic rate constants for inducer binding to the monomer were largely independent of pH; thus, the quaternary structure of the wild-type repressor is required for the pH-associated effects on inducer binding. Although ultraviolet absorbance difference spectra indicated that inducer binding to T-41 protein elicited different changes in the environment of aromatic residues from those generated in wild-type repressor, the shift in the fluorescence emission maximum in response to inducer binding was similar for T-41 and wild-type repressors. Similarity in 1-anilinonaphthalene-8-sulfonic acid binding to monomer and tetramer suggests that this fluorophore does not bind at subunit interfaces. Modification of Cys-281 with methyl methanethiosulfonate was observed at low molar ratios of reagent per T-41 monomer (4-fold). This result is in contrast to data observed for tetrameric wild-type repressor which requires high molar ratios for this cysteine to react. We conclude that Cys-281, adjacent to the site of the T-41 mutation, is located on the surface of the monomer in this region crucial for subunit interaction.
D000911:Antibodies, Monoclonal; D004926:Escherichia coli / Q000235:genetics*; D005798:Genes, Bacterial; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology; D007700:Kinetics; D008741:Methyl Methanesulfonate / Q000031:analogs & derivatives / Q000494:pharmacology; D008970:Molecular Weight; D009154:Mutation; D012097:Repressor Proteins / Q000235:genetics* / Q000302:isolation & purification; D014157:Transcription Factors / Q000235:genetics*
D000911:Antibodies, Monoclonal; D012097:Repressor Proteins; D014157:Transcription Factors; C014674:methyl methanethiosulfonate; D007544:Isopropyl Thiogalactoside; D008741:Methyl Methanesulfonate