THERMODYNAMICS OF CYCLIC-NUCLEOTIDE BINDING TO THE CAMP RECEPTOR PROTEIN AND ITS T127L MUTANT

The thermodynamics of the binding of cyclic adenosine monophosphate (c AMP) and its non-functional analog, cyclic guanosine monophosphate (cG MP), to cyclic AMP receptor protein (CRP) and its T127L mutant were in vestigated by isothermal titration calorimetry (ITC) in 0.2 and 0.5 M KCl phosphate buffer (pH 7.0) at 24 and 39 degrees C. Although, the bi nding of the first cAMP molecule to CRP is exothermic with an enthalpy change (Delta H-b) of -6 kJ mol(-1), a heat capacity change (Delta C- p) of -0.300 kJ mol(-1) K-1, and an entropy increase (Delta S-b) 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 t he affinity for the second one by more than an order of magnitude at 2 4 degrees C. The binding of the second cAMP molecule is accompanied by large changes of 48.1 kJ mol(-1) in Delta H-b, of -1.4 kJ mol(-1) K-1 in Delta C-p, and of 255 J mol(-1) K-1 in Delta S-b at 24 degrees C a nd 0.5 M KCl phosphate buffer. In contrast, the overall binding of cGM P to CRP is exothermic and non-cooperative with Delta H-b, Delta C-p, and Delta S-b, values close to the those values for binding of the fir st cAMP molecule to CRP. The point mutation, T127L, switches off the c ooperativity between the cAMP ligated binding sites without affecting the binding constant of cAMP and changes the specificity of the protei n so that transcription is now activated only upon cGMP binding. All t he binding reactions to CRP and the mutant are mainly entropically dri ven at 24 degrees C.