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


Abstract

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. Study holds ProTherm entries: 8402, 8403, 8404, 8405, 8406, 8407, 8408, 8409, 8410, 8411, 8412, 8413, 8414, 8415, 8416, 8417, 8418, 8419, 8420, 8421, 8422, 8423, 8424, 8425, 8426, 8427, 8428, 8429 Extra Details: dimer; F plasmid; Let; plasmid addiction; post-segregational killing

Submission Details

ID: AEmFw26i3

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:36 p.m.

Version: 1

Publication Details
Dao-Thi MH;Messens J;Wyns L;Backmann J,J. Mol. Biol. (2000) The thermodynamic stability of the proteins of the ccd plasmid addiction system. PMID:10873460
Additional Information

Number of data points 76
Proteins Toxin CcdB ; Toxin CcdB ; Antitoxin CcdA
Unique complexes 2
Assays/Quantities/Protocols Experimental Assay: Cm prot_conc:5.73 micro M, temp:30.0 C, buffers:phosphate: 50 mM, pH:7.0 ; Experimental Assay: m prot_conc:5.73 micro M, temp:30.0 C, buffers:phosphate: 50 mM, pH:7.0 ; Experimental Assay: dG_H2O prot_conc:5.73 micro M, temp:30.0 C, buffers:phosphate: 50 mM, pH:7.0 ; Experimental Assay: Cm prot_conc:5.73 micro M, buffers:phosphate: 50 mM, temp:25.0 C, pH:7.0 ; Experimental Assay: m prot_conc:5.73 micro M, buffers:phosphate: 50 mM, temp:25.0 C, pH:7.0 ; Experimental Assay: dG_H2O prot_conc:5.73 micro M, buffers:phosphate: 50 mM, temp:25.0 C, pH:7.0 ; Experimental Assay: Cm prot_conc:5.73 micro M, temp:20.0 C, buffers:phosphate: 50 mM, pH:7.0 ; Experimental Assay: m prot_conc:5.73 micro M, temp:20.0 C, buffers:phosphate: 50 mM, pH:7.0 ; Experimental Assay: dG_H2O prot_conc:5.73 micro M, temp:20.0 C, buffers:phosphate: 50 mM, pH:7.0 ; Experimental Assay: Cm prot_conc:5.73 micro M, temp:15.0 C, buffers:phosphate: 50 mM, pH:7.0 ; Experimental Assay: m prot_conc:5.73 micro M, temp:15.0 C, buffers:phosphate: 50 mM, pH:7.0 ; Experimental Assay: dG_H2O prot_conc:5.73 micro M, temp:15.0 C, buffers:phosphate: 50 mM, pH:7.0 ; Experimental Assay: Cm prot_conc:5.73 micro M, buffers:phosphate: 50 mM, temp:10.0 C, pH:7.0 ; Experimental Assay: m prot_conc:5.73 micro M, buffers:phosphate: 50 mM, temp:10.0 C, pH:7.0 ; Experimental Assay: dG_H2O prot_conc:5.73 micro M, buffers:phosphate: 50 mM, temp:10.0 C, pH:7.0 ; Experimental Assay: Cm prot_conc:5.73 micro M, buffers:phosphate: 50 mM, temp:5.0 C, pH:7.0 ; Experimental Assay: m prot_conc:5.73 micro M, buffers:phosphate: 50 mM, temp:5.0 C, pH:7.0 ; Experimental Assay: dG_H2O prot_conc:5.73 micro M, buffers:phosphate: 50 mM, temp:5.0 C, pH:7.0 ; Experimental Assay: Tm pH:8.0, buffers:Tris-HCl: 25 mM ; Experimental Assay: Tm buffers:phosphate: 50 mM, pH:7.0 ; Experimental Assay: Tm buffers:cacodylate: 25 mM, pH:6.5 ; Experimental Assay: Tm buffers:Sodium citrate: 25 mM, pH:5.6 ; Experimental Assay: Cm prot_conc:4.92 micro M, temp:20.0 C, pH:8.0, buffers:Tris-HCl: 50 mM ; Experimental Assay: m prot_conc:4.92 micro M, temp:20.0 C, pH:8.0, buffers:Tris-HCl: 50 mM ; Experimental Assay: dG_H2O prot_conc:4.92 micro M, temp:20.0 C, pH:8.0, buffers:Tris-HCl: 50 mM ; Experimental Assay: Cm prot_conc:5.26 micro M, temp:20.0 C, buffers:phosphate: 50 mM, pH:7.0 ; Experimental Assay: m prot_conc:5.26 micro M, temp:20.0 C, buffers:phosphate: 50 mM, pH:7.0 ; Experimental Assay: dG_H2O prot_conc:5.26 micro M, temp:20.0 C, buffers:phosphate: 50 mM, pH:7.0 ; Experimental Assay: Cm temp:20.0 C, buffers:Sodium cacodylate: 50 mM, prot_conc:5.65 micro M, pH:6.5 ; Experimental Assay: m temp:20.0 C, buffers:Sodium cacodylate: 50 mM, prot_conc:5.65 micro M, pH:6.5 ; Experimental Assay: dG_H2O temp:20.0 C, buffers:Sodium cacodylate: 50 mM, prot_conc:5.65 micro M, pH:6.5 ; Experimental Assay: Cm temp:20.0 C, buffers:Sodium citrate: 50 mM, prot_conc:4.84 micro M, pH:5.6 ; Experimental Assay: m temp:20.0 C, buffers:Sodium citrate: 50 mM, prot_conc:4.84 micro M, pH:5.6 ; Experimental Assay: dG_H2O temp:20.0 C, buffers:Sodium citrate: 50 mM, prot_conc:4.84 micro M, pH:5.6 ; Experimental Assay: Cm temp:20.0 C, prot_conc:8.55 micro M, pH:8.25, buffers:Tris-HCl: 50 mM ; Experimental Assay: m temp:20.0 C, prot_conc:8.55 micro M, pH:8.25, buffers:Tris-HCl: 50 mM ; Experimental Assay: dG_H2O temp:20.0 C, prot_conc:8.55 micro M, pH:8.25, buffers:Tris-HCl: 50 mM ; Experimental Assay: Cm temp:20.0 C, prot_conc:8.55 micro M, pH:8.0, buffers:Tris-HCl: 50 mM ; Experimental Assay: m temp:20.0 C, prot_conc:8.55 micro M, pH:8.0, buffers:Tris-HCl: 50 mM ; Experimental Assay: dG_H2O temp:20.0 C, prot_conc:8.55 micro M, pH:8.0, buffers:Tris-HCl: 50 mM ; Experimental Assay: Cm pH:7.5, temp:20.0 C, prot_conc:8.55 micro M, buffers:Tris-HCl: 50 mM ; Experimental Assay: m pH:7.5, temp:20.0 C, prot_conc:8.55 micro M, buffers:Tris-HCl: 50 mM ; Experimental Assay: dG_H2O pH:7.5, temp:20.0 C, prot_conc:8.55 micro M, buffers:Tris-HCl: 50 mM ; Experimental Assay: Cm temp:35.0 C, prot_conc:8.55 micro M, buffers:phosphate: 50 mM, pH:7.0 ; Experimental Assay: m temp:35.0 C, prot_conc:8.55 micro M, buffers:phosphate: 50 mM, pH:7.0 ; Experimental Assay: dG_H2O temp:35.0 C, prot_conc:8.55 micro M, buffers:phosphate: 50 mM, pH:7.0 ; Experimental Assay: Cm prot_conc:8.55 micro M, temp:30.0 C, buffers:phosphate: 50 mM, pH:7.0 ; Experimental Assay: m prot_conc:8.55 micro M, temp:30.0 C, buffers:phosphate: 50 mM, pH:7.0 ; Experimental Assay: dG_H2O prot_conc:8.55 micro M, temp:30.0 C, buffers:phosphate: 50 mM, pH:7.0 ; Experimental Assay: Cm prot_conc:8.55 micro M, temp:20.0 C, buffers:phosphate: 50 mM, pH:7.0 ; Experimental Assay: m prot_conc:8.55 micro M, temp:20.0 C, buffers:phosphate: 50 mM, pH:7.0 ; Experimental Assay: dG_H2O prot_conc:8.55 micro M, temp:20.0 C, buffers:phosphate: 50 mM, pH:7.0 ; Experimental Assay: Cm prot_conc:8.55 micro M, buffers:phosphate: 50 mM, temp:10.0 C, pH:7.0 ; Experimental Assay: m prot_conc:8.55 micro M, buffers:phosphate: 50 mM, temp:10.0 C, pH:7.0 ; Experimental Assay: dG_H2O prot_conc:8.55 micro M, buffers:phosphate: 50 mM, temp:10.0 C, pH:7.0 ; Experimental Assay: Cm prot_conc:8.55 micro M, buffers:phosphate: 50 mM, temp:5.0 C, pH:7.0 ; Experimental Assay: m prot_conc:8.55 micro M, buffers:phosphate: 50 mM, temp:5.0 C, pH:7.0 ; Experimental Assay: dG_H2O prot_conc:8.55 micro M, buffers:phosphate: 50 mM, temp:5.0 C, pH:7.0 ; Experimental Assay: Cm temp:20.0 C, pH:6.5, buffers:Sodium cacodylate: 50 mM, prot_conc:1.71 micro M ; Experimental Assay: m temp:20.0 C, pH:6.5, buffers:Sodium cacodylate: 50 mM, prot_conc:1.71 micro M ; Experimental Assay: dG_H2O temp:20.0 C, pH:6.5, buffers:Sodium cacodylate: 50 mM, prot_conc:1.71 micro M ; Experimental Assay: Cm temp:20.0 C, pH:6.5, buffers:Sodium cacodylate: 50 mM, prot_conc:4.27 micro M ; Experimental Assay: m temp:20.0 C, buffers:Sodium cacodylate: 50 mM, prot_conc:4.27 micro M, pH:6.5 ; Experimental Assay: dG_H2O temp:20.0 C, pH:6.5, buffers:Sodium cacodylate: 50 mM, prot_conc:4.27 micro M ; Experimental Assay: Cm temp:20.0 C, prot_conc:8.55 micro M, buffers:Sodium cacodylate: 50 mM, pH:6.5 ; Experimental Assay: m temp:20.0 C, prot_conc:8.55 micro M, buffers:Sodium cacodylate: 50 mM, pH:6.5 ; Experimental Assay: dG_H2O temp:20.0 C, prot_conc:8.55 micro M, buffers:Sodium cacodylate: 50 mM, pH:6.5 ; Experimental Assay: Cm temp:20.0 C, pH:5.6, prot_conc:8.55 micro M, buffers:Sodium citrate: 50 mM ; Experimental Assay: m temp:20.0 C, pH:5.6, prot_conc:8.55 micro M, buffers:Sodium citrate: 50 mM ; Experimental Assay: dG_H2O temp:20.0 C, pH:5.6, prot_conc:8.55 micro M, buffers:Sodium citrate: 50 mM
Libraries Mutations for sequence MKQRITVTVDSDSYQLLKAYDVNISGLVSTTMQNEARRLRAERWKAENQEGMAEVARFIEMNGSFADENRDW ; Mutations for sequence MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDKVSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI
Sequence Assay Result Units