Subunit dissociation affects DNA binding in a dimeric lac repressor produced by C-terminal deletion

Area of Science:

• Biochemistry
• Molecular Biology
• Protein Dynamics

Background:

• Dimeric lac repressor protein binding affinity is influenced by C-terminal interactions.
• Thermodynamic linkage between dimer-monomer and protein-DNA equilibria is proposed to affect binding.
• Investigating these linkages requires understanding protein dissociation and unfolding.

Purpose of the Study:

• To explore the thermodynamic linkage in dimeric lac repressor proteins.
• To determine the free energy changes associated with dimer dissociation and monomer unfolding.
• To quantify the dissociation constants (Kd) for specific lac repressor mutants.

Main Methods:

• Urea denaturation studies were performed on two dimeric lac repressor mutants (-11 aa and R3).
• The free energy change for dimer to unfolded monomer conversion was determined.
• Free energy change for protomer unfolding was measured using a monomeric mutant (Y282D).

Main Results:

• Dimer dissociation and monomer unfolding were found to be concerted processes under denaturing conditions.
• The dissociation constant (Kd) for the -11 aa dimer mutant was 7.7 x 10(-8) M.
• The dissociation constant (Kd) for the R3 protein mutant was 3.2 x 10(-11) M.

Conclusions:

• The study quantifies the thermodynamic linkage between dimer dissociation and monomer unfolding in lac repressor proteins.
• The results provide a detailed understanding of how structural changes affect protein-DNA binding affinity.
• Specific mutations significantly alter the stability and dissociation characteristics of the dimeric lac repressor.