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. Author manuscript; available in PMC: 2013 Dec 28.
Published in final edited form as: Science. 2013 Jun 28;340(6140):10.1126/science.1237864. doi: 10.1126/science.1237864

Figure 1. Isolation and characterization of the ColE9 translocon from the OM of Escherichia coli.

Figure 1

(A) Strategy for the capture and isolation of the ColE9 translocon. Nuclease colicins such as ColE9 (brown) contain a hairpin receptor-binding (R−) domain, an N-terminal translocation (T−) domain that includes the intrinsically unstructured translocation domain (IUTD), shown as a dotted line, and a C-terminal cytotoxic DNase domain to which is bound the immunity protein Im9 (yellow). The engineered cysteines for disulfide bond formation between the TolB binding epitope of ColE9 and histidine-tagged TolB (purple) are shown as red circles. (B) BN-PAGE of the isolated translocon showing the complex contains the ColE9-Im9 complex, BtuB, OmpF and TolB. (C) 12% SDS-PAGE of the translocon ± β-mercaptoethanol (β-Me) confirming the presence of the disulfide between ColE9 and TolB. (D) Native-state ESI-MS spectrum of the ColE9 translocon. Coloured inserts give assignments for species observed in the spectrum, all devoid of detergent: orange hexagon, the intact complex that includes a single molecule of lipopolysaccharide; red circle, translocon from which BtuB and lipopolysaccharide have dissociated in the gas phase; blue square, dissociated BtuB. See Table S2 for masses.