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

Study Summary

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

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P
 

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)


Relevant PDB Entries

Structure ID Release Date Resolution Structure Title
2ADL 2006-08-22 Solution structure of the bacterial antitoxin CcdA: Implications for DNA and toxin binding
2H3A 2006-11-21 Structural basis for nucleic acid and toxin recognition of the bacterial antitoxin CcdA
2ADN 2006-08-22 Solution structure of the bacterial antitoxin CcdA: Implications for DNA and toxin binding
2H3C 2006-11-21 Structural basis for nucleic acid and toxin recognition of the bacterial antitoxin CcdA
3VUB 1998-06-17 1.4 CCDB, A TOPOISOMERASE POISON FROM E. COLI
4VUB 1998-10-14 1.45 CCDB, A TOPOISOMERASE POISON FROM ESCHERICHIA COLI
3HPW 2009-08-11 1.45 CcdB dimer in complex with one C-terminal CcdA domain
3TCJ 2012-08-15 1.93 CcdB dimer from V. fisheri in complex with one C-terminal domain of F-plasmid CcdA
3G7Z 2009-08-11 2.35 CcdB dimer in complex with two C-terminal CcdA domains
2VUB 1998-06-17 2.45 CCDB, A TOPOISOMERASE POISON FROM E. COLI
1VUB 1998-07-15 2.6 CCDB, A TOPOISOMERASE POISON FROM E. COLI
1X75 2005-05-17 2.8 CcdB:GyrA14 complex

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
98.0 Toxin CcdB Q46996 CCDB2_ECOLX
100.0 Toxin CcdB P62554 CCDB_ECOLI
100.0 Toxin CcdB P62555 CCDB_ECO57
100.0 Toxin CcdB Q52043 CCDB4_ECOLX
100.0 Toxin CcdB Q52042 CCDB3_ECOLX
90.3 Antitoxin CcdA Q46995 CCDA2_ECOLX
100.0 Antitoxin CcdA P62552 CCDA_ECOLI
100.0 Antitoxin CcdA P62553 CCDA_ECO57