Article:Mosbahi, Khédidja, Lemaître, Christelle, Mobasheri, Hamid et al. (6 more authors) (2002) The cytotoxic domain of colicin E9 is a channel-forming endonuclease. Nature Structural Biology. pp. 476-484. ISSN 1545-9985 https://doi.org/10.1038/nsb797 eprints@whiterose.ac.uk https://eprints.whiterose.ac.uk/ Reuse Items deposited in White Rose Research Online are protected by copyright, with all rights reserved unless indicated otherwise. They may be downloaded and/or printed for private study, or other acts as permitted by national copyright laws. The publisher or other rights holders may allow further reproduction and re-use of the full text version. This is indicated by the licence information on the White Rose Research Online record for the item. TakedownIf you consider content in White Rose Research Online to be in breach of UK law, please notify us by emailing eprints@whiterose.ac.uk including the URL of the record and the reason for the withdrawal request. White Rose Consortium ePrints Repositoryhttp://eprints.whiterose.ac.uk/ This is an author produced version of a paper published in Nature Structural Biology. This paper has been peer-reviewed but does not include final publisher proof-corrections or journal pagination.White Rose Repository URL for this paper: http://eprints.whiterose.ac.uk/archive/00001066/ Citation for the published paper Mosbahi, Khédidja and Lemaître, Christelle and Keeble, Anthony H. and Mobasheri, Hamid and Morel, Bertrand and James, Richard and Moore, Geoffrey R. and Lea, Edward J. A. and Kleanthous, Colin (2002) The cytotoxic domain of colicin E9 is a channel-forming endonuclease. Nature Structural Biology, 9 (6). pp. 476-484. Citation for this paperTo refer to the repository paper, the following format may be used: Mosbahi, Khédidja and Lemaître, Christelle and Keeble, Anthony H. and Mobasheri, Hamid and Morel, Bertrand and James, Richard and Moore, Geoffrey R. and Lea, Edward J. A. and Kleanthous, Colin (2002) Colicin E9 is a 60 kDa toxin that is normally released from colicinogenic bacteria in the form of a heterodimeric complex with its 9.5 kDa immunity protein, Im9 (ref. 14). The immunity protein protects the colicin-producing bacterium from the activity of its own toxin but is jettisoned on entry of the colicin into a susceptible cell 15 . Hence, the form of the toxin tested in the bilayer experiments had the immunity protein removed (see Materials andMethods section). Previous work from our laboratory has shown that this form of the toxin retains complete biological activity 14 . Immunity-free colicin E9 (2 nM) was added to the cis chamber of a bilayer apparatus in 10 mM Tris/HCl buffer at pH 7.5, containing 0.1 M NaCl and 10 mM CaCl 2 , and a potential difference (p.d.) applied across the membrane. Random, fluctuating current was observed that showed evidence of opening and closing events with conductance of the order of ~100 pS, although larger conductance states were also seen ( Fig. 1a). In order to identify the region(s) of the protein responsible for this a...
A detergent-solubilized fraction of skin mucus of carp (Cyprinus carpio) induced ion channels after reconstitution into planar lipid bilayers. A differential extraction using a non-ionic detergent followed by electrophoretic separation led to the isolation of two hydrophobic 31 -kDa and 27-kDa proteins. In contrast to the 27-kDa protein, which was glycosylated, the 31-kDa did not bind to concanavalin A. The reconstitution of these proteins into a planar lipid bilayer restored the ionophore behavior already observed with the crude mucus. The main unit conductance levels were about 900 pS for the 27-kDa protein and 500 pS for the 31-kDa protein, and selectivity measurements gave P J P , ratios of 0.6 and 1.0, respectively. These proteins had large potent microbicidal activities (0.018 -0.1 8 pM) against different strains of gramnegative and gram-positive bacteria. This behavior can be compared with insect defensins that are known to form large ion channels in the bacterial membrane. To exclude the eventuality of bacterial origin, the bacterial flora of the crude mucus were analysed and the following were identified: Pseudomonas cepacia ; Micrococcus luteus; Micrococcus roseus; Flavobacterium sp. ; Aeromonas hydrophila. Antibacterial assays with both proteins were performed against these specific strains and revealed good growth inhibition activities. Furthermore, microsequencing analysis showed that the 31-kDa protein was protected on its N-terminal extremity in contrast to the 27-kDa protein, which had a 19-amino-acid sequence. This last sequence, when compared with sequences in protein data banks, did not reveal any significant similarities to other proteins. These results suggest that these novel proteins could be involved in antibacterial defense processes in fish.
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