The thermodynamic stability of the proteins of the ccd plasmid addiction system

The two opponents, toxin (CcdB, LetB or LetD, protein G, LynB) and antidote (CcdA, LetA, protein H, LynA), in the plasmid addiction system ccd of the F plasmid were studied by different biophysical methods. The thermodynamic stability was measured at different temperatures combining denaturant and t hermally induced unfolding. It was found that both proteins denature in a t wo-state equilibrium (native dimer versus unfolded monomer) and that CcdA h as a significantly lower thermodynamic stability. Using a numerical model, which was developed earlier by us, and on the basis of the determined therm odynamic parameters the concentration dependence of the denaturation transi tion temperature was obtained for both proteins. This concentration depende nce may be of physiological significance, as the concentration of both ccd addiction proteins cannot exceed a certain limit because their expression i s controlled by autoregulation. The influence of DNA on the thermal stability of the two proteins was probe d. It was found that cognate DNA increases the melting temperature of CcdA. In the presence of non-specific DNA the thermal stability was not changed. The melting temperature of CcdB was not influenced by the applied double-s tranded oligonucleotides, neither cognate nor unspecific. (C) 2000 Academic Press.