Tetranitromethane modification of the tyrosine residues of the lactose repressor.
Repressor protein modified with N-ethylmaleimide has been used to determine the exclusive effects of tyrosine nitration by tetranitromethane. Since modification of proteins with tetranitromethane generally results in both cysteine oxidation and tyrosine nitration, N-ethylmaleimide has been used to protect the cysteines in the repressor against oxidation in subsequent tetranitromethane reactions. Nitration of tyrosine residues in repressor previously reacted with N-ethylmaleimide results in loss of both specific and nonspecific DNA-binding activities. Na2S2O4 reduction of tetranitromethane-modified protein restores partial operator DNA-binding and complete nonspecific DNA-binding capability. Residues primarily affected are tyrosines 7 and 17, which are both in the NH2 terminus. Inter- and intramolecular cross-links which are observed in the modified protein can be minimized by altering reaction conditions; the cross-links present occur between sites located in the NH2 termini. Modification of the core protein also results in loss of the operator DNA-binding capacity, and subsequent reduction restores partial operator-binding activity. Both operator and nonspecific DNA-binding capabilities of the repressor protein are protected by the presence of nonspecific DNA during the tetranitromethane modification, and simultaneously the extent of nitration is decreased.
D001665:Binding Sites; D004247:DNA / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D005033:Ethylmaleimide / Q000494:pharmacology; D007700:Kinetics; D046911:Macromolecular Substances; D008697:Methane / Q000031:analogs & derivatives*; D011485:Protein Binding; D012097:Repressor Proteins / Q000378:metabolism*; D013774:Tetranitromethane / Q000494:pharmacology*; D014157:Transcription Factors / Q000378:metabolism*; D014443:Tyrosine
D046911:Macromolecular Substances; D012097:Repressor Proteins; D014157:Transcription Factors; D014443:Tyrosine; D004247:DNA; D013774:Tetranitromethane; D005033:Ethylmaleimide; D008697:Methane