in-vitro-investigation-mexab-efflux-pump-from-pseudomonas

<em>In vitro</em> Investigation of the MexAB Efflux Pump From <em>Pseudomonas aeruginosa</em>

There is an emerging scientific need for reliable tools for monitoring membrane protein transport. We present a methodology leading to the reconstitution ...

CNRS & Université Paris Descartes

The kinetics of charge recombination following photoexcitation by a laser pulse have been analyzed in the reaction center-light harvesting complex 1 (RC-LH1) purified from the photosynthetic bacterium Rhodobacter sphaeroides. In RC-LH1 core complexes isolated from photosynthetically grown cells P(+)Q(B)(-) recombines with an average rate constant, k approximately 0.3 s(-1), more than three times smaller than that measured in RC deprived of the LH1 (k approximately 1 s(-1)). A comparable, slowed recombination kinetics is observed in RC-LH1 complexes purified from a pufX-deleted strain. Slowing of the charge recombination kinetics is even more pronounced in RC-LH1 complexes isolated from wild-type semiaerobically grown cells (k approximately 0.2 s(-1)). Since the kinetics of P(+)Q(A)(-) recombination is unaffected by the presence of the antenna, the P(+)Q(B)(-) state appears to be energetically stabilized in core complexes. Determinations of the ubiquinone-10 (UQ(10)) complement associated with the purified RC-LH1 complexes always yield UQ(10)/RC ratios larger than 10. These quinone molecules are functionally coupled to the RC-LH1 complex, as judged from the extent of exogenous cytochrome c(2) rapidly oxidized under continuous light excitation. Analysis of P(+)Q(B)(-) recombination, based on a kinetic model which considers fast quinone equilibrium at the Q(B) binding site, indicates that the slowing down of charge recombination kinetics observed in RC-LH1 complexes cannot be explained solely by a quinone concentration effect and suggests that stabilization of the light-induced charge separation is predominantly due to interaction of the Q(B) site with the LH1 complex. The high UQ(10) complements detected in RC-LH1 core complexes, but not in purified light-harvesting complex 2 and in RC, are proposed to reflect an in vivo heterogeneity in the distribution of the quinone pool within the chromatophore bilayer.

Light-harvesting complex 1 stabilizes P+QB- charge separation in reaction centers of Rhodobacter sphaeroides.

The bacterial reaction center (RC), a membrane photosynthetic protein, has been adsorbed onto a glass surface by alternating deposition with the cationic polymer poly(dimethyldiallylammonium chloride) (PDDA) obtaining as an end result an ordinate polyelectrolyte multilayer (PEM) where the protein retains its integrity and photoactivity over a period of several months. Such a system has been characterized from the functional point of view by checking the protein photoactivity at different hydration conditions, from extensive drought to full hydration. The kinetic analysis of charge recombination indicates that incorporation of RCs into dehydrated PEM hinders the conformational dynamics gating QA- to QB electron-transfer leaving unchanged the protein relaxation that stabilizes the primary charge separated state P+QA-. The herbicide-induced inhibition of the QB activity was studied in some detail. By dipping the PEM in herbicide solutions for short times, kinetics of herbicide binding and release have been determined; binding isotherms have been studied using PEM immersed in herbicide solution. QB functionality of RC has been restored by rinsing the PEM with water, thus allowing the reuse of the same sample. This last point has been exploited to design a simple optical biosensor for herbicides. A suitable kinetic model has been proposed to describe the interplay between forward and back electron-transfer processes upon continuous illumination, and the use of the PDDA-RC multilayers in herbicide bioassays was successfully tested.

Functionality of photosynthetic reaction centers in polyelectrolyte multilayers: toward an herbicide biosensor.

The reaction center-light harvesting complex 1 (RC-LH1) purified from the photosynthetic bacterium Rhodobacter sphaeroides has been studied with respect to the kinetics of charge recombination and to the phospholipid and ubiquinone (UQ) complements tightly associated with it. In the antenna-RC complexes, at 6.5<pH<9.0, P(+)Q(B)(-) recombines with a pH independent average rate constant <k> more than three times smaller than that measured in LH1-deprived RCs. At increasing pH values, for which <k> increases, the deceleration observed in RC-LH1 complexes is reduced, vanishing at pH >11.0. In both systems kinetics are described by a continuous rate distribution, which broadens at pH >9.5, revealing a strong kinetic heterogeneity, more pronounced in the RC-LH1 complex. In the presence of the antenna the Q(A)Q(B)(-) state is stabilized by about 40 meV at 6.5<pH<9.0, while it is destabilized at pH >11. The phospholipid/RC and UQ/RC ratios have been compared in chromatophore membranes, in RC-LH1 complexes and in the isolated peripheral antenna (LH2). The UQ concentration in the lipid phase of the RC-LH1 complexes is about one order of magnitude larger than the average concentration in chromatophores and in LH2 complexes. Following detergent washing RC-LH1 complexes retain 80-90 phospholipid and 10-15 ubiquinone molecules per monomer. The fractional composition of the lipid domain tightly bound to the RC-LH1 (determined by TLC and (31)P-NMR) differs markedly from that of chromatophores and of the peripheral antenna. The content of cardiolipin, close to 10% weight in chromatophores and LH2 complexes, becomes dominant in the RC-LH1 complexes. We propose that the quinone and cardiolipin confinement observed in core complexes reflects the in vivo heterogeneous distributions of these components. Stabilization of the charge separated state in the RC-LH1 complexes is tentatively ascribed to local electrostatic perturbations due to cardiolipin.

Stabilization of charge separation and cardiolipin confinement in antenna-reaction center complexes purified from Rhodobacter sphaeroides.

Picosecond time-resolved fluorescence spectroscopy has been used in order to compare the fluorescence kinetics of detergent-solubilized and membrane-reconstituted light-harvesting 2 (LH2) complexes from the purple bacteria Rhodopseudomonas (Rps.) acidophila and Rhodobacter (Rb.) sphaeroides. LH2 complexes were reconstituted in phospholipid model membranes at different lipid:protein-ratios and all samples were studied exciting with a wide range of excitation densities. While the detergent-solubilized LH2 complexes from Rps. acidophila showed monoexponential decay kinetics (tau(f )= 980 ps) for excitation densities of up to 3.10(13) photons/(pulse.cm(2)), the membrane-reconstituted LH2 complexes showed multiexponential kinetics even at low excitation densities and high lipid:protein-ratios. The latter finding indicates an efficient clustering of LH2 complexes in the phospholipid membranes. Similar results were obtained for the LH2 complexes from Rb. sphaeroides.

Comparison of the fluorescence kinetics of detergent-solubilized and membrane-reconstituted LH2 complexes from Rps. acidophila and Rb. sphaeroides.

Trehalose is a nonreducing disaccharide of glucose found in organisms, which can survive adverse conditions such as extreme drought and high temperatures. Furthermore, isolated structures, as enzymes or liposomes, embedded in trehalose are preserved against stressing conditions [see, e.g., Crowe, L. M. Comp. Biochem. Physiol. A 2002, 131, 505-513]. Among other hypotheses, such protective effect has been suggested to stem, in the case of proteins, from the formation of a water-mediated, hydrogen bond network, which anchors the protein surface to the water-sugar matrix, thus coupling the internal degrees of freedom of the biomolecule to those of the surroundings [Giuffrida, S.; et al. J. Phys. Chem. B 2003, 107, 13211-13217]. Analogous protective effect is also accomplished by other saccharides, although with a lower efficiency. Here, we studied the recombination kinetics of the primary, light-induced charge separated state (P(+)Q(A)(-)) and the thermal stability of the photosynthetic reaction center (RC) of Rhodobacter sphaeroides in trehalose-water and in sucrose-water matrixes of decreasing water content. Our data show that, in sucrose, at variance with trehalose, the system undergoes a "nanophase separation" when the water/sugar mole fraction is lower than the threshold level approximately 0.8. We rationalize this result assuming that the hydrogen bond network, which anchors the RC surface to its surrounding, is formed in trehalose but not in sucrose. We suggest that both the couplings, in the case of trehalose, and the nanophase separation, in the case of sucrose, start at low water content when the components of the system enter in competition for the residual water.

Protein-matrix coupling/uncoupling in "dry" systems of photosynthetic reaction center embedded in trehalose/sucrose: the origin of trehalose peculiarity.

The core complex formed by the reaction center and the light harvesting complex 1 (RC-LH1) was purified from the photosynthetic bacterium Rhodobacter sphaeroides. We analyzed the lipid and ubiquinone (UQ) complements copurifying with the RC-LH1 complex and with the peripheral antenna (LH2). In RC-LH1 UQ was almost ten times more concentrated than in the LH2 and in the native membranes from which the complexes were extracted. The fractional lipid composition of the RC-LH1 complex also differed from that of the intact membranes, exhibiting a marked increase in cardiolipin concentration. We propose that the confinement of cardiolipin and ubiquinone observed within the RC-LH1 complex, plays a role in vivo in the stabilization of the light-induced charge separation catalyzed by the RC.

Confinement of cardiolipin and ubiquinone in reaction-center core complexes purified from the photosynthetic bacterium Rhodobacter sphaeroides.

ABCG2 is a human membrane ATP-binding cassette half-transporter that hydrolyzes ATP to efflux a large number of chemotherapeutic agents. Several oligomeric states of ABCG2 from homodimers to dodecamers have been reported depending on the overexpression systems and/or the protocols used for purification. Here, we compared the oligomeric state of His(6)-ABCG2 expressed in Sf9 insect cells and in human Flp-In-293/ABCG2 cells after solubilization in mild detergents. His(6)-ABCG2 was purified through a new approach involving its specific recognition onto a functionalized lipid layer containing a Ni-NTA lipid. This approach allowed the purification of His-ABCG2 in presence of all solubilized membrane components that might be involved in the stabilisation of native oligomers and without requiring any additional washing or concentration passages. ABCG2 purified onto the NiNTA lipid surfaces were directly analyzed by electron microscopy and by biochemical assays. Altogether, our data are consistent with a tetrameric organization of ABCG2 when expressed in either heterologous Sf9 insect cells or in human homologous cells.

The multidrug resistance half-transporter ABCG2 is purified as a tetramer upon selective extraction from membranes.

We present a detailed AFM study on multilayered dry lipid films prepared from aqueous vesicle suspensions. Different preparation techniques were applied in order to optimize the preparation of homogeneous lipid films of various film thicknesses. Suspensions of preformed DOPC/DPPC vesicles were adsorbed onto indium tin oxide-coated glass coverslips, a substrate also commonly employed for the formation of giant liposomes. We found that the homogeneity of the lipid films could substantially be improved when applying a spin-coating step during the film preparation. These films were much more homogeneous than those prepared by conventional drop-casting and in addition the film thickness could be controlled. When using a combination of vesicle adsorption and spin-coating the quality and thickness of the films depended crucially on the lipid concentration of the vesicle suspension, the adsorption temperature and the adsorption time. For lipid films prepared by direct spin-coating the lipid concentration and the applied spin-coating sequence were critical parameters for the quality and thickness of the deposited lipid films.

AFM characterization of spin-coated multilayered dry lipid films prepared from aqueous vesicle suspensions.

Monolayer of functionalized lipid spread at the air/water interface is used for the structural analysis of soluble and membrane proteins by electron crystallography and single particle analysis. This powerful approach lacks of a method for the screening of the binding of proteins to the surface of the lipid layer. Here, we described an optical method based on the use of reflected light microscopy to image, without the use of any labeling, the lipid layer at the surface of buffers in the Teflon wells used for 2D crystallization. Images revealed that the lipid layer was made of a monolayer coexisting with liposomes or aggregates of lipids floating at the surface. Protein binding led to an increase of the contrast and the decrease of the fluidity of the lipid surface, as demonstrated with the binding of soluble Shiga toxin B subunit, of purple membrane and of solubilized His-BmrA, a bacterial ABC transporter. Moreover the reconstitution of membrane proteins bound to the lipidic surface upon detergent removal can be followed through the appearance of large recognizable domains at the surface. Proteins binding and reconstitution were further confirmed by electron microcopy. Overall, this method provides a quick evaluation of the monolayer trials, a significant reduction in screening by transmission electron microscopy and new insights in the proteins binding and 2D crystallogenesis at the lipid surface.

Binding, reconstitution and 2D crystallization of membrane or soluble proteins onto functionalized lipid layer observed in situ by reflected light microscopy.

The lipid-layer technique allows reconstituting transmembrane proteins at a high density in microns size planar membranes and suspended to a lipid monolayer at the air/water interface. In this paper, we transferred these membranes onto two hydrophobic substrates for further structural analysis of reconstituted proteins by Atomic Force Microscopy (AFM). We used a mica sheet covered by a lipid monolayer or a sheet of highly oriented pyrolytic graphite (HOPG) to trap the lipid monolayer at the interface and the suspended membranes. In both cases, we succeeded in the transfer of large membrane patches containing densely packed or 2D-crystallized proteins. As a proof of concept, we transferred and imaged the soluble Shiga toxin bound to its lipid ligand and the ATP-binding cassette (ABC) transporter BmrA reconstituted into a planar bilayer. AFM imaging with a lateral resolution in the nanometer range was achieved. Potential applications of this technique in structural biology and nanobiotechnology are discussed.

Transfer on hydrophobic substrates and AFM imaging of membrane proteins reconstituted in planar lipid bilayers.

Optimized protocols for achieving high-yield expression, purification and reconstitution of membrane proteins are required to study their structure and function. We previously reported high-level expression in Escherichia coli of active BmrC and BmrD proteins from Bacillus subtilis, previously named YheI and YheH. These proteins are half-transporters which belong to the ABC (ATP-Binding Cassette) superfamily and associate in vivo to form a functional transporter able to efflux drugs. In this report, high-yield purification and functional reconstitution were achieved for the heterodimer BmrC/BmrD. In contrast to other detergents more efficient for solubilizing the transporter, dodecyl-ß-D-maltoside (DDM) maintained it in a drug-sensitive and vanadate-sensitive ATPase-competent state after purification by affinity chromatography. High amounts of pure proteins were obtained which were shown either by analytical ultracentrifugation or gel filtration to form a monodisperse heterodimer in solution, which was notably stable for more than one month at 4°C. Functional reconstitution using different lipid compositions induced an 8-fold increase of the ATPase activity (k(cat)∼5 s(-1)). We further validated that the quality of the purified BmrC/BmrD heterodimer is suitable for structural analyses, as its reconstitution at high protein densities led to the formation of 2-D crystals. Electron microscopy of negatively stained crystals allowed the calculation of a projection map at 20 Å resolution revealing that BmrC/BmrD might assemble into oligomers in a lipidic environment.

Optimized purification of a heterodimeric ABC transporter in a highly stable form amenable to 2-D crystallization.

Giant unilamellar vesicles (GUVs) are convenient biomimetic systems of the same size as cells that are increasingly used to quantitatively address biophysical and biochemical processes related to cell functions. However, current approaches to incorporate transmembrane proteins in the membrane of GUVs are limited by the amphiphilic nature or proteins. Here, we report a method to incorporate transmembrane proteins in GUVs, based on concepts developed for detergent-mediated reconstitution in large unilamellar vesicles. Reconstitution is performed either by direct incorporation from proteins purified in detergent micelles or by fusion of purified native vesicles or proteoliposomes in preformed GUVs. Lipid compositions of the membrane and the ionic, protein, or DNA compositions in the internal and external volumes of GUVs can be controlled. Using confocal microscopy and functional assays, we show that proteins are unidirectionally incorporated in the GUVs and keep their functionality. We have successfully tested our method with three types of transmembrane proteins. GUVs containing bacteriorhodopsin, a photoactivable proton pump, can generate large transmembrane pH and potential gradients that are light-switchable and stable for hours. GUVs with FhuA, a bacterial porin, were used to follow the DNA injection by T5 phage upon binding to its transmembrane receptor. GUVs incorporating BmrC/BmrD, a bacterial heterodimeric ATP-binding cassette efflux transporter, were used to demonstrate the protein-dependent translocation of drugs and their interactions with encapsulated DNA. Our method should thus apply to a wide variety of membrane or peripheral proteins for producing more complex biomimetic GUVs.

Manuela Dezi

Laboratoire de Cristallographie & RMN Biologiques

CNRS & Université Paris Descartes

In vitro Investigation of the MexAB Efflux Pump From Pseudomonas aeruginosa

Manuela Dezi

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CNRS & Université Paris Descartes

In vitro Investigation of the MexAB Efflux Pump From Pseudomonas aeruginosa

Laboratoire de Cristallographie & RMN Biologiques