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

Bajaj, Kanika and Chakshumathi, Ghadiyaram and Bachhawat-Sikder, Kiran and Surolia, Avadhesha and Varadarajan, Raghavan (2004) Thermodynamic characterization of monomeric and dimeric forms of CcdB (controller of cell division or death B protein). In: Biochemical Journal, 380 (2). 409–417.

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Abstract

The protein CcdB (controller of cell division or death B)is an Fplasmid-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. Sizeexclusion 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 formswas characterized by isothermal denaturant unfolding and calorimetry. The free energies of unfolding were found to be 9.2 kcal · $mol^−^1$ (1 cal≈4.184 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 $(C_m)$ of the dimer is dependent on protein concentration,whereas the $C_m$ of the monomer is independent of protein 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 $\Delta C_p$ (representing the change in excess heat capacity upon protein denaturation) is $2.8\pm 0.2$ kcal · $mol^−^1$ ·$K^−^1$ for unfolding of dimeric CcdB, and only has a weak dependence on denaturant concentration.

Item Type:	Journal Article
Publication:	Biochemical Journal
Publisher:	Biochemical Scociety
Additional Information:	This article copyright belongs to Biochemical Society.
Keywords:	CcdB (controller of cell division or death B protein);DSC (differential scanning calorimetry);GdmCl (guanidinium chloride);stability curve
Department/Centre:	Division of Biological Sciences > Molecular Biophysics Unit
Date Deposited:	27 Jun 2007
Last Modified:	16 Jan 2012 05:39
URI:	http://eprints.iisc.ac.in/id/eprint/11236

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