Membrane topology of the colicin A pore-forming domain analyzed by disulfide bond engineering.
Four colicin A double-cysteine mutants possessing a disulfide bond in their pore-forming domain were constructed to study the translocation and the pore formation of colicin A. The disulfide bonds connected alpha-helices 1 and 2, 2 and 10, 3 and 9, or 3 and 10 of the pore-forming domain. The disulfide bonds did not prevent the colicin A translocation through the Escherichia coli envelope. However, the mutated colicins were able to exert their in vivo channel activity only after reduction of their disulfide bonds. In vitro studies with brominated phospholipid vesicles and planar lipid bilayers revealed that the disulfide bond that connects the alpha-helices 2 and 10 prevented the colicin A membrane insertion, whereas the other double-cysteine mutants inserted into lipid vesicles. The disulfide bonds that connect either the alpha-helices 1 and 2 or 3 and 10 were unable to prevent the formation of a conducting channel in presence of membrane potential. These results indicate that alpha-helices 1, 2, 3, and 10 remain at the membrane surface after application of a membrane potential.
Duché
D
D
Laboratoire d'Ingénierie et Dynamique des Systèmes Membranaires, Institut de Biologie Structurale et Microbiologie du CNRS, Marseille, France. Biochemistry and Molecular Biology, Faculty of.
Izard
J
J
González-Mañas
J M
JM
Parker
M W
MW
Crest
M
M
Chartier
M
M
Baty
D
D
D003087:Colicins / Q000737:chemistry*; D003545:Cysteine / Q000737:chemistry; D004220:Disulfides; D004926:Escherichia coli / Q000737:chemistry; D015640:Ion Channel Gating; D007473:Ion Channels / Q000737:chemistry*; D008051:Lipid Bilayers; D008565:Membrane Proteins / Q000737:chemistry*; D008567:Membranes, Artificial; D016297:Mutagenesis, Site-Directed; D017433:Protein Structure, Secondary; D013329:Structure-Activity Relationship
D003087:Colicins; D004220:Disulfides; D007473:Ion Channels; D008051:Lipid Bilayers; D008565:Membrane Proteins; D008567:Membranes, Artificial; D003545:Cysteine