title: Determination of RNase A/2'-cytidine monophosphate binding affinity and enthalpy by a global fit of thermal unfolding curves.
abstract: A spectropolarimeter was used to measure the thermal response curves of RNase A in the presence and absence of the ligand cytidine 2'-monophosphate. A coupled equilibrium model was used to describe the dissociation of the protein-ligand complex (NL<==>N + L) and the thermal unfolding of the free protein (N<==>U). The unfolding curves of the protein in the presence of several different concentrations of ligand were fit to this coupled equilibrium model using global linkage analysis. The best-fitted values for the thermal unfolding of the apo-protein were 60.9 +/- 0.2 degrees C (T(m)) and 105.5 +/- 1.4 kcal/mol (DeltaH), while the fitted values for the dissociation of the protein-ligand complex were 1.6 +/- 0.4 microM (K(D)) and 18.7 +/- 1.0 kcal mol(-1) (DeltaH(L)). These values were in excellent agreement with values obtained by other methods. The sensitivity of the data fitting was compared using linear or quadratic temperature dependence for the response curves of the free ligand (L), native apo-protein (N), native ligand-bound protein (NL), and unfolded apo-protein (U). There was no significant improvement in the precision of the fitted data when the temperature-dependent response for each species (N, L, NL, and U) was expressed as quadratic functions of temperature.
authors.affiliation: Department of Chemistry, University of Alabama at Birmingham, 901 South 14th Street, Alabama 35294, USA.
authors.forename: Cecil L
authors.initials: CL
authors.lastname: Jones
authors.forename: Fred
authors.initials: F
authors.lastname: Fish
authors.forename: Donald D
authors.initials: DD
authors.lastname: Muccio
mesh_terms: D001665:Binding Sites; D002151:Calorimetry / Q000379:methods; D002942:Circular Dichroism; D003568:Cytidine Monophosphate / Q000378:metabolism*; D017510:Protein Folding; D012259:Ribonuclease, Pancreatic / Q000378:metabolism*; D013696:Temperature; D013816:Thermodynamics
chemical_list: D012259:Ribonuclease, Pancreatic; D003568:Cytidine Monophosphate