Thermodynamic characterization of monomeric and dimeric forms of CcdB (controller of cell division or death B protein).
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 x mol(-1) (1 cal approximately 4.184 J) and 21 kcal x 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 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 Delta C (p) (representing the change in excess heat capacity upon protein denaturation) is 2.8+/-0.2 kcal x mol(-1) x K(-1) for unfolding of dimeric CcdB, and only has a weak dependence on denaturant concentration.
Bajaj
Kanika
K
Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560 012, India.
Chakshusmathi
Ghadiyaram
G
Bachhawat-Sikder
Kiran
K
Surolia
Avadhesha
A
Varadarajan
Raghavan
R
D001426:Bacterial Proteins / Q000737:chemistry*; D001427:Bacterial Toxins / Q000737:chemistry*; D019281:Dimerization; D029968:Escherichia coli Proteins / Q000737:chemistry; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D013816:Thermodynamics
D001426:Bacterial Proteins; D001427:Bacterial Toxins; C068356:CcdB protein, Plasmid F; D029968:Escherichia coli Proteins