Thermodynamics of cyclic nucleotide binding to the cAMP receptor protein and its T127L mutant.
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 degrees C. Although, the binding of the first cAMP molecule to CRP is exothermic with an enthalpy change (delta Hb) of -6 kJ mol-1, a heat capacity change (delta Cp) of -0.300 kJ mol-1 K-1, and an entropy increase (delta Sb) 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 degrees C. The binding of the second cAMP molecule is accompanied by large changes of 48.1 kJ mol-1 in delta Hb, of -1.4 kJ mol-1 K-1 in delta Cp, and of 255 J mol-1 K-1 in delta Sb at 24 degrees C and 0.5 M KCl phosphate buffer. In contrast, the overall binding of cGMP to CRP is exothermic and non-cooperative with delta Hb, delta Cp, and delta Sb 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 degrees C.
Gorshkova
I
I
Center for Advanced Research in Biotechnology, National Institute of Standards and Technology, Rockville, Maryland 20850, USA.
Moore
J L
JL
McKenney
K H
KH
Schwarz
F P
FP
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism; D002151:Calorimetry; D002352:Carrier Proteins; D000242:Cyclic AMP / Q000378:metabolism*; D002373:Cyclic AMP Receptor Protein / Q000737:chemistry* / Q000378:metabolism*; D006152:Cyclic GMP / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism; D007700:Kinetics; D008433:Mathematics; D008962:Models, Theoretical; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013816:Thermodynamics
D001426:Bacterial Proteins; D002352:Carrier Proteins; D002373:Cyclic AMP Receptor Protein; D011994:Recombinant Proteins; D000242:Cyclic AMP; D006152:Cyclic GMP