(A) Ribbon diagram description of OmpF trimer determined at 1.6 Å resolution. The structure is tilted forward 20° from a vertical position of the trimer threefold symmetry axis; monomers are coloured in yellow, blue and green; two molecules of the detergent, octyl-tetraoxyethylene, are bound per monomer. (B) Superimposed ribbon diagrams of the structure of the monomer of the OmpF porin with the 1.6 Å resolution of wild-type porin obtained in this study and 2.2 Å resolution structure of the Tyr106Phe mutant (Phale et al, 2001), viewed parallel to the plane of the membrane. The r.m.s.d. between the backbone atoms of the two structures is 0.26 Å. The 1.6 and 2.2 Å structures are coloured green and blue (barrel), and orange and purple (loop 3), respectively.

Occlusion of OmpF channels by T83. Planar bilayer membranes were formed of DOPC and DOPE (1:1, w/w) dissolved in n-decane. OmpF in 0.7% octyl-POE was added to the cis-side of the membrane (ca. 10 pg/ml). The sign of the trans-membrane potential is shown for the cis-side of the membrane. Trans-membrane current (grey traces) was (A) +156 pA (+50 mV) and (B) −192 pA (−50 mV) implying opening of 12 OmpF channels (four trimers). After recording OmpF channels, T83 (0.2 μg/ml) was added to the trans-compartment. Recording started at −50 mV revealed an occluded state of all channels with sporadic opening of 1 or 2 channels (lower panel). (A) Switching the potential to +50 mV partially restored the open state of the OmpF channels.

Immunodetection of the complex between BtuB, OmpF and colicin E3. E. coli membranes were incubated with colicin E3, 0.26 μM, and then cross-linked with 1% formaldehyde. Proteins were extracted with 3% OG. Complexes containing colicin E3 were immunoprecipitated using anti-C-domain antibodies conjugated with CNBr-activated Sepharose beads, subjected to SDS–PAGE, transferred to a PVDF membrane and immunodetected using antibodies against BtuB, OmpF and colicin E3. Immunoblot samples: 1, protein standards; 2, 4 and 6, isolated OmpF, BtuB and colicin E3, respectively; 3, 5 and 7, membrane protein extract. Samples 2 and 3, 4 and 5, 6 and 7 were incubated with anti-OmpF, anti-BtuB and anti-colicin E3 antibodies, respectively.

(A) F_o–F_c difference map in the 1.6 Å OmpF structure showing the electron density of a hexa-H₂O-coordinated Mg²⁺ atom (orange) near Asp113, Leu115 and Glu117 in the L3 loop of the selectivity filter. Surrounding OmpF barrel region, green; loop 3, purple. Also shown are Arg42, Arg82, Arg132, Lys16, Lys80 and Glu62 on the opposite side of the limiting filter aperture. Other charged and Tyr residues that define the boundaries of the limiting filter aperture are Lys312, Glu117, Tyr102 and Tyr106. The cross-sectional dimensions of the limiting aperture are 7.8 Å (Arg82NH–Asp113OD2)—8.8 Å (Lys16NZ–Leu115O) × 14.4 Å (Tyr310OH–Tyr106OH, or Tyr310OH–Tyr102OH). (B) Hydrogen-bonding pattern of (H₂O)₆–Mg²⁺ to Asp113OD1 and to the backbone carbonyls of Leu115 and Glu117. Residues removed for purposes of clarity in Figure 2B are 25–34 in the loop 1, 161–170 in loop 4, 197–209 in loop 5, 237–251 in loop 6 and 318–329 in loop 8.

Complex of OmpF and colicin E3 N-terminal T83 peptide detected by size-exclusion chromatography. (A) OmpF was passed through a Superdex 200 (10/300) column (thin line) in 0.7% octyl-POE buffer as described in Materials and methods. OmpF trimer (fractions 10–12.5 ml from previous run), 9.2 nmol, was mixed with T83, 25 nmol, and was passed through the same column (bold line). The absence of T83 oligomers, which could mimic formation of the OmpF/T83 complex, was confirmed by the elution of T83 alone, 130 nmol, through the same column (dashed line). All data are normalized to a common maximum ordinate. (B) SDS–PAGE of proteins eluted from Superdex 200 column. (1) Protein standards; (2) OmpF trimer (peak 11.1 ml from the first run); (3) OmpF–T83 complex (second run, peak 11.0 ml); (4) OmpF monomer (second run, peak 14.6 ml); (5) T83 (peak 17.8 ml). SDS–PAGE was run in 15% polyacrylamide. (C) Estimate of T83 content complexed to OmpF trimer. SDS–PAGE: 1, OmpF/T83 from elution peak at 11.0 ml; 2, 3, 4 and 5, T83, 0.125, 0.25, 0.5 and 1.0 μg, respectively. The content of the OmpF trimer in the elution peak at 11.0 ml (5.3 μM) was determined spectrophotometrically, using a molar extinction coefficient (ɛ_M) at 280 nm of 1.63 × 10⁵, and neglecting the contribution of T83 (ɛ_M=1.65 × 10⁴) to the absorbance of the OmpF trimer. Assuming that the intensity of the Coomassie stain of T83 bands in samples 1 and 3 is similar, a 10 μl aliquot applied to SDS–PAGE contained ∼0.25 μg T83, implying that each OmpF trimer in the peak at 11.0 ml bound 1–2 molecules of the T83 peptide.

Interaction of an incremental electron density (magenta) associated with a peptide segment of T83 bound in the OmpF pore. (A) Stereo view of OmpF and the F_o−F_c map of the additional electron density attributed to a segment of the T83 peptide. L3 loop is shown in yellow with its helical sequences in red. The proximity of the peptide density to the binding site of a Mg²⁺ ion is shown. The additional density attributed to the T83 peptide consists of at least seven residues and extends over a distance of at least 18 Å. Top, extracellular, and bottom, periplasmic side of OmpF. (B) View along axis of selectivity filter (cf., Figure 2B) showing superimposed difference densities of the peptide and the bound Mg²⁺ ion seen in the absence of T83. (C) Region of interaction of T83 peptide segment (electron density map in blue), loop 3 of OmpF and bound Mg²⁺ ion, shown in enlarged format.