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

Ghoshal, Alokesh K. ; Swaminathan, Chittoor P. ; Thomas, Celestine J. ; Surolia, Avadhesha ; Varadarajan, Raghavan (1999) Thermodynamic and kinetic analysis of the Escherichia coli thioredoxin-C' fragment complementation system Biochemical Journal, 339 . pp. 721-727. ISSN 0264-6021

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Escherichia coli thioredoxin was cleaved with CNBr at its single Met residue at position 37, which lies in the middle of a longα -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 ΔCp, ΔH0 and ΔG0 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 ΔH0 was linearly dependent on temperature from 8-40 °C, suggesting that ΔCp is independent of temperature. The values for ΔCp and ΔG0 are very similar to the corresponding values for the unfolding of intact thioredoxin at 25 °C. However, both ΔH0 and ΔS are significantly more positive for dissociation of the complex, suggesting a decreased hydrophobic stabilization of the complex relative to the situation for intact thioredoxin.

Item Type:Article
Source:Copyright of this article belongs to Portland Press.
Keywords:Heat Capacity; Isothermal Titration Calorimetry; Peptide Fragments; Stopped-flow Kinetics
ID Code:57299
Deposited On:26 Aug 2011 04:35
Last Modified:13 Dec 2011 12:02

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