Journal of Biological Chemistry
Volume 271, Number 26, Issue of June 28, 1996 pp. 15401-15406
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
ISSN 0021-9258

Membrane Topology of the Colicin A Pore-forming Domain Analyzed by Disulfide Bond Engineering

Denis Duché , Jacques Izard , Juan M. González-Mañas , Michael W. Parker § , Marcel Crest ¶ , Martine Chartier and Daniel Baty

From the Laboratoire d'Ingénierie et Dynamique des Systèmes Membranaires, Institut de Biologie Structurale et Microbiologie du CNRS, 31 chemin J. Aiguier, 13402 Marseille Cedex 20, France, the Department of Biochemistry and Molecular Biology, Faculty of Science, P.O. Box 644, University of the Basque Country, 48080 Bilbao, Spain, the § St. Vincent's Institute of Medical Research, 41 Victoria Parade, Fitzroy, Victoria 3065, Australia, and the ¶ Laboratoire de Neurobiologie, 31 chemin J. Aiguier, 13402 Marseille Cedex 20, France

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 -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.