Thermodynamic and kinetic analysis of the Escherichia coli thioredoxin-C 'fragment complementation system

Escherichia coli thioredoxin was cleaved with CNBr at its single Met residu e at position 37, which lies in the middle of a long alpha-helix. The two f ragments, 1-37 and 38-108, were purified and characterized by using CD and fluorescence spectroscopy. Both fragments lack structure at neutral pH and room temperature. The secondary and tertiary structural contents of the non covalent complex formed on the mixing of the two peptide fragments are 47 % and 35 % of the intact protein respectively. The thermodynamics and kineti cs of fragment association were characterized by titration calorimetry and stopped-flow fluorescence spectroscopy. Single phases were observed for bot h association and dissociation, with rate constants at 298 K of k(on) = 497 1 +/- 160 M-1 s(-1) and k(off) = 0.063 +/- 0.009 s(-1) respectively. The ra tio k(on)/k(off) was very similar to the binding constant determined by tit ration calorimetry, suggesting that binding is a two-state process. The val ues for Delta C-p, Delta H-0 and Delta G(0) at 298 K for dissociation of th e complex were 5.7 kJ mol(-1) K-1, 45.3 kJ mol(-1) and 29.8 kJ mol(-1) resp ectively. The value for Delta H-0 was linearly dependent on temperature fro m 8-40 degrees C, suggesting that Delta C-p is independent of temperature. The values for Delta C-p and Delta G(0) are very similar to the correspondi ng values for the unfolding of intact thioredoxin at 25 degrees C. However, both Delta H-0 and Delta S are significantly more positive for dissociatio n of the complex, suggesting a decreased hydrophobic stabilization of the c omplex relative to the situation for intact thioredoxin.