title: Comparative thermodynamic analysis of cyclic nucleotide binding to protein kinase A.
abstract: We have investigated the thermodynamic parameters and binding of a regulatory subunit of cAMP-dependent protein kinase (PKA) to its natural low-molecular-weight ligand, cAMP, and analogues thereof. For analysis of this model system, we compared side-by-side isothermal titration calorimetry (ITC) with surface plasmon resonance (SPR). Both ITC and SPR analyses revealed that binding of the protein to cAMP or its analogues was enthalpically driven and characterised by similar free energy values (DeltaG=-9.4 to -10.7 kcal mol-1) for all interactions. Despite the similar affinities, binding of the cyclic nucleotides used here was characterised by significant differences in the contribution of entropy (-TDeltaS) and enthalpy (DeltaH) to DeltaG. The comparison of ITC and SPR data for one cAMP analogue further revealed deviations caused by the method. These equilibrium parameters could be complemented by thermodynamic data of the transition state (DeltaHnot equal, DeltaGnot equal, DeltaSnot equal) for both association and dissociation measured by SPR. This direct comparison of ITC and SPR highlights method-specific advantages and drawbacks for thermodynamic analyses of protein/ligand interactions.
authors.affiliation: Department of Biochemistry, University of Kassel, Heinrich-Plett-Str. 40, D-34132 Kassel, Germany.
authors.forename: Daniela
authors.initials: D
authors.lastname: Moll
authors.forename: Sonja
authors.initials: S
authors.lastname: Schweinsberg
authors.forename: Christian
authors.initials: C
authors.lastname: Hammann
authors.forename: Friedrich W
authors.initials: FW
authors.lastname: Herberg
mesh_terms: D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive / Q000502:physiology; D002151:Calorimetry / Q000379:methods; D002417:Cattle; D000242:Cyclic AMP / Q000032:analysis* / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000032:analysis* / Q000378:metabolism; D008024:Ligands; D008956:Models, Chemical; D008968:Molecular Conformation; D008970:Molecular Weight; D012680:Sensitivity and Specificity; D020349:Surface Plasmon Resonance / Q000379:methods; D013816:Thermodynamics; D013997:Time Factors
chemical_list: D008024:Ligands; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases