THERMODYNAMICS OF LIGAND-BINDING TO TRP REPRESSOR

The thermodynamics of L-tryptophan and operator DNA binding to the try ptophan repressor of Escherichia coli were analyzed by titration micro calorimetry and van't Hoff analysis of footprinting titrations, respec tively. At 25-degrees-C in 10 mM sodium phosphate, pH 7.6, and 0.1 M N aCl, the binding of L-tryptophan to the repressor is characterized by values of DELTAG-degrees = -6.04, DELTAH-degrees = - 14.7, and TDELTAS -degrees = -8.67 kcal/mol. The temperature dependence of DELTAH-degree s yields DELTAC(p)degrees = -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 o perator DNA to the repressor is characterized by values of DELTAG-degr ees = -13.3 kcal/mol, DELTAH-degrees = -1.55 kcal/mol, TDELTAS-degrees = 11.8 kcal/mol, and DELTAC(p)degrees = -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 experimenta lly observed DELTAC(p)degrees values were compared with DELTAC(p)degre es values calculated according to several methods based on various pro posed relationships between surface area changes and heat capacity cha nges. Regardless of which method is used, we find poor agreement betwe en the calorimetric results for L-tryptophan binding and the surface a reas calculated from X-ray data; the direction of the discrepancy is t hat the X-ray data underestimate the value of DELTAC(p)degrees. Better agreement is obtained by incorporating solution data on repressor fle xibility, suggesting that DELTAC(p)degrees measurements may report on protein dynamical transitions accompanying ligand binding. For the cas e of DNA binding there is apparently fortuitous agreement between the measured and calculated DELTAC(p)degrees values, despite clear limitat ions in calculating DELTAC(p)degrees for this type of reaction.