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

Escherichia coli thioredoxin was cleaved with CNBr at its single Met residue at position 37, which lies in the middle of a long α-helix. The two fragments, 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 kinetics of fragment association were characterized by titration calorimetry and stopped-flow fluorescence spectroscopy. Single phases were observed for both association and dissociation, with rate constants at 298 K of k_on = 4971±160 M^-1·s ^-1 and k_off = 0.063±0.009 s^-1 respectively. The ratio k_on/k_off was very similar to the binding constant determined by titration calorimetry, suggesting that binding is a two-state process. The values for ∆C_p, ∆H⁰ and ∆G⁰ at 298 K for dissociation of the complex were 5.7 kJ·mol^-1·K^-1, 45.3 kJ·mol^-1 and 29.8 kJ·mol^-1 respectively. The value for ∆H⁰ was linearly dependent on temperature from 8-40 °C, suggesting that ∆C_p is independent of temperature. The values for ∆C_p and ∆G⁰ are very similar to the corresponding values for the unfolding of intact thioredoxin at 25 °C. However, both ∆H⁰ and ∆S are significantly more positive for dissociation of the complex, suggesting a decreased hydrophobic stabilization of the complex relative to the situation for intact thioredoxin.