Subunit Dissociation Affects DNA Binding in a Dimeric Lac Repressor Produced by C-Terminal Deletion

Jie Chen, Kathleen S. Matthews

Research output: Contribution to journalArticle

Abstract

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 × 10−8 M, and the corresponding value for R3 protein was 3.2 × 10∓11 M. Using these dimer-monomer dissociation constants, the intrinsic equilibrium binding constant for DNA could be derived by fits to operator binding isotherms; for both −11 aa and R3, these values were very similar and are comparable to the corresponding apparent equilibrium dissociation constant for the wild-type protein under the conditions employed (∼ 10−10 M). These data demonstrate directly that the monomer-dimer and dimer-operator equilibria are thermodynamically linked for the −11 aa dimer and presumably for other dimeric proteins that display reduced apparent operator binding. Linkage of protein-protein, protein-DNA, and protein-ligand binding is an essential feature of genetic regulatory proteins.

Original languageEnglish (US)
Pages (from-to)8728-8735
Number of pages8
JournalBiochemistry
Volume33
Issue number29
DOIs
StatePublished - Jul 1 1994
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry

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