Thermodynamic characterization of monomeric and dimeric forms of CcdB (controller of cell division or death B protein)

Bajaj, Kanika ; Chakshusmathi, Gadhiyaram ; Bachhawat-Sikder, Kiran ; Surolia, Avadhesha ; Varadarajan, Raghvan (2004) Thermodynamic characterization of monomeric and dimeric forms of CcdB (controller of cell division or death B protein) Biochemical Journal, 380 . pp. 409-417. ISSN 0264-6021

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The protein CcdB (̲controller of ̲cell division or ̲death ̲B) is an F-plasmid-encoded toxin that acts as an inhibitor of Escherichia coli DNA gyrase. The stability and aggregation state of CcdB have been characterized as a function of pH and temperature. Size-exclusion chromatography revealed that the protein is a dimer at pH 7.0, but a monomer at pH 4.0. CD analysis and fluorescence spectroscopy showed that the monomer is well folded, and has similar tertiary structure to the dimer. Hence intersubunit interactions are not required for folding of individual subunits. The stability of both forms was characterized by isothermal denaturant unfolding and calorimetry. The free energies of unfolding were found to be 9.2 kcal·mol−1 (1 cal4≈184 J) and 21 kcal·mol−1 at 298 K for the monomer and dimer respectively. The denaturant concentration at which one-half of the protein molecules are unfolded (Cm) of the dimer is dependent on protein concentration, whereas the Cm of the monomer is independent of protein concentration, as expected. Although thermal unfolding of the protein in aqueous solution is irreversible at neutral pH, it was found that thermal unfolding is reversible in the presence of GdmCl (guanidinium chloride). Differential scanning calorimetry in the presence of low concentrations of GdmCl in combination with isothermal denaturation melts as a function of temperature were used to derive the stability curve for the protein. The value of ΔCp (representing the change in excess heat capacity upon protein denaturation) is 2.8±0.2 kcal·mol−1·K−1 for unfolding of dimeric CcdB, and only has a weak dependence on denaturant concentration.

Item Type:Article
Source:Copyright of this article belongs to Portland Press.
Keywords:CcdB (controller of Cell Division or Death B Protein); DSC (differential Scanning Calorimetry); GdmCl (guanidinium Chloride); Stability Curve
ID Code:57298
Deposited On:26 Aug 2011 04:24
Last Modified:21 Dec 2011 06:20

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