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

Abstract

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 kon = 4971±160 M-1·s -1 and koff = 0.063±0.009 s-1 respectively. The ratio kon/koff was very similar to the binding constant determined by titration calorimetry, suggesting that binding is a two-state process. The values for ∆Cp, ∆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 ∆Cp is independent of temperature. The values for ∆Cp 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.