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 thermally induced unfolding. It was found that both proteins denature in a two-state equilibrium (native dimer versus unfolded monomer) and that CcdA has a significantly lower thermodynamic stability. Using a numerical model, which was developed earlier by us, and on the basis of the determined thermodynamic parameters the concentration dependence of the denaturation transition temperature was obtained for both proteins. This concentration dependence may be of physiological significance, as the concentration of both ccd addiction proteins cannot exceed a certain limit because their expression is controlled by autoregulation. The influence of DNA on the thermal stability of the two proteins was probed. 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-stranded oligonucleotides, neither cognate nor unspecific.
Dao-Thi
M H
MH
Dienst Ultrastruktuur, Vrije Universiteit Brussel, Paardenstraat 65, Sint-Genesius-Rode, B-1640, Belgium.
Messens
J
J
Wyns
L
L
Backmann
J
J
D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001427:Bacterial Toxins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002152:Calorimetry, Differential Scanning; D002942:Circular Dichroism; D004247:DNA / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019281:Dimerization; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics; D005453:Fluorescence; D005798:Genes, Bacterial / Q000235:genetics*; D019791:Guanidine / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D009838:Oligodeoxyribonucleotides / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D009875:Operator Regions, Genetic / Q000235:genetics; D010957:Plasmids / Q000235:genetics*; D011489:Protein Denaturation / Q000187:drug effects; D017510:Protein Folding; D013379:Substrate Specificity; D013696:Temperature; D013816:Thermodynamics; D014508:Urea / Q000494:pharmacology
D001426:Bacterial Proteins; D001427:Bacterial Toxins; C068355:CcdA protein, Bacteria; C068356:CcdB protein, Plasmid F; D004268:DNA-Binding Proteins; D009838:Oligodeoxyribonucleotides; D014508:Urea; D004247:DNA; D019791:Guanidine