Wild-type operator binding and altered cooperativity for inducer binding of lac repressor dimer mutant R3

Area of Science:

• Molecular biology
• Protein engineering
• Biochemistry

Background:

• The lactose repressor is a tetrameric protein crucial for regulating lactose metabolism in bacteria.
• Understanding protein quaternary structure and its impact on function is vital for molecular biology.

Purpose of the Study:

• To investigate the functional consequences of altering the lactose repressor's oligomeric state.
• To characterize a novel dimeric lactose repressor variant (R3) and compare its DNA binding and allosteric properties to the wild-type.

Main Methods:

• Protein purification of the R3 variant.
• Gel retardation assays for DNA binding analysis.
• Molecular sieve chromatography for determining Stokes radius and molecular mass.
• Inducer binding assays at varying pH and in the presence of operator DNA.

Main Results:

• The R3 protein, a dimer, exhibits wild-type equilibrium and kinetic DNA binding affinity.
• R3 shows altered inducer binding affinity and cooperativity, particularly at elevated pH and when bound to operator DNA.
• Higher inducer concentrations are required to release operator DNA from R3 compared to wild-type repressor.

Conclusions:

• Substitution of the C-terminal region with a dimerization domain alters the allosteric behavior of the lactose repressor.
• The subunit interface plays a critical role in modulating the repressor's response to inducers and operator binding.