Hervé Celia

Carbon nanotubes as templates for polymerized lipid assemblies

Amphiphilic molecules-molecules that have both hydrophobic and hydrophilic properties-can self-assemble in water to form diverse structures such as micelles, vesicles and tubes, and these nanostructures can be used for delivering drugs, stabilizing membrane proteins or as nanoreactors. We have previously shown that lipids can self-organize on the surface of single-walled carbon nanotubes into regular ring-shaped assemblies. Here we show that these lipid assemblies can be polymerized and isolated from the nanotube template by application of an electric field. We also demonstrate that these assemblies are monodispersed, water-soluble, and can dissolve various hydrophobic rylene dyes, fullerenes and membrane proteins. The stability of these constructs and their diverse applications will be useful in the fields of cosmetics, medicine and material sciences.

The Metal Dependence of Pyoverdine Interactions with Its Outer Membrane Receptor FpvA

To acquire iron, Pseudomonas aeruginosa secretes the fluorescent siderophore pyoverdine (Pvd), which chelates iron and shuttles it into the cells via the specific outer membrane transporter FpvA. We studied the role of iron and other metals in the binding and transport of Pvd by FpvA and conclude that there is no significant affinity between FpvA and metal-free Pvd. We found that the fluorescent in vivo complex of iron-free FpvA-Pvd is in fact a complex with aluminum (FpvA-Pvd-Al) formed from trace aluminum in the growth medium. When Pseudomonas aeruginosa was cultured in a medium that had been treated with a metal affinity resin, the in vivo formation of the FpvA-Pvd complex and the recycling of Pvd on FpvA were nearly abolished. The accumulation of Pvd in the periplasm of Pseudomonas aeruginosa was also reduced in the treated growth medium, while the addition of 1 microM AlCl(3) to the treated medium restored the effects of trace metals observed in standard growth medium. Using fluorescent resonance energy transfer and surface plasmon resonance techniques, the in vitro interactions between Pvd and detergent-solubilized FpvA were also shown to be metal dependent. We demonstrated that FpvA binds Pvd-Fe but not Pvd and that Pvd did not compete with Pvd-Fe for FpvA binding. In light of our finding that the Pvd-Al complex is transported across the outer membrane of Pseudomonas aeruginosa, a model for siderophore recognition based on a metal-induced conformation followed by redox selectivity for iron is discussed.

Fpva Bound to Non-Cognate Pyoverdines: Molecular Basis of Siderophore Recognition by an Iron Transporter.

The first step in the specific uptake of iron via siderophores in Gram‐negative bacteria is the recognition and binding of a ferric siderophore by its cognate receptor. We investigated the molecular basis of this event through structural and biochemical approaches. FpvA, the pyoverdine–Fe transporter from Pseudomonas aeruginosa ATCC 15692 (PAO1 strain), is able to transport ferric–pyoverdines originating from other species, whereas most fluorescent pseudomonads are only able to use the one they produce among the more than 100 known different pyoverdines. We solved the structure of FpvA bound to non‐cognate pyoverdines of high‐ or low‐affinity and found a close correlation between receptor–ligand structure and the measured affinities. The structure of the first amino acid residues of the pyoverdine chain distinguished the high‐ and low‐affinity binders while the C‐terminal portion of the pyoverdines, often cyclic, does not appear to contribute extensively to the interaction between the siderophore and its transporter. The specificity of the ferric–pyoverdine binding site of FpvA is conferred by the structural elements common to all ferric–pyoverdines, i.e. the chromophore, iron, and its chelating groups.

Structural study of the yeast RNA polymerase A: Electron microscopy of lipid-bound molecules and two-dimensional crystals

Two-dimensional crystals of yeast RNA polymerase A (I) were obtained by interaction with positively charged lipid layers. The analysis of single molecular images of lipid-bound RNA polymerases showed that the enzyme was preferentially oriented by the lipid phase, which probably facilitated crystallization. Electron micrographs of the crystals revealed a rectangular unit cell 25.8 nm by 45.6 nm in size containing four RNA polymerase dimers related by P22(1)2(1) symmetry. The projection map showed, at about 2.5 nm resolution, two different views of the enzyme characterized by two bent arms, which appeared to cross at one end. These arms are likely to contain the A190 and A135 subunits and delimit a 3 to 4 nm wide groove. Additional structural features were observed and compared to the Escherichia coli enzyme.

Interaction of TonB with the Outer Membrane Receptor FpvA of Pseudomonas aeruginosa

Pyoverdine-mediated iron uptake by the FpvA receptor in the outer membrane of Pseudomonas aeruginosa is dependent on the inner membrane protein TonB1. This energy transducer couples the proton-electrochemical potential of the inner membrane to the transport event. To shed more light upon this process, a recombinant TonB1 protein lacking the N-terminal inner membrane anchor (TonB(pp)) was constructed. This protein was, after expression in Escherichia coli, purified from the soluble fraction of lysed cells by means of an N-terminal hexahistidine or glutathione S-transferase (GST) tag. Purified GST-TonB(pp) was able to capture detergent-solubilized FpvA, regardless of the presence of pyoverdine or pyoverdine-Fe. Targeting of the TonB1 fragment to the periplasm of P. aeruginosa inhibited the transport of ferric pyoverdine by FpvA in vivo, indicating an interference with endogenous TonB1, presumably caused by competition for binding sites at the transporter or by formation of nonfunctional TonB heterodimers. Surface plasmon resonance experiments demonstrated that the FpvA-TonB(pp) interactions have apparent affinities in the micromolar range. The binding of pyoverdine or ferric pyoverdine to FpvA did not modulate this affinity. Apparently, the presence of either iron or pyoverdine is not essential for the formation of the FpvA-TonB complex in vitro.

The pyoverdin receptor FpvA, a TonB-dependent receptor involved in iron uptake by Pseudomonas aeruginosa (Review)

Iron is an important element, essential for the growth of almost all living cells. Because of the high insolubility of iron(III) in aerobic conditions, many gram-negative bacteria produce, under iron limitation, small iron-chelating compounds called siderophores, together with new outer-membrane proteins, which function as receptors for the ferrisiderophores. Pseudomonas aeruginosa, an important human opportunistic pathogen, produces at least three known siderophores when grown in irondeficient conditions: pyochelin, salicylate and pyoverdin. This reviewfocuses on pyoverdin and on the ability of FpvA to bind iron-free and ferric-PaA pyoverdin, in the light of recent information gained from biochemical and biophysical studies and of the recently solved 3D-structures of the related ferrichrome FhuA and enterobactin FepA receptors in Escherichia coli.Iron is an important element, essential for the growth of almost all living cells. Because of the high insolubility of iron(III) in aerobic conditions, many gram-negative bacteria produce, under iron limitation, small iron-chelating compounds called siderophores, together with new outer-membrane proteins, which function as receptors for the ferrisiderophores. Pseudomonas aeruginosa, an important human opportunistic pathogen, produces at least three known siderophores when grown in iron-deficient conditions: pyochelin, salicylate and pyoverdin. This review focuses on pyoverdin and on the ability of FpvA to bind iron-free and ferric-PaA pyoverdin, in the light of recent information gained from biochemical and biophysical studies and of the recently solved 3D-structures of the related ferrichrome FhuA and enterobactin FepA receptors in Escherichia coli.

Crystallization and X-ray diffraction analyses of the outer membrane pyochelin receptor FptA from Pseudomonas aeruginosa

FptA, the pyochelin outer membrane receptor from Pseudomonas aeruginosa, is a siderophore receptor involved in iron uptake when the bacterium grows under iron limitation. Two crystal forms of the FptA-pyochelin complex were obtained under different crystallization conditions. They belong to space groups P1 and P2(1)2(1)2(1) and data sets were collected for both crystal forms. The triclinic crystals diffract to 3.2 A resolution and the orthorhombic crystals show a 1.9 A resolution limit. A data set at the peak of the iron K edge was also collected at 3.1 A resolution.

Mapping the interactions between Escherichia coli TolQ transmembrane segments.

The tolQRAB-pal operon is conserved in Gram-negative genomes. The TolQRA proteins of Escherichia coli form an inner membrane complex in which TolQR uses the proton-motive force to regulate TolA conformation and the in vivo interaction of TolA C-terminal region with the outer membrane Pal lipoprotein. The stoichiometry of the TolQ, TolR, and TolA has been estimated and suggests that 4–6 TolQ molecules are associated in the complex, thus involving interactions between the transmembrane helices (TMHs) of TolQ, TolR, and TolA. It has been proposed that an ion channel forms at the interface between two TolQ and one TolR TMHs involving the TolR-Asp23, TolQ-Thr145, and TolQ-Thr178 residues. To define the organization of the three TMHs of TolQ, we constructed epitope-tagged versions of TolQ. Immunodetection of in vivo and in vitro chemically cross-linked TolQ proteins showed that TolQ exists as multimers in the complex. To understand how TolQ multimerizes, we initiated a cysteine-scanning study. Results of single and tandem cysteine substitution suggest a dynamic model of helix interactions in which the hairpin formed by the two last TMHs of TolQ change conformation, whereas the first TMH of TolQ forms intramolecular interactions.

Crystallization and preliminary X-ray analysis of the outer membrane pyoverdine receptor FpvA from Pseudomonas aeruginosa

FpvA, the pyoverdine outer-membrane receptor from Pseudomonas aeruginosa, is involved in iron uptake when bacteria grow under iron limitation. Crystals of the in vivo pyoverdine-loaded FpvA were obtained under several crystallization conditions using different detergents. A native data set was collected at 3.6 A resolution and a three-wavelength MAD data set was collected at 3.6 A resolution using crystals of selenomethionine-substituted protein. The crystals grew under similar conditions and both belong to space group C2, but have different unit-cell parameters.

Visualization of interactions between siderophore transporters and the energizing protein TonB by native PAGE.

Horizontal nondenaturing electrophoresis of proteins in polyacrylamide gels was used to observe specific interactions between membrane proteins. The method was particularly well suited for solubilized transporters of the outer membrane of Gram‐negative bacteria, and allowed specific complexes of transporter and the inner‐membrane protein TonB to be isolated. We have used this method to investigate the interactions between four different outer‐membrane transporters, and the TonB proteins from two different organisms. The results show that a stable complex can be isolated on gels for all the proteins studied, but can depend in some cases of the detergent used for solubilization. Furthermore, we observe cross‐species interaction as TonB from a given organism can interact with transporters from another organism.