title: Multi-method global analysis of thermodynamics and kinetics in reconstitution of monellin.
abstract: 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 degrees 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 x 10(3) M(-1) s(-1) and a dissociation rate constant, k(off) of 3.1 x 10(-4) s(-1). The equilibrium constant K(A) is 2.8 x 10(7) M(-1) corresponding to a standard free energy of association, DeltaG degrees , of -42.5 kJ/mol. The enthalpic component, DeltaH degrees , is -18.7 kJ/mol and the entropic contribution, DeltaS degrees , is 79.8 J mol(-1) K(-1) (-TDeltaS degrees = -23.8 kJ/mol). The association of monellin is therefore a bimolecular intra-protein association whose energetics are slightly dominated by entropic factors.
authors.affiliation: Department of Biophysical Chemistry, Lund University, Lund, Sweden.
authors.forename: Wei-Feng
authors.initials: WF
authors.lastname: Xue
authors.forename: Jannette
authors.initials: J
authors.lastname: Carey
authors.forename: Sara
authors.initials: S
authors.lastname: Linse
mesh_terms: D002151:Calorimetry; D019277:Entropy; D006358:Hot Temperature; D007700:Kinetics; D016018:Least-Squares Analysis; D029583:Menispermaceae / Q000737:chemistry*; D008958:Models, Molecular; D010940:Plant Proteins / Q000737:chemistry* / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D020349:Surface Plasmon Resonance; D017186:Titrimetry
chemical_list: D010940:Plant Proteins; C002541:monellin protein, Dioscoreophyllum cumminsii