Biomedical subjects

Arnaud Baslé

Publications and source records attributed to Arnaud Baslé.

5 recordsLinked to original sources

PubMed2006

Crystal structure of osmoporin OmpC from E. coli at 2.0 A.

Porins form transmembrane pores in the outer membrane of Gram-negative bacteria with matrix porin OmpF and osmoporin OmpC from Escherichia coli being differentially expressed depending on environmental conditions. The three-dimensional structure of OmpC has been determined to 2.0 A resolution by X-ray crystallography. As expected from the high sequence similarity, OmpC adopts the OmpF-like 16-stranded hollow beta-barrel fold with three beta-barrels associated to form a tight trimer. Unlike in OmpF, the extracellular loops form a continuous wall at the perimeter of the vestibule common to the three pores, due to a 14-residues insertion in loop L4. The pore constriction and the periplasmic outlet are very similar to OmpF with 74% of the pore lining residues being conserved. Overall, only few ionizable residues are exchanged at the pore lining. The OmpC structure suggests that not pore size, but electrostatic pore potential and particular atomic details of the pore linings are the critical parameters that physiologically distinguish OmpC from OmpF.

Amino Acid Sequence↗

PubMed2006

Conductance and selectivity fluctuations in D127 mutants of the bacterial porin OmpF.

A recent molecular dynamics study questioned the protonation state and physiological role of aspartate 127 (D127) of E. coli porin OmpF. To address that question we isolated two OmpF mutants with D127 either neutralized (D127N) or replaced by a positively charged lysine (D127K). The charge state of the residue at position 127 has clear effects on both conductance and selectivity. The D127K but not the D127N mutant expresses resilient conductance and selectivity fluctuations. These fluctuations reflect, we think, either changes in the ionization state of K127 and/or transitions between unstable subconformations as induced by the electrostatic repulsion between two positively charged residues, K127 and the nearby R167. Our results slightly favor the view that in WT OmpF residue D127 is deprotonated. As for the role of D127 in OmpF functionality, the gating of both mutants shows very similar sensitivity toward voltage as WT OmpF. Moreover, the current fluctuations of the D127K mutant were observed also in the absence of an applied electric field. We therefore dismiss D127 as a key residue in the control mechanism of the voltage-dependent gating of OmpF.

Bacterial Outer Membrane Proteins↗

PubMed2004

Deletions of single extracellular loops affect pH sensitivity, but not voltage dependence, of the Escherichia coli porin OmpF.

The molecular basis for the voltage and pH dependence of the Escherichia coli OmpF porin activity remains unknown. The L3 loop was previously shown not be involved in voltage dependence. Here we used seven OmpF mutants where single extracellular loops, except L3, were deleted one at a time. The proteins are expressed at levels comparable to wild-type and purified as trimers. Wild-type and mutant proteins were inserted into planar lipid bilayers for electrophysiological measurement of their activity. Current-voltage relationships show the typical porin channel closure at voltages greater than the critical voltage. Measurements of critical voltages for the seven deletion mutants showed no significant differences relative to wild-type, hence eliminating the role of single loops in voltage sensitivity. However, deletions of loops L1, L7 or L8 affected the tendency of channels to close at acidic pH. Wild-type channels close more readily at acidic pH and their open probability is decreased by approximately 60% at pH 4.0 relative to pH 7.0. For mutants lacking loop L1, L7 or L8, the channel open probability was found not to be significantly different at pH 4.0 than at pH 7.0. The other deletion mutants retained a pH sensitivity similar to the wild-type channel. Possible mechanistic scenarios for the voltage- and pH dependence of E.coli OmpF porin are discussed based on these results.

Amino Acid Motifs↗

PubMed2004

Subconductance states in OmpF gating.

Discrepancies were noted in the published conductance of the Escherichia coli porin OmpF. Results from various papers are hard to compare because of the use of different channel preparations, salt types and concentrations, and electrophysiological techniques (black lipid membrane (BLM) vs. patch clamp). To reconcile these data, we present a side-by-side comparison of OmpF activity studied with the two techniques on the same preparation of pure protein, and in the same low salt concentrations (150 mM KCl). The novel aspect of OmpF porin behavior revealed by this comparison is the ubiquitous existence of states of smaller conductance than the monomeric conductance (subconductance states), regardless of the techniques or experimental conditions used, and the drastic enhancement of subconductance gating by polyamines. Transitions to subconductance states have received little attention in previous publications, in particular when BLM electrophysiology was used. Monomeric closures are rare in recordings at clamped potentials, at least at voltages lower than approximately 100-120 mV. Most closing activity is in the form of subconductance gating, which becomes more dominant in the presence of spermine, with a more frequent and prolonged occupation of these substates. A discussion of the molecular basis for this hallmark behavior of porin is presented.

Cell Membrane↗

PubMed2003

The Vibrio cholerae porins OmpU and OmpT have distinct channel properties.

Numerous environmental signals regulate the production of virulence factors and the composition of the outer membrane of Vibrio cholerae. In particular, bile promotes the ToxR-dependent expression of the porin OmpU. Strains expressing solely OmpU are more resistant to bile, are better able to colonize the intestine, and produce more cholera toxin than strains expressing solely the OmpT porin. To gain some understanding in the physiological relevance and the molecular mechanism underlying these porin-dependent phenotypes, we have undertaken a thorough electrophysiological characterization of the channel properties of the two porins. Purified OmpU or OmpT was reconstituted in liposomes suitable for patch clamp and in planar lipid bilayers. The high resolution of the patch clamp technique allowed us to analyze in detail the behavior of single OmpU and OmpT channels. Both channels exhibit closing transitions to various conductance states. OmpT is a much more dynamic channel than OmpU, displaying frequent and prolonged closures, even at low transmembrane potentials. With a critical voltage for closure V(c) of approximately +/-90 mV, OmpT is much more voltage-sensitive than OmpU (with a V(c) of approximately +/-160 mV), a feature that is also readily apparent in the voltage dependence of closing probability observed in patch clamp in the +/-100 mV range. OmpT has low ionic selectivity (P(K)/P(Cl) = approximately 4), whereas OmpU is more cation-selective (P(K)/P(Cl) = approximately 14). The distinct functional properties of the two porins are likely to play an integrated role with environmental regulation of their expression. For example, the higher selectivity of OmpU for cations provides a possible explanation for the protective role played by this porin in a bile-containing environment, because this type of selectivity would restrict the flux of anionic bile salts through the outer membrane and thus would reduce the exposure of the cytoplasmic membrane to this natural detergent.

Adhesins, Bacterial↗