Ion channel formation by N-terminal domain: a common feature of OprFs of Pseudomonas and OmpA of Escherichia coli

Saint, N

doi:10.1016/s0378-1097(00)00345-1

Cited by 24 publications

(36 citation statements)

References 19 publications

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“…In any case, we did not observe the diffusion of the smallest organic solutes we could test in the liposome swelling assay (e.g. glycine) through the N-terminal domain of OprF, and we suggest that the N-terminal eight-stranded ␤-barrel domain should be thought of as a completely closed channel despite the suggestions from other laboratories (8,28) as well as from a computer modeling study of its homolog OmpA (30). In conclusion, our results as well as results from other laboratories suggest strongly that the large channel is produced by the folding of the entire OprF sequence to produce a ␤-barrel of many strands, certainly more than eight and perhaps close to 16, that are found in classical porins (see Fig.…”

Section: Discussioncontrasting

confidence: 67%

Pseudomonas aeruginosa Porin OprF

Nestorovich

Sugawara

Nikaido

et al. 2006

Journal of Biological Chemistry

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Using ion channel reconstitution in planar lipid bilayers, we examined the channel-forming activity of subfractions of Pseudomonas aeruginosa OprF, which was shown to exist in two different conformations: a minority single domain conformer and a majority two-domain conformer (Sugawara, E., Nestorovich, E. M., Bezrukov, S. M., and Nikaido, H. (2006) J. Biol. Chem. 281, 16220 -16229). With the fraction depleted for the single domain conformer, we were unable to detect formation of any channels with well defined conductance levels. With the unfractionated OprF, we saw only rare channel formation. However, with the single domainenriched fraction of OprF, we observed regular insertion of channels with highly reproducible conductances. Single OprF channels demonstrate rich kinetic behavior exhibiting spontaneous transitions between several subconformations that differ in ionic conductance and radius measured in polymer exclusion experiments. Although we showed that the effective radius of the most conductive conformation exceeds that of the general outer membrane porin of Escherichia coli, OmpF, we also found that a single OprF channel mainly exists in weakly conductive subconformations and switches to the fully open state for a short time only. Therefore, the low permeability of OprF reported earlier may be due to two factors: mainly to the paucity of the single domain conformer in the OprF population and secondly to the predominance of weakly conductive subconformations within the single domain conformer.The channel properties of OprF, the major nonspecific porin of Pseudomonas aeruginosa, have been studied by several methods. Early studies (1, 2), utilizing the near equilibrium redistribution of radiolabeled solutes initially trapped in reconstituted liposomes, have suggested that the channel was large. OprF allows nearly complete outward diffusion of polysaccharides of 2,000 -3,000 daltons in contrast to the Escherichia coli general porin channel that is permeable to sugars of only up to 600 daltons. Kinetic studies of solute diffusion (3, 4), using osmotic swelling of proteoliposomes, have shown that OprF has much lower permeability (i.e. allows much slower permeation of the same test solute) than the classical trimeric porins of E. coli but forms channels that are wider than the channels of E. coli porins because the diffusion rates are much less influenced by the size of the oligosaccharide solutes. Intact E. coli cells expressing OprF porin from plasmid-coded gene are capable of growing on raffinose (5), which is too large (505 daltons) to serve as an effective carbon source for the wild-type E. coli.A number of studies of OprF have been carried out with planar bilayer systems. The first study by Benz and Hancock (6) has already shown that the addition of OprF produces channels of large single channel conductance (several nanosiemens (nS) 2 in 1 M KCl or NaCl) that are also large by other criteria, such as indifference to the nature of the permeating ions and proportionality of channel conductance to the conce...

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Section: Discussioncontrasting

confidence: 67%

Pseudomonas aeruginosa Porin OprF

Nestorovich

Sugawara

Nikaido

et al. 2006

Journal of Biological Chemistry

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“…A survey of the literature shows that barrels with fewer or with more ␤-strands than the predicted 10 for HMW1B form pores with smaller conductance values than what we report here. For example, the conductance of the eight ␤-stranded N-terminal domain barrel of OmpA is ϳ110 pS in 1 M KCl (44). The conductance appears to increase for pores containing a larger number of ␤ strands (for example, 800 pS for the monomeric OmpG (14 strands) (45) or 1200 pS for FhaC (16 strands) (18)), but one has to exercise caution because the conductance is not only determined by the pore diameter but also by the charges and the electric field within the pore as well as by the access resistance of the pore (46).…”

Section: Discussionmentioning

confidence: 99%

The TpsB Translocator HMW1B of Haemophilus influenzae Forms a Large Conductance Channel

Duret

Szymanski

Choi

et al. 2008

Journal of Biological Chemistry

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The Haemophilus influenzae HMW1 adhesin is secreted via the two-partner secretion pathway and requires HMW1B for translocation across the outer membrane. HMW1B belongs to the Omp85-TpsB superfamily of transporters and consists of two structural domains, a C-terminal transmembrane ␤-barrel and an N-terminal periplasmic domain. We investigated the electrophysiological properties of the purified full-length HMW1B and the C-terminal domain using planar lipid bilayers. Both the full-length and the truncated proteins formed conductive pores with a low open probability, two well defined conductance states, and other substates. The kinetic patterns of the two conductance states were distinct, with rapid and frequent transitions to the small conductance state and occasional and more prolonged openings to the large conductance state. The channel formed by the full-length HMW1B showed selectivity for cations, which decreased when measured at pH 5.2, suggesting the presence of acidic residues in the pore. The C-terminal domain of HMW1B was less stable and required reconstitution into liposomes prior to insertion in the bilayer. It formed a channel of smaller conductance but a similar gating pattern as the full-length protein, demonstrating the ability of the last 312 C-terminal amino acids to form a pore and suggesting that the periplasmic domain is not involved in occluding the pore, nor in controlling the inherent basal kinetics of the channel. The HMW1 pro-piece containing the secretion domain, although binding to the channel with high affinity, did not induce channel opening.Secretion of proteins in the external medium from the cytosol of a Gram-negative bacterium is a complex mechanism. Because of the compartmented structure of the cell envelope, a periplasm limited by the inner membrane and the outer membrane, secreted proteins may need to undergo two translocation processes. Seven main types of mechanisms exist to translocate proteins through the cell envelope (1-6). Some of these pathways export the protein directly from the cytosol to the extracellular milieu, whereas others include a periplasmic intermediate step. In this case, independent translocation pathways exist through the inner membrane and comprise the Sec pathway and the TAT pathway (7). The type V secretion pathway is one of those two-step processes, where proteins are first exported to the periplasm in a Sec-dependent manner (5). The second step involves the translocation through the outer membrane of a passenger peptide through a transporter. In many cases, the passenger and the transporter domains belong to the same polypeptide, thus defining an auto-transporter type of mechanism. The two-partner secretion pathway is distinct in that the secreted exoprotein (TpsA) and its cognate transporter protein (TpsB) are translated as two separate proteins but are coded on the same operon or the same locus. Each TpsB transporter is devoted to the translocation of one specific TpsA. For example, the Haemophilus influenzae HMW1/HMW1B pair represents a prototype t...

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“…Additionally, an operon formed by a hypothetical protein BT2437 from Bacteroides thetaiotaomicron VPI-5482 was found that codes for a putative lipoprotein (Chang et al, 1999). Proteins from this family are always associated with channelforming eight-stranded beta-barrel proteins from the OprF family (Saint et al, 2000) (Figure 5c). The list of hypothetical proteins and predicted functions can be found in Supplementary Table S11.…”

Section: Analysis Of Unknown Hypothetical Proteinsmentioning

confidence: 99%

Shotgun metaproteomics of the human distal gut microbiota

et al. 2008

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The human gut contains a dense, complex and diverse microbial community, comprising the gut microbiome. Metagenomics has recently revealed the composition of genes in the gut microbiome, but provides no direct information about which genes are expressed or functioning. Therefore, our goal was to develop a novel approach to directly identify microbial proteins in fecal samples to gain information about the genes expressed and about key microbial functions in the human gut. We used a non-targeted, shotgun mass spectrometry-based whole community proteomics, or metaproteomics, approach for the first deep proteome measurements of thousands of proteins in human fecal samples, thus demonstrating this approach on the most complex sample type to date. The resulting metaproteomes had a skewed distribution relative to the metagenome, with more proteins for translation, energy production and carbohydrate metabolism when compared to what was earlier predicted from metagenomics. Human proteins, including antimicrobial peptides, were also identified, providing a non-targeted glimpse of the host response to the microbiota. Several unknown proteins represented previously undescribed microbial pathways or host immune responses, revealing a novel complex interplay between the human host and its associated microbes.

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Ion channel formation by N-terminal domain: a common feature of OprFs of Pseudomonas and OmpA of Escherichia coli

Cited by 24 publications

References 19 publications

Pseudomonas aeruginosa Porin OprF

Pseudomonas aeruginosa Porin OprF

The TpsB Translocator HMW1B of Haemophilus influenzae Forms a Large Conductance Channel

Shotgun metaproteomics of the human distal gut microbiota

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