Characterization of mutants affecting the KRK sequence in the carboxyl-terminal domain of lac repressor.
The lac repressor carboxyl-terminal region is required for tetramer assembly and protein stability. To further investigate this region, especially the unusual sequence KRK, four deletion mutants eliminating the carboxyl-terminal 34, 35, 36, and 39 amino acids and five substitution mutants at the position of Arg-326, R326K, R326A, R326E, R326L, and R326W, were constructed using site-specific mutagenesis. The -34-amino-acid (aa) mutant, missing the most carboxyl-proximal lysine from the KRK sequence, exhibited lower affinity for both operator and inducer and lower protein stability than dimeric proteins studied previously. The -35-aa mutant with RK missing, as well as -36 aa and -39 aa, for which the entire KRK sequence was deleted, yielded inactive polypeptides that could be detected only by monoclonal antibody for lac repressor. In the Arg-326 mutant proteins, operator binding affinity was decreased by approximately 6-fold, the shift in inducer binding at elevated pH was diminished, and protein stability was decreased. Dramatic decreases in protein expression and stability occurred with substitution at position 326 by glutamate, leucine, or tryptophan. These results suggest that Arg-326 plays an important role in the formation of the proper tertiary structure necessary for inducer and operator affinity and for protein stability.
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Department of Biochemistry & Cell Biology, Rice University, Houston, Texas 77251, USA.
Matthews
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D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics; D004269:DNA, Bacterial / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D007544:Isopropyl Thiogalactoside / Q000378:metabolism; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009875:Operator Regions, Genetic; D010449:Peptide Mapping; D011485:Protein Binding; D011489:Protein Denaturation; D012097:Repressor Proteins / Q000737:chemistry* / Q000235:genetics; D017384:Sequence Deletion; D013329:Structure-Activity Relationship; D014357:Trypsin / Q000494:pharmacology; D014508:Urea
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D046911:Macromolecular Substances; D012097:Repressor Proteins; D007544:Isopropyl Thiogalactoside; D014508:Urea; D014357:Trypsin