Disulfide bond tethering of extracellular loops does not affect the closure of OmpF porin at acidic pH

Wager, Beau; Baslé, Arnaud; Delcour, Anne H.

doi:10.1002/prot.22807

Cited by 20 publications

(23 citation statements)

References 29 publications

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“…Recently, we showed that introducing a pool of negative charges within a robust β-barrel protein pore can induce current fluctuations in the form of well-defined transient current closures, which are otherwise absent in the native protein (28;29). Conformational fluctuations of the L3 extracellular loop that folds back into the pore lumen is a hallmark in the spontaneous gating of trimeric porins, including the outer membrane proteins C (OmpC) and F (OmpF) of E. coli (20;23;30–33). Liu and Delcour (1998) showed that single-site mutations that alter a putative hydrogen bond between loop L3 and the barrel wall, along with ion-pair interactions at the root of the L3 loop impact the spontaneous gating activity of OmpC (20).…”

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confidence: 99%

Analysis of Gating Transitions among the Three Major Open States of the OpdK Channel

2011

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OpdK is an outer membrane protein of the pathogenic bacterium Pseudomonas aeruginosa. The recent crystal structure of this protein revealed a monomeric, 18-stranded β-barrel with a kidney-shaped pore, whose constriction features a diameter of 8 Å. Using systematic single-channel electrical recordings of this protein pore reconstituted into planar lipid bilayers under a broad range of ion concentrations, we were able to probe its discrete gating kinetics involving three major and functionally distinct conformations, in which a dominant open sub-state O2 is accompanied by less thermodynamically stable sub-states O1 and O3. Single-channel electrical data enabled us to determine the alterations in the energetics and kinetics of the OpdK protein when experimental conditions were changed. In the future, such a semi-quantitative analysis might provide a better understanding on the dynamics of current fluctuations of other β-barrel membrane protein channels.

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confidence: 99%

Analysis of Gating Transitions among the Three Major Open States of the OpdK Channel

2011

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“…[14][15][16][17][18] However, given the absence of energy in the OM, the energy provided by thermal fluctuations is likely the source to support spontaneous changes during the transition between conducting and non-conducting states in OM porins. [19][20][21][22][23][24] The most abundant OM porin in Escherichia coli, OmpF, is an ideal model to study such conformational changes; OmpF is known for spontaneously fluctuating between highly stable, conducting (open) and less stable, non-conducting (closed) states, as observed in electrophysiological measurements. 21,22,25 However, the molecular mechanism by which OmpF undergoes these spontaneous gating processes is still not well understood.…”

Section: Introductionmentioning

confidence: 99%

“…[19][20][21][22][23][24] The most abundant OM porin in Escherichia coli, OmpF, is an ideal model to study such conformational changes; OmpF is known for spontaneously fluctuating between highly stable, conducting (open) and less stable, non-conducting (closed) states, as observed in electrophysiological measurements. 21,22,25 However, the molecular mechanism by which OmpF undergoes these spontaneous gating processes is still not well understood.…”

Section: Introductionmentioning

confidence: 99%

Investigation of gating in outer membrane porins provides new perspectives on antibiotic resistance mechanisms

Vasan

Haloi

Ulrich

et al. 2021

Preprint

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Gram-negative bacteria pose a serious public health concern, primarily due to a higher frequency of antibiotic resistance conferred to them as a result of low permeability of their outer membrane (OM). Antibiotics capable of traversing the OM typically permeate through OM porins; thus, understanding the permeation properties of these porins is instrumental to the development of new antibiotics. A common macroscopic feature of many OM porins is their ability to transition between functionally distinct open and closed states that regulate transport properties and rate. To obtain a molecular basis for these processes, we performed tens of microseconds of molecular dynamics simulations of E. coli OM porin, OmpF. We observed that large-scale motion of the internal loop, L3, leads to widening and narrowing of the pore, suggesting its potential role in gating. Furthermore, Markov state analysis revealed multiple energetically stable conformations of L3 corresponding to open and closed states of the porin. Dynamics between these functional states occurs on the time scale of tens of microseconds and are mediated by the movement of highly conserved acidic residues of L3 to form H-bonds with opposing sides of the barrel wall of the pore. To validate our mechanism, we mutated key residues involved in the gating process that alter the H-bond pattern in the open/closed states and performed additional simulations. These mutations shifted the dynamic equilibrium of the pore towards open or closed states. Complementarily, the mutations favoring the open/closed states lead to increased/decreased accumulation of multiple antibiotics in our whole-cell accumulation assays. Notably, porins containing one of the mutations favoring the closed state has previously been found in antibiotic resistant bacterial strains. Overall, our 180 μs of simulation data (wild type and mutants) with concerted experiments suggests that regulation of the dynamic equilibrium between open and closed states of OM porins could be a mechanism by which Gram-negative bacteria acquire antibiotic resistance.

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“…Each monomer features an hourglass shape due to a central constriction formed by loop L3, which is folded into the barrel. Several studies have shown that OmpF voltage-induced gating changes with the properties of the bathing electrolyte, namely concentration, salt type, and solution pH [5,19,20,21,22,23]. These results suggest that the gating mechanism involves a global reorganization of the channel conformation, rather than just a residue-specific or loop-dependent effect [5,24].…”

Section: Introductionmentioning

confidence: 99%

Lipid Headgroup Charge and Acyl Chain Composition Modulate Closure of Bacterial β-Barrel Channels

Perini

Alcaraz

Queralt-Martín

2019

IJMS

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The outer membrane of Gram-negative bacteria contains β-barrel proteins that form high-conducting ion channels providing a path for hydrophilic molecules, including antibiotics. Traditionally, these proteins have been considered to exist only in an open state so that regulation of outer membrane permeability was accomplished via protein expression. However, electrophysiological recordings show that β-barrel channels respond to transmembrane voltages by characteristically switching from a high-conducting, open state, to a so-called ‘closed’ state, with reduced permeability and possibly exclusion of large metabolites. Here, we use the bacterial porin OmpF from E. coli as a model system to gain insight on the control of outer membrane permeability by bacterial porins through the modulation of their open state. Using planar bilayer electrophysiology, we perform an extensive study of the role of membrane lipids in the OmpF channel closure by voltage. We pay attention not only to the effects of charges in the hydrophilic lipid heads but also to the contribution of the hydrophobic tails in the lipid-protein interactions. Our results show that gating kinetics is governed by lipid characteristics so that each stage of a sequential closure is different from the previous one, probably because of intra- or intermonomeric rearrangements.

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Disulfide bond tethering of extracellular loops does not affect the closure of OmpF porin at acidic pH

Cited by 20 publications

References 29 publications

Analysis of Gating Transitions among the Three Major Open States of the OpdK Channel

Analysis of Gating Transitions among the Three Major Open States of the OpdK Channel

Investigation of gating in outer membrane porins provides new perspectives on antibiotic resistance mechanisms

Lipid Headgroup Charge and Acyl Chain Composition Modulate Closure of Bacterial β-Barrel Channels

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