Abstract
VOLTAGE-GATED channels undergo a conformational change in response to changes in transmembrane voltage. Here we use site-directed biotinylation to create conformation-sensitive sites on col-icin la, a bacteriocidal protein that forms a voltage-sensitive mem-brane channel, which can be monitored by electrophysiological methods1,2. We investigated a model of gating developed for the partly homologous colicin El that is based on the insertion of regions of the protein into the membrane in response to cis-positive voltages3–6. Site-directed cysteine mutagenesis, followed by chemi-cal modification, was used to attach a biotin molecule covalently to a series of unique sites on colicin la. The modified protein was incorporated into planar lipid membranes, where the introduced biotin moiety served as a site to bind the water-soluble protein streptavidin, added to one side of the membrane or the other. Our results show that colicin gating is associated with the translocation across the membrane of a segment of the protein of at least 31 amino acids.
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Slatin, S., Qiu, XQ., Jakes, K. et al. Identification of a translocated protein segment in a voltage-dependent channel. Nature 371, 158–161 (1994). https://doi.org/10.1038/371158a0
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DOI: https://doi.org/10.1038/371158a0
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