Unfolding of colicin A during its translocation through the Escherichia coli envelope as demonstrated by disulfide bond engineering.
Three double cysteine mutants, each possessing a disulfide bond in its pore-forming domain, were used to study the translocation of colicin A through the Escherichia coli envelope. These mutated colicins were able to exert their in vivo channel activity only after their disulfide bonds had been reduced by dithiothreitol. In solution, the reduction of the disulfide bonds by dithiothreitol was a slow process whose kinetics depended on the position of the disulfide bond (t1/2 varying between 35 and 100 s). This t1/2 was strongly decreased (t1/2 = 8-9 s) upon predenaturation of the mutated colicins with urea. The t1/2 values of reduction of the mutants bound to E. coli-sensitive cells were similar to those of predenatured colicins. This suggested that the interaction of the oxidized double cysteine mutants with the E. coli envelope triggered their unfolding. The disulfide bonds did not prevent but delayed the translocation of the colicins. The amplitude of the delay and the time at which it occurred during translocation depended on the position of the disulfide bond. We could discriminate between the delays accumulated during binding to the receptor and those during the translocation via OmpF and the Tol proteins.
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Laboratoire d'Ingénierie et de Dynamique des Systèmes Membranaires, Centre de Biochimie et de Biologie Moleculaire, Marseille.
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D001425:Bacterial Outer Membrane Proteins; D001692:Biological Transport; D003087:Colicins / Q000737:chemistry* / Q000378:metabolism; D004220:Disulfides; D004229:Dithiothreitol / Q000494:pharmacology; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins; D026901:Membrane Transport Proteins; D009154:Mutation; D017510:Protein Folding; D018000:Receptors, Peptide / Q000378:metabolism
D001425:Bacterial Outer Membrane Proteins; C044592:BtuB protein, E coli; D003087:Colicins; D004220:Disulfides; D029968:Escherichia coli Proteins; D026901:Membrane Transport Proteins; D018000:Receptors, Peptide; D004229:Dithiothreitol