title: Thermodynamic evaluation of binding interactions in the methionine repressor system of Escherichia coli using isothermal titration calorimetry.
abstract: 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 Cp = -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 Cp = -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.
authors.affiliation: Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710.
authors.forename: D E
authors.initials: DE
authors.lastname: Hyre
authors.forename: L D
authors.initials: LD
authors.lastname: Spicer
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D002151:Calorimetry; D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins; D008954:Models, Biological; D008969:Molecular Sequence Data; D011485:Protein Binding; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism*; D012436:S-Adenosylmethionine / Q000378:metabolism; D013816:Thermodynamics
chemical_list: D001426:Bacterial Proteins; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D012097:Repressor Proteins; C060486:methionine repressor protein, Bacteria; C489220:methionine repressor protein, E coli; D012436:S-Adenosylmethionine