Subunit dissociation affects DNA binding in a dimeric lac repressor produced by C-terminal deletion.
The reduction in apparent operator binding affinity found for dimeric lac repressor proteins produced by disruption of the C-terminal coiled-coil interaction has been proposed to derive from thermodynamic linkage between dimer-monomer and protein-DNA equilibria [Brenowitz et al. (1991) J. Biol. Chem. 266, 1281]. To explore this linkage, we have employed two dimeric proteins, a deletion mutant (-11 aa) missing 11 amino acids at the C-terminus that has diminished apparent operator binding affinity [Chen & Matthews (1992) J. Biol. Chem. 267, 13843] and a mutant (R3) that binds to operator with wild-type affinity in which the C-terminal leucine heptad repeats of lac repressor were replaced by the GCN4 dimerization sequence [Alberti et al. (1993) EMBO J. 12, 3227; Chen et al. (1994) J. Biol. Chem. (in press)]. To avoid the complexities of working at the low concentrations of protein required by the high affinity between the monomer subunits, urea denaturation studies were undertaken to determine the free energy change(s) for dissociation and/or unfolding. Under denaturing conditions, dimer dissociation and monomer unfolding were found to be concerted processes, and the free energy change for the overall process of dimer to unfolded monomer was derived from these experiments for the two dimeric proteins. A monomeric mutant (Y282D) of the lactose repressor was examined to determine the free energy change of protomer unfolding. From the combination of these data, the Kd for -11 aa dimer dissociation was determined to be 7.7 x 10(-8) M, and the corresponding value for R3 protein was 3.2 x 10(-11) M.(ABSTRACT TRUNCATED AT 250 WORDS)
Chen
J
J
Department of Biochemistry and Cell Biology, Rice University, Houston, Texas 77251.
Matthews
K S
KS
D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D046911:Macromolecular Substances; D009875:Operator Regions, Genetic; D011485:Protein Binding; D011489:Protein Denaturation; D011994:Recombinant Proteins; D012097:Repressor Proteins / Q000737:chemistry* / Q000378:metabolism; D017384:Sequence Deletion; D013329:Structure-Activity Relationship
D004268:DNA-Binding Proteins; D046911:Macromolecular Substances; D011994:Recombinant Proteins; D012097:Repressor Proteins; D004247:DNA