Abstract
Colicin E1 belongs to a group of bacteriocins whose cytotoxicity toward Escherichia coli is exerted through formation of ion channels that depolarize the cytoplasmic membrane. The lipid dependence of colicin single-channel conductance demonstrated intimate involvement of lipid in the structure of this channel. The colicin formed "small" conductance 60-picosiemens (pS) channels, with properties similar to those previously characterized, in 1,2-dieicosenoyl-sn-glycero-3-phosphocholine (C20) or thinner membranes, whereas it formed a novel "large" conductance 600-pS state in thicker 1,2-dierucoyl-sn-glycero-3-phosphocholine (C22) bilayers. Both channel states were anion-selective and voltage-gated and displayed a requirement for acidic pH. Lipids having negative spontaneous curvature inhibited the formation of both channels but increased the ratio of open 600 pS to 60 pS conductance states. Different diameters of small and large channels, 12 and 16 A, were determined from the dependence of single-channel conductance on the size of nonelectrolyte solute probes. Colicin-induced lipid "flip-flop" and the decrease in anion selectivity of the channel in the presence of negatively charged lipids implied a significant contribution of lipid to the structure of the channel, most readily described as toroidal organization of lipid and protein to form the channel pore.
Highlights
Channel-forming proteins can require specific lipids, presumably because of a combination of charge and curvature parameters, to form active channels
Histograms of the channel current (Fig. 1B) and lifetime (Fig. 1C) show the high conductance state to be characterized by a single-channel conductance of 590 Ϯ 10 pS and a lifetime of 120 Ϯ 30 ms, whereas small channels have a conductance of 65 Ϯ 2 pS (n ϭ 13) and a lifetime of 4 Ϯ 1 s (n ϭ 18)
Both conductance states displayed the same voltage dependence; they were opened by a cis-positive voltage and closed when the sign of the voltage was reversed (Fig. 1D), which is characteristic for colicin-induced macroscopic current [51]
Summary
The 178-residue C-terminal colicin E1 channel polypeptide, P178, was prepared by thermolysin proteolysis of intact colicin E1 [56]. Liposomes—Liposomes were prepared by evaporation under a stream of nitrogen of a 2% solution of a DPhPC/DPhPG mixture (70%/ 30%) in chloroform (Merck, Darmstadt, Germany) followed by hydration with a buffer solution containing 10 mM -alanine, 0.12 M KCl, pH 4.0. Incorporation of pyPC into the outer membrane leaflet was followed by measuring the ratio of fluorescence intensities of monomers (IM) and excimers (IE) at 395 and 495 nm, respectively. C-terminal colicin peptide (P178) or intact colicin E1 was added to, and defined, the cis side of the membrane. Agar-agar bridges connecting the electrodes and the membrane bathing solutions were used in selectivity measurements
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