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

摘要

Escherichia coli thioredoxin was cleaved with CNBr at its single Met residue at position 37, which lies in the middle of a long lpha -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 $k_o_n = 4971±160 M^-^1·s ^-^1$ and $k_o_f_f = 0.063±0.009 s^-^1$ respectively. The ratio $k_o_n/k_o_f_f$ was very similar to the binding constant determined by titration calorimetry, suggesting that binding is a two-state process. The values for $Delta C_p$, $Delta H^0$ and $Delta G^0$ 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 $Delta H^0$ was linearly dependent on temperature from 8-40 °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 corresponding values for the unfolding of intact thioredoxin at 25 °C. However, both $Delta H^0$ and $Delta S$ are significantly more positive for dissociation of the complex, suggesting a decreased hydrophobic stabilization of the complex relative to the situation for intact thioredoxin.