THERMODYNAMIC EVALUATION OF BINDING INTERACTIONS IN THE METHIONINE REPRESSOR SYSTEM OF ESCHERICHIA-COLI USING ISOTHERMAL TITRATION CALORIMETRY

The binding interactions of the methionine repressor protein, MetJ, from Escherichia coli with its cognate, metbox DNA sequence and corepressor S-adenosylmethionine were examined using calorimetric methods. A detailed thermodynamic characterization of this system which exhibits the recently reported (beta alpha alpha)(2) binding motif provides values for Delta G, Delta H, and Delta S for each step in the repressor binding cycle. These studies show that, in the presence of corepressor, MetJ binds to a single metbox operator site with Delta G = -7.7 kcal . mol(-1), whereas in the absence of corepressor, the free energy of interaction with a single site is -5.8 kcal . mol(-1). Cooperative interactions between two repressor molecules bound to two adjacent sites contribute an additional free energy of -1.3 kcal . mol(-1) to binding at the second site. Binding is enthalpically unfavorable in the absence of the corepressor with Delta H = +2.6 kcal . mol(-1) but becomes exothermic with Delta H = -4.6 kcal . mol(-1) when corepressor is present. The heat capacity for the system decreases significantly by Delta C-p = -290 cal . mol(-1). K-1 on a per site basis when the protein binds to DNA, and interactions between repressor molecules bound to adjacent sites contribute a Delta C-p = -800 cal . mol(-1). K-1 indicating that solvent exclusion plays a significant role in binding in this system. The corepressor binds to the unbound repressor protein with a free energy of Delta G = -6.0 kcal . mol(-1) and to the MetJ-operator complex with Delta G = -6.95 kcal.mol(-1). Repressor binding to random-sequence DNA was estimated to occur with a free energy of -5.7 kcal . mol(-1) in the presence of corepressor. These data clearly indicate that MetJ repressor dimer binds specifically to the central region of its 8 bp cognate metbox operator but recognizes partial operator sequences as short as 6 bp. Cooperativity in binding of adjacent MetJ dimers to a double metbox sequence is demonstrated to be important in determining the energetics of the interaction. Finally, the corepressor S-adenosylmethionine enhances the affinity of MetJ for its recognition site DNA by a factor of 25 and contributes significantly to the net exothermicity of repressor binding.