Thermodynamics of ligand binding to trp repressor

Abstract

The thermodynamics of L-tryptophan and operator DNA binding to the tryptophan repressor of Escherichia coli were analyzed by titration microcalorimetry and van't Hoff analysis of footprinting titrations, respectively. At 25 degrees C in 10 mM sodium phosphate, pH 7.6, and 0.1 M NaCl, the binding of L-tryptophan to the repressor is characterized by values of delta G degrees = -6.04, delta H degree = -14.7, and T delta S degree = -8.67 kcal/mol. The temperature dependence of delta H degree yields delta Cp degree = -0.46 +/- 0.08 kcal/(mol.K) per dimer. The binding is noncooperative at all temperatures studied. At 23 degrees C in 2.5 mM sodium phosphate, pH 7.6, and 25 mM NaCl, the binding of operator DNA to the repressor is characterized by values of delta G degree = -13.3 kcal/mol, delta H degree = -1.55 kcal/mol, T delta S degree = 11.8 kcal/mol, and delta Cp degree = -0.54 +/- 0.10 kcal/(mol.K). Changes in water-accessible surface areas upon binding of L-tryptophan or DNA were calculated from X-ray crystal structures. The experimentally observed delta Cp degree values were compared with delta Cp degree values calculated according to several methods based on various proposed relationships between surface area changes and heat capacity changes. Regardless of which method is used, we find poor agreement between the calorimetric results for L-tryptophan binding and the surface areas calculated from X-ray data; the direction of the discrepancy is that the X-ray data underestimate the value of delta Cp degree.(ABSTRACT TRUNCATED AT 250 WORDS)

Interaction of the DNA‐binding domain of Drosophila heat shock factor with its cognate DNA site: A thermodynamic analysis using analytical ultracentrifugation

“…Therefore, although the near-0 value of AC:,o for the wild-type DNA binding to dHSF(33-163) suggests that minimal changesin polypeptide conformation occur upon DNA binding, this is probably not the case for the binding of the mutant DNA. The near-0 value of AC: for the binding of the wild-type DNA is in contrast to recent studies on sequence-specific protein-DNA interactions, which do show such temperature dependence Jin et al, 1993;Lundback et al, 1993;Spolar & Record, 1994). It should be noted, however, that reactants used in these studies were of different size and had different stoichiometries than those studied here.…”

Calorimetric studies of the energetics of protein—DNA interactions in the E. coli methionine repressor (MetJ) system

“…Control experiments for nonspecific (non-operator sequence) DNA effects on MetJ (+ SAM) showed no effect on stability of the protein in DSC, and only weak (LIH = -8 kJ.mol-') non-stoichiometric binding in ITC measurements. Unlike several other protein-DNA systems [12,20,21], the enthalpies of formation of the MetJ: SAM : DNA complex show no significant variation with temperature over a 1040°C range (Table 1; AC, = -0.2 f 0.3 kJ.K-'.mol-').…”

“…The only other equivalent system in which relevant thermodynamic measurements have been reported to date for protein-DNA interactions is the Cro protein-DNA complex, studied by pulsed-flow microcalorimetry under saturating conditions to give the heats of complex formation [20]. The thermodynamics of co-repressor and operator DNA binding by the E. coli TrpR repressor have also been studied [21], although the protein-DNA complex data could only be obtained by indirect Van? Hoff methods.…”

Thermodynamic and structural insights into CSL‐DNA complexes

“…28,29 Our calculated values tended to underestimate the DCp of binding by as much as 0.1 kcal mol À1 K À1 . Because there are no structures of CSL in the absence of DNA, our calculations cannot account for folding of CSL coupled to DNA binding, which may account for the discrepancy in the experimentally determined and calculated values of DCp.…”