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Trimeric porins in the outer membrane (OM) of Gram-negative bacteria are the conduits by which nutrients and antibiotics diffuse passively into cells. The narrow gateways that porins form in the OM are also exploited by bacteriocins to translocate into cells by a poorly understood process. Here, using single-channel electrical recording in planar lipid bilayers in conjunction with protein engineering, we explicate the mechanism by which the intrinsically unstructured N-terminal translocation domain (IUTD) of the endonuclease bacteriocin ColE9 is imported passively across the Escherichia coli OM through OmpF. We show that the import is dominated by weak interactions of OmpF pores with binding epitopes within the IUTD that are orientationally biased and result in the threading of over 60 amino acids through 2 subunits of OmpF. Single-molecule kinetic analysis demonstrates that the IUTD enters from the extracellular side of OmpF and translocates to the periplasm where the polypeptide chain does an about turn in order to enter a neighboring subunit, only for some of these molecules to pop out of this second subunit before finally re-entering to form a stable complex. These intimately linked transport/binding processes generate an essentially irreversible, hook-like assembly that constrains an import activating peptide epitope between two subunits of the OmpF trimer.

Original publication




Journal article


J Am Chem Soc

Publication Date





12157 - 12166