Thermodynamics of Cyclic Nucleotide Binding to the cAMP Receptor Protein and Its T127L Mutant

Abstract

The thermodynamics of the binding of cyclic adenosine monophosphate (cAMP) and its non-functional analog, cyclic guanosine monophosphate (cGMP), to cyclic AMP receptor protein (CRP) and its T127L mutant were investigated by isothermal titration calorimetry (ITC) in 0.2 and 0.5 M KCl phosphate buffer (pH 7.0) at 24 and 39°C. Although, the binding of the first cAMP molecule to CRP is exothermic with an enthalpy change (ΔHb) of −6 kJ mol−1, a heat capacity change (ΔCp) of −0.300 kJ mol−1 K−1, and an entropy increase (ΔSb) of 72 J mol−1 K−1, the overall binding of cAMP to CRP is endothermic and positively cooperative: binding of the first cAMP molecule increases the affinity for the second one by more than an order of magnitude at 24°C. The binding of the second cAMP molecule is accompanied by large changes of 48.1 kJ mol−1 in ΔHb, of −1.4 kJ mol−1 K−1 in ΔCp, and of 255 J mol−1 K−1in ΔSb at 24°C and 0.5 M KCl phosphate buffer. In contrast, the overall binding of cGMP to CRP is exothermic and non-cooperative with ΔHb, ΔCp, and ΔSb values close to the those values for binding of the first cAMP molecule to CRP. The point mutation, T127L, switches off the cooperativity between the cAMP ligated binding sites without affecting the binding constant of cAMP and changes the specificity of the protein so that transcription is now activated only upon cGMP binding. All the binding reactions to CRP and the mutant are mainly entropically driven at 24°C. The thermodynamics of the binding of cyclic adenosine monophosphate (cAMP) and its non-functional analog, cyclic guanosine monophosphate (cGMP), to cyclic AMP receptor protein (CRP) and its T127L mutant were investigated by isothermal titration calorimetry (ITC) in 0.2 and 0.5 M KCl phosphate buffer (pH 7.0) at 24 and 39°C. Although, the binding of the first cAMP molecule to CRP is exothermic with an enthalpy change (ΔHb) of −6 kJ mol−1, a heat capacity change (ΔCp) of −0.300 kJ mol−1 K−1, and an entropy increase (ΔSb) of 72 J mol−1 K−1, the overall binding of cAMP to CRP is endothermic and positively cooperative: binding of the first cAMP molecule increases the affinity for the second one by more than an order of magnitude at 24°C. The binding of the second cAMP molecule is accompanied by large changes of 48.1 kJ mol−1 in ΔHb, of −1.4 kJ mol−1 K−1 in ΔCp, and of 255 J mol−1 K−1in ΔSb at 24°C and 0.5 M KCl phosphate buffer. In contrast, the overall binding of cGMP to CRP is exothermic and non-cooperative with ΔHb, ΔCp, and ΔSb values close to the those values for binding of the first cAMP molecule to CRP. The point mutation, T127L, switches off the cooperativity between the cAMP ligated binding sites without affecting the binding constant of cAMP and changes the specificity of the protein so that transcription is now activated only upon cGMP binding. All the binding reactions to CRP and the mutant are mainly entropically driven at 24°C.