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

ABSTRACT: 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.

SUBMITTER: Bajaj K

PROVIDER: S-EPMC1224168 | biostudies-other | 2004 Jun

REPOSITORIES: biostudies-other

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Project description:The F(1)F(0)-adenosine triphosphate (ATP) synthase rotational motor synthesizes most of the ATP required for living from adenosine diphosphate, Pi, and a proton electrochemical gradient across energy-transducing membranes of bacteria, chloroplasts, and mitochondria. However, as a reversible nanomotor, it also hydrolyzes ATP during de-energized conditions in all energy-transducing systems. Thus, different subunits and mechanisms have emerged in nature to control the intrinsic rotation of the enzyme to favor the ATP synthase activity over its opposite and commonly wasteful ATPase turnover. Recent advances in the structural analysis of the bacterial and mitochondrial ATP synthases are summarized to review the distribution and mechanism of the subunits that are part of the central rotor and regulate its gyration. In eubacteria, the epsilon subunit works as a ratchet to favor the rotation of the central stalk in the ATP synthase direction by extending and contracting two alpha-helixes of its C-terminal side and also by binding ATP with low affinity in thermophilic bacteria. On the other hand, in bovine heart mitochondria, the so-called inhibitor protein (IF(1)) interferes with the intrinsic rotational mechanism of the central gamma subunit and with the opening and closing of the catalytic beta-subunits to inhibit its ATPase activity. Besides its inhibitory role, the IF(1) protein also promotes the dimerization of the bovine and rat mitochondrial enzymes, albeit it is not essential for dimerization of the yeast F(1)F(0) mitochondrial complex. High-resolution electron microscopy of the dimeric enzyme in its bovine and yeast forms shows a conical shape that is compatible with the role of the ATP synthase dimer in the formation of tubular the cristae membrane of mitochondria after further oligomerization. Dimerization of the mitochondrial ATP synthase diminishes the rotational drag of the central rotor that would decrease the coupling efficiency between rotation of the central stalk and ATP synthesis taking place at the F(1) portion. In addition, F(1)F(0) dimerization and its further oligomerization also increase the stability of the enzyme to natural or experimentally induced destabilizing conditions.

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

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