Multi-method global analysis of thermodynamics and kinetics in reconstitution of monellin

Accurate and precise determinations of thermodynamic parameters of binding are important steps toward understanding many biological mechanisms. Here, a multi-method approach to binding analysis is applied and a detailed error analysis is introduced. Using this approach, the binding thermodynamics and kinetics of the reconstitution of the protein monellin have been quantitatively determined in detail by simultaneous analysis of data collected with fluorescence spectroscopy, surface plasmon resonance and isothermal titration calorimetry at 25°C, pH 7.0 and 150 mM NaCl. Monellin is an intensely sweet protein composed of two peptide chains that form a single globular domain. The kinetics of the reconstitution reaction are slow, with an association rate constant, k_on of 8.8 × 10³ M^-1 s ^-1 and a dissociation rate constant, k_off of 3.1 × 10^-4 s^-1. The equilibrium constant K_A is 2.8 × 10⁷ M^-1 corresponding to a standard free energy of association, ΔG°, of -42.5 kJ/mol. The enthalpic component, ΔH°, is -18.7 kJ/mol and the entropic contribution, ΔS°, is 79.8 J mol^-1 K^-1 (-TΔS° = -23.8 kJ/mol). The association of monellin is therefore a bimolecular intra-protein association whose energetics are slightly dominated by entropie factors.