Fig 1.
Coomassie stained, 12% SDS-PAGE of different protein preparations of C. crescentus.
Lane 1, Crude membrane preparation. Lane 2, PBS-EDTA extract, not boiled prior to loading. Lane 3, PBS-EDTA extract, boiled prior to loading. The star highlights the position of OmpW.
Fig 2.
A. Single-channel recordings of a PC/n-decane membrane in the presence of enriched outer membranes of C. crescentus. The aqueous phase contained 1 M KCl and 100 ng ml−1 protein from enriched outer membranes treated with 0.5% LDAO. The applied membrane potential was 20 mV; T = 20°C. B. Histogram of the probability P(G) for the occurrence of a given conductivity unit observed with membranes formed of PC/n-decane in the presence of enriched outer membranes of C. crescentus. P(G) is the probability that a given conductance increment G is observed in the single-channel experiments. It was calculated by dividing the number of fluctuations with a given conductance increment by the total number of conductance fluctuations. The aqueous phase contained 1 M KCl. The applied membrane potential was 20 mV; T = 20°C. A Gaussian function was applied to the histogram and the maximum of the curve was taken as average single-channel conductance. It was 113 (± 22) pS for 105 single-channel events (left-hand maximum).
Fig 3.
Coomassie stained, 15% SDS-PAGE of OmpW of C. crescentus obtained by elution of the 20 kDa band from preparative SDS-PAGE.
Lane 1: Molecular mass. Lane 2: OmpW solubilized at room temperature for 10 min in 5μl sample buffer. Lane 3: OmpW solubilized at 100°C for 10 min in 5 μl sample buffer.
Fig 4.
A. Single-channel recordings of a PC/n-decane membrane in the presence of purified OmpW of C. crescentus. The aqueous phase contained 1 M KCl and 20 ng ml−1 OmpW dissolved in 1% Genapol. The applied membrane potential was 20 mV; T = 20°C. B. Histogram of the probability P(G) for the occurrence of a given conductivity unit observed with membranes formed of PC/n-decane in the presence of OmpW of C. crescentus. P(G) is the probability that a given conductance increment G is observed in the single-channel experiments. It was calculated by dividing the number of fluctuations with a given conductance increment by the total number of conductance fluctuations. The aqueous phase contained 1 M KCl and about 20 ng/ml OmpW. The applied membrane potential was 20 mV; T = 20°C. A Gaussian function was applied to the histogram and the maximum of the curve was taken as average single-channel conductance. It was 117 (± 19) pS for 95 single-channel events.
Table 1.
Average single-channel conductance, G, of OmpW of C. crescentus in different salt solutions and radii, hydrated radii and limiting molar conductivity of the cations.
Fig 5.
Coomassie stained, 12% SDS PAGE of PBS-EDTA extractions from wildtype and C. crescentus ΔompW.
Lane 1, C. crescentus PBS-EDTA extract. Lane 2, C. crescentus ΔompW PBS-EDTA extract. While PBS-EDTA extraction was a convenient method to enrich for OmpW, there was some variability between extractions, which can be seen in comparing protein profiles in lanes 1 and 2. Equal volume loadings were used. The star highlights the position of OmpW.
Fig 6.
Single-channel recording of a PC/n-decane membrane in the presence of enriched outer membranes of C. crescentus ΔompW mutant.
The aqueous phase contained 1 M KCl and 100 ng ml−1 protein from enriched outer membranes treated with 0.5% LDAO. The applied membrane potential was 50 mV; T = 20°C. Note that the recording starts at the lower trace about 2 minutes after addition of the membrane extract and then continues in the upper trace.
Fig 7.
Single-channel conductance of OmpW in 1 M salt solution as a function of the ionic radii of monovalent cations.
The data were taken from Table 1. The single-channel conductance of LamB of E. coli is given for comparison [45].
Fig 8.
Amino acid sequence alignment of OprG of P. aeruginosa, OmpW of E. coli and OmpW of C. crescentus.
The alignment was performed using Pole Bioinformatique Lyonnaise Network Protein Sequence Analysis (http://npsa-pbil.ibcp.fr). Amino acids identical in all three proteins are highlighted in red, strongly similar amino acids (:) are given in green and weakly similar ones (.) in blue. The replacement of W155 of OmpW of E. coli and W170 of OprG of P. aeruginosa by K160 of C. crescentus is given in green color and is indicated by an arrow. The eight beta strands in OprG of P. aeruginosa and in OmpW of E. coli are numbered and indicated by blue bars [18, 19]. The yellow highlighted sequence was found by mass spectrometric analysis of tryptic peptides using Mascot N-terminal sequencing of OmpW of C. crescentus (http://www.matrixscience.com/).
Fig 9.
Structural properties of OmpW of C. crescentus.
A. Comparison of structural features between a) OmpW C. crescentus, b) OmpW E. coli and c) OprG P. aeruginosa. Residues that are a part of a putative hydrophobic gate in E. coli (W155 and L56) and P. aeruginosa (W170 and V65) channels are shown as spheres. The corresponding residues in OmpW C. crescentus (K159 and I65) are also shown. Moreover, all channels are shown using a surface representation and color coded according to the residue type (Green: hydrophilic, white: hydrophobic, red: acidic, blue: basic). The channels are cut from the front to enable visualization of the channel surface interior. The black colored box indicates a putative ion transport pathway across the channel. EC and PC denote the extracellular and periplasmic sides of the channel, respectively. The models of the three OmpW homologs were modeled using the program Modeller [32], taking the structure of E. coli OmpW as a template [18]. B. Comparison of channel radii between C. crescentus OmpW (blue), E. coli OmpW (red) and OprG of P. aeruginosa (green) along the channel axis. The channel radii were calculated using the program HOLE [51].