Designed disulfide between N-terminal domains of lactose repressor disrupts allosteric linkage.
Substitution of Cys for Val at position 52 of the lac repressor was designed to permit disulfide bond formation between the two N-terminal DNA binding domains that comprise an operator DNA binding site. This position marks the closest approach of these domains based on the x-ray crystallographic structures of the homologous purine holorepressor-operator complex and lac repressor-operator complex (Schumacher, M. A., Choi, K. Y., Zalkin, H., and Brennan, R. G. (1994) Science 266, 763-770; Lewis, M., Chang, G., Horton, N.C., Kercher, M. A., Pace, H. C., Schumacher, M. A., Brennan, R. G., and Lu, P. (1996) Science 271, 1247-1254). The V52C mutation was generated by site-specific methods, and the mutant protein was purified and characterized. In the reduced form, V52C bound operator DNA with slightly increased affinity. Exposure to oxidizing conditions resulted in disulfide bond formation, and the oxidized protein bound operator DNA with approximately 6-fold higher affinity than wild-type protein. Inducer binding for both oxidized and reduced forms of V52C was comparable to wild-type lac repressor. In the presence of inducer, the reduced protein exhibited wild-type, diminished DNA binding. In contrast, DNA binding for the oxidized form was unaffected by inducer, even at 1 mM. Thus, the formation of the designed disulfide between Cys52 side chains within each dimer renders the protein-operator complex unresponsive to sugar binding, presumably by disrupting the allosteric linkage between operator and inducer binding.
Falcon
C M
CM
Department of Biochemistry & Cell Biology, Rice University, Houston, Texas 77005-1892, USA.
Swint-Kruse
L
L
Matthews
K S
KS
D000494:Allosteric Regulation; D000495:Allosteric Site; D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D018360:Crystallography, X-Ray; D003545:Cysteine; D004269:DNA, Bacterial / Q000737:chemistry / Q000378:metabolism; D004220:Disulfides; D029968:Escherichia coli Proteins; D007700:Kinetics; D007763:Lac Operon; D056968:Lac Repressors; D008961:Models, Structural; D016297:Mutagenesis, Site-Directed; D009690:Nucleic Acid Conformation; D010084:Oxidation-Reduction; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012097:Repressor Proteins / Q000737:chemistry* / Q000378:metabolism; D014633:Valine
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004220:Disulfides; D029968:Escherichia coli Proteins; D056968:Lac Repressors; D011994:Recombinant Proteins; D012097:Repressor Proteins; D014633:Valine; D003545:Cysteine