Modulation of<i>Vibrio cholerae</i>Porin Function by Acidic pH

Duret, Guillaume; Simonet, Valérie; Delcour, Anne H.

doi:10.4161/chan.3983

Cited by 19 publications

(29 citation statements)

References 38 publications

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“…However, this view has been challenged by studies which showed that the open state probability of the OmpF and OmpC pores in E. coli is regulated by pH, voltage and by small molecules which stabilize a closed pore conformation [34], [35], [36]. Although regulatory events of MspA channel activity are not known yet, it is likely that they exist considering the necessity of bacteria to adequately respond to rapid changes in environmental conditions such as osmolarity or presence of toxic solutes [37]. In this regard, it is tempting to speculate that the wt MspA pore might respond to these environmental signals by more or faster closing events than a pore made from subunit dimers.…”

Section: Discussionmentioning

confidence: 99%

MspA Nanopores from Subunit Dimers

et al. 2012

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Mycobacterium smegmatis porin A (MspA) forms an octameric channel and represents the founding member of a new family of pore proteins. Control of subunit stoichiometry is important to tailor MspA for nanotechnological applications. In this study, two MspA monomers were connected by linkers ranging from 17 to 62 amino acids in length. The oligomeric pore proteins were purified from M. smegmatis and were shown to form functional channels in lipid bilayer experiments. These results indicated that the peptide linkers did not prohibit correct folding and localization of MspA. However, expression levels were reduced by 10-fold compared to wild-type MspA. MspA is ideal for nanopore sequencing due to its unique pore geometry and its robustness. To assess the usefulness of MspA made from dimeric subunits for DNA sequencing, we linked two M1-MspA monomers, whose constriction zones were modified to enable DNA translocation. Lipid bilayer experiments demonstrated that this construct also formed functional channels. Voltage gating of MspA pores made from M1 monomers and M1-M1 dimers was identical indicating similar structural and dynamic channel properties. Glucose uptake in M. smegmatis cells lacking porins was restored by expressing the dimeric mspA M1 gene indicating correct folding and localization of M1-M1 pores in their native membrane. Single-stranded DNA hairpins produced identical ionic current blockades in pores made from monomers and subunit dimers demonstrating that M1-M1 pores are suitable for DNA sequencing. This study provides the proof of principle that production of single-chain MspA pores in M. smegmatis is feasible and paves the way for generating MspA pores with altered stoichiometries. Subunit dimers enable better control of the chemical and physical properties of the constriction zone of MspA. This approach will be valuable both in understanding transport across the outer membrane in mycobacteria and in tailoring MspA for nanopore sequencing of DNA.

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Section: Discussionmentioning

confidence: 99%

MspA Nanopores from Subunit Dimers

et al. 2012

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“…It is possible that it stems from an enhanced voltage-sensitivity, as documented [63]. In OmpU, channels are immediately stabilized in a closed configuration, and surprisingly, individual closures of three monomers are not observed, but rather closing events of an apparent single channel of increasingly large size as the pH is decreased [64]. In OmpF, extracellular loops L1, L7 and L8 have been implicated in the conformational changes that might lead to acidic pH-induced channel closures [62].…”

Section: Porin-mediated Om Permeabilitymentioning

confidence: 99%

Outer membrane permeability and antibiotic resistance

Delcour

2009

Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics

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SummaryTo date most antibiotics are targeted at intracellular processes, and must be able to penetrate the bacterial cell envelope. In particular, the outer membrane of Gram-negative bacteria provides a formidable barrier that must be overcome. There are essentially two pathways that antibiotics can take through the outer membrane: a lipid-mediated pathway for hydrophobic antibiotics, and general diffusion porins for hydrophilic antibiotics. The lipid and protein compositions of the outer membrane have a strong impact on the sensitivity of bacteria to many types of antibiotics, and drug resistance involving modifications of these macromolecules is common. This review will describe the molecular mechanisms for permeation of antibiotics through the outer membrane, and the strategies that bacteria have deployed to resist antibiotics by modifications of these pathways.

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“…The proteins were purified as described [21,23]. Briefly, porin was extracted with 1% octyl-POE and purified by ion exchange chromatography (MonoQ HR 10/10), followed when necessary by size exclusion chromatography (Hiload 26/20 Superdex 200).…”

Section: Protein Purificationmentioning

confidence: 99%

Altered pore properties and kinetic changes in mutants of theVibrio choleraePorin OmpU

Lauman

Pagel

Delcour

2008

Molecular Membrane Biology

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The electrophysiological technique of patch-clamp was used to characterize the pore properties of site-directed mutants in the Vibrio cholerae general diffusion porin OmpU. Changes in conductance and selectivity were observed, thus confirming the predicted pore location of these residues, based on homology with the Escherichia coli porins OmpF and OmpC. Some mutants acquire a weak selectivity for cations, which mirrors the properties of the homologous, deoxycholic acid sensitive, OmpT porin of V. cholerae. However, the mutants remain insensitive to deoxycholic acid, like wildtype OmpU. This result suggests that channel selectivity is not an important determinant in the sensitivity to this drug, and is in agreement with our finding that the neutral deoxycholic acid, and not deoxycholate, is the actual active form in channel block. Modifications in the kinetics of spontaneous closures were also noted, and are similar to those found for the E. coli channels. In addition, mutants at the D116 residue on the L3 loop display marked transitions to sub-conductance states. The results reported here are compared to a phenotypical characterization of the mutants in terms of permeability to maltodextrins and beta-lactam antibiotic sensitivity. No strict correlations are observed, suggesting that distinct, but somewhat overlapping, molecular determinants control electrophysiological properties and substrate permeability.

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Modulation ofVibrio choleraePorin Function by Acidic pH

Cited by 19 publications

References 38 publications

MspA Nanopores from Subunit Dimers

MspA Nanopores from Subunit Dimers

Outer membrane permeability and antibiotic resistance

Altered pore properties and kinetic changes in mutants of theVibrio choleraePorin OmpU

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