Effects of pore mutations and permeant ion concentration on the spontaneous gating activity of OmpC porin

Liu, Nazhen; Samartzidou, Hrissi; Lee, Keun Woo; Briggs, James M.; Delcour, Anne H.

doi:10.1093/protein/13.7.491

Cited by 27 publications

(38 citation statements)

References 53 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The electrophysiological properties of these mutant porins were examined by patch clamp of liposome blisters containing multiple porin channels. Some of the mutant porins displayed a dramatic increase in the closing rate and decrease in open probability relative to wildtype OmpC (17,18). These changes were interpreted to be the result of changes in the flexibility of loop 3 within the barrel, caused by the loss of hydrogen bonds and/or salt bridges between loop 3 and the outer barrel.…”

Section: Discussionmentioning

confidence: 97%

“…The increased channel noise and increased time spent in subconductance states observed in the variant PIBs are reminiscent of the changes in the channel properties of the OmpC porin with mutations designed to disrupt the interaction of loop 3 with the outer barrel (17,18). The electrophysiological properties of these mutant porins were examined by patch clamp of liposome blisters containing multiple porin channels.…”

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Porin-Mediated Antibiotic Resistance in Neisseria gonorrhoeae : Ion, Solute, and Antibiotic Permeation through PIB Proteins with penB Mutations

et al. 2006

View full text Add to dashboard Cite

Neisseria gonorrhoeae has two porins, PIA and PIB, whose genes (porA and porB, respectively) are alleles of a single por locus. We recently demonstrated that penB mutations at positions 120 and 121 in PIB, which are presumed to reside in loop 3 that forms the pore constriction zone, confer intermediate-level resistance to penicillin and tetracycline (M. Olesky, M. Hobbs, and R. A. Nicholas, Antimicrob. Agents Chemother. 46: 2811-2820, 2002). In the present study, we investigated the electrophysiological properties as well as solute and antibiotic permeation rates of recombinant PIB proteins containing penB mutations (G120K, G120D/A121D, G120P/A121P, and G120R/A121H). In planar lipid bilayers, the predominant conducting state of each porin variant was 30 to 40% of the wild type, even though the anion selectivity and maximum channel conductance of each PIB variant was similar to that of the wild type. Liposome-swelling experiments revealed no significant differences in the permeation of sugars or ␤-lactam antibiotics through the wild type or PIB variants. Although these results are seemingly contradictory with the ability of these variants to increase antibiotic resistance, they are consistent with MIC data showing that these porin mutations confer resistance only in strains containing an mtrR mutation, which increases expression of the MtrC-MtrD-MtrE efflux pump. Moreover, both the mtrR and penB mutations were required to decrease in vivo permeation rates below those observed in the parental strain containing either mtrR or porin mutations alone. Thus, these data demonstrate a novel mechanism of porin-mediated resistance in which mutations in PIB have no affect on antibiotic permeation alone but instead act synergistically with the MtrC-MtrD-MtrE efflux pump in the development of antibiotic resistance in gonococci.In the 1970s and early 1980s, Neisseria gonorrhoeae, the etiological agent of the sexually transmitted infection gonorrhea, gradually became more resistant to penicillin and tetracycline until these antibiotics were discontinued as a first-line therapy. Resistance to these antibiotics can be either plasmid mediated or chromosomally mediated. Plasmid-mediated resistance to penicillin or tetracycline involves expression of a TEM-1-like ␤-lactamase (8, 30) or the TetM protein (25), respectively. In contrast, chromosomally mediated resistant N. gonorrhoeae (CMRNG) strains arise from cumulative mutations in endogenous genes or loci that gradually increase resistance until treatment failure occurs (3).Four genes or loci (penA, mtrR, penB, and ponA) are known to be involved in high-level penicillin resistance (MICs, Ն2 g/ml) in CMRNG strains, while the existence of a fifth resistance gene is inferred but not yet identified (3, 9, 32). penA (38, 39) and ponA (32, 33) encode alterations in the two essential penicillin-binding proteins (PBP 2 and PBP 1, respectively) that decrease their rates of acylation by ␤-lactam antibiotics. A single-base-pair deletion in the mtrR promoter abolishes transcription of the mtrR...

show abstract

Section: Discussionmentioning

confidence: 97%

Section: Discussionmentioning

confidence: 99%

Porin-Mediated Antibiotic Resistance in Neisseria gonorrhoeae : Ion, Solute, and Antibiotic Permeation through PIB Proteins with penB Mutations

et al. 2006

View full text Add to dashboard Cite

show abstract

“…The gating of OmpA is associated with breaking and rearrangement of the salt bridges in the center of the barrel (21,23). In the case of the trimeric porin OmpC, it has been proposed that spontaneous gating is related to the positioning or flexibility of loop 3, which folds to form an eyelet (constriction zone) within the pore (24,25). It has been previously suggested that, under acidic conditions, L6 is implicated in the pH-dependent gating of OmpG (7).…”

Section: Discussionmentioning

confidence: 99%

Outer membrane protein G: Engineering a quiet pore for biosensing

Chen

Khalid

Sansom

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

163

236

View full text Add to dashboard Cite

Bacterial outer membrane porins have a robust ␤-barrel structure and therefore show potential for use as stochastic sensors based on single-molecule detection. The monomeric porin OmpG is especially attractive compared with multisubunit proteins because appropriate modifications of the pore can be easily achieved by mutagenesis. However, the gating of OmpG causes transient current blockades in single-channel recordings that would interfere with analyte detection. To eliminate this spontaneous gating activity, we used molecular dynamics simulations to identify regions of OmpG implicated in the gating. Based on our findings, two approaches were used to enhance the stability of the open conformation by site-directed mutagenesis. First, the mobility of loop 6 was reduced by introducing a disulfide bond between the extracellular ends of strands ␤12 and ␤13. Second, the interstrand hydrogen bonding between strands ␤11 and ␤12 was optimized by deletion of residue D215. The OmpG porin with both stabilizing mutations exhibited a 95% reduction in gating activity. We used this mutant for the detection of adenosine diphosphate at the single-molecule level, after equipping the porin with a cyclodextrin molecular adapter, thereby demonstrating its potential for use in stochastic sensing applications.gating ͉ MD simulation ͉ OmpG ͉ stochastic sensor E ngineered protein pores can be used as stochastic sensors for single-molecule detection (1). The ionic current flowing through a pore under an applied potential is altered when an analyte binds within the lumen (1). Measurement of the frequency of occurrence of the binding events allows the determination of the concentration of an analyte, while the nature of the events (e.g., their amplitude or duration) aids in analyte identification. To date, studies of proteinaceous stochastic sensing elements have mostly focused on staphylococcal ␣-hemolysin (␣HL), a ␤-barrel pore-forming toxin (1, 2). However, the stoichiometry and symmetry of the heptameric ␣HL pore generate a large number of combinations and permutations when more than one type of subunit is used (3), which makes it difficult to fine-tune the properties of the pore. Therefore, it would be highly desirable to design a stochastic sensor based on a monomeric ␤-barrel.OmpG is a monomeric porin from the outer membrane of Escherichia coli that presents an attractive alternative to ␣HL for stochastic sensing (4-7). Previous studies have shown that OmpG undergoes pH-dependent, voltage-dependent, and spontaneous gating (4, 5). Specifically, the pore tends to close when the pH decreases to below 7 or when the voltage is higher than Ϯ100 mV (5). At neutral pH and an applied potential lower than Ϯ100 mV, OmpG exhibits a spontaneous gating behavior-i.e., it rapidly switches between open and closed states (Fig. 1A). Such spontaneous gating interferes with the application of a pore as a biosensor. Therefore, eliminating or significantly reducing the intrinsic gating activity of OmpG would be an important step in the development of an al...

show abstract

“…For instance, the constriction loop in OmpC has been suggested to be important in the gating process. 12,50 Correspondingly, in OpdK the loops L3 or L7 are likely very important for the multiple substates. Hence, we have performed a structural analysis of these loops and compared their flexibility.…”

Section: Simulations Of Ion Conductancementioning

confidence: 99%

Theoretical analysis of ion conductance and gating transitions in the OpdK (OccK1) channel

Pothula

Kleinekathöfer

2015

Analyst

View full text Add to dashboard Cite

Electrophysiological measurements have shown that the channel protein OpdK, also known as OccK1, from Pseudomonas aeruginosa shows three conductance substates. Although several experimental studies have been performed, a description of the gating transitions at the molecular level remains elusive. In the present investigation, molecular dynamics simulations have been employed to elucidate the conductance and gating properties of the OpdK channel and loop deletion mutant thereof. Our results suggest that switching between different substates are coupled to conformational changes in the constriction loop L7 which is in accord with the experimental results. Unbiased simulations at different temperatures are analyzed and residues R284 and F291 on loop L7 have been identified to be key in the gating transitions. A plausible mechanism of gating for this channel is discussed. The obtained molecular level description might have important implications for understanding the functional properties of OpdK channel in vitro as well as within a cellular environment.

show abstract

Effects of pore mutations and permeant ion concentration on the spontaneous gating activity of OmpC porin

Cited by 27 publications

References 53 publications

Porin-Mediated Antibiotic Resistance in Neisseria gonorrhoeae : Ion, Solute, and Antibiotic Permeation through PIB Proteins with penB Mutations

Porin-Mediated Antibiotic Resistance in Neisseria gonorrhoeae : Ion, Solute, and Antibiotic Permeation through PIB Proteins with penB Mutations

Outer membrane protein G: Engineering a quiet pore for biosensing

Theoretical analysis of ion conductance and gating transitions in the OpdK (OccK1) channel

Contact Info

Product

Resources

About