Abstract
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.
Highlights
The lac operon is a well-studied system for discerning principles that govern regulation of genetic expression [1, 2]
The small hinge sequence that connects the N-terminal and core domains must be involved in this communication, since the x-ray crystallographic structure of the protein-operator complex reveals few direct contacts between these distinct domains [10]
Based on the crystal structures of the highly homologous purine repressor, PurR holorepressor-DNA [30], and LacI-DNA complexes [10], a mutation was designed at Val52 within the hinge region to generate a protein in which a disulfide bond could be formed between the N-terminal domains (Fig. 1)
Summary
Mutagenesis—Plasmid pJC1 [20] contains the complete LacI gene and was used as the mutagenesis vector. The Chameleon double-stranded mutagenesis protocol from Stratagene (La Jolla, CA) was followed using a selection oligonucleotide that converts the PstI site in pJC1 to an XhoI site. The oligonucleotides were annealed to denatured plasmid DNA, and extension and ligation were performed using T7 polymerase (New England Biolabs, Beverly, MA), T4 ligase, and singlestranded binding protein (Promega, Madison, WI). After inactivation of T4 ligase, an initial digestion with PstI was followed by transformation into XLmutS cells (Stratagene). Plasmid DNA purified from these cells using the Wizard preparation protocol (Promega) was digested with PstI and transformed into DH5␣ cells (Life Technologies, Inc.).
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