Biomedical subjects

Alain Brisson

Publications and source records attributed to Alain Brisson.

11 recordsLinked to original sources

PubMed2006

Protein crystallography under xenon and nitrous oxide pressure: comparison with in vivo pharmacology studies and implications for the mechanism of inhaled anesthetic action.

In contrast with most inhalational anesthetics, the anesthetic gases xenon (Xe) and nitrous oxide (N(2)O) act by blocking the N-methyl-d-aspartate (NMDA) receptor. Using x-ray crystallography, we examined the binding characteristics of these two gases on two soluble proteins as structural models: urate oxidase, which is a prototype of a variety of intracellular globular proteins, and annexin V, which has structural and functional characteristics that allow it to be considered as a prototype for the NMDA receptor. The structure of these proteins complexed with Xe and N(2)O were determined. One N(2)O molecule or one Xe atom binds to the same main site in both proteins. A second subsite is observed for N(2)O in each case. The gas-binding sites are always hydrophobic flexible cavities buried within the monomer. Comparison of the effects of Xe and N(2)O on urate oxidase and annexin V reveals an interesting relationship with the in vivo pharmacological effects of these gases, the ratio of the gas-binding sites' volume expansion and the ratio of the narcotic potency being similar. Given these data, we propose that alterations of cytosolic globular protein functions by general anesthetics would be responsible for the early stages of anesthesia such as amnesia and hypnosis and that additional alterations of ion-channel membrane receptor functions are required for deeper effects that progress to "surgical" anesthesia.

Anesthetics↗

PubMed2006

Phosphorylcholine-based pH-responsive diblock copolymer micelles as drug delivery vehicles: light scattering, electron microscopy, and fluorescence experiments.

The micellization behavior of a diblock copolymer comprising a highly hydrophilic and biocompatible poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC) corona-forming block and a pH-sensitive poly(2-(diisopropylamino)ethyl methacrylate) (PDPA) core-forming block (PMPC-b-PDPA) has been studied by static and dynamic light scattering (SDLS), transmission electron microscopy (TEM), and potentiometry. Self-assembly of PMPC-b-PDPA copolymers with two different DPA volume fractions (phi(DPA)) leads to narrowly distributed and structurally distinct spherical micelles, as evidenced by their molecular weight (M(w,mic)), aggregation number (N(agg)), hydrodynamic radius (R(H)), corona width (W), and core radius (R(c)). The excellent potential of these pH-responsive micelles as nanosized drug delivery vehicles was illustrated by the encapsulation of dipyridamole (DIP), a model hydrophobic drug that dissolves in acid solutions and becomes insoluble above pH 5.8, which is comparable to the pK(a) of the PDPA block. The influence of micelle structure (namely M(w,mic), N(agg), R(H), W, and R(c)) on drug loading content, drug loading efficiency, partition coefficient, and release kinetics was investigated and confirmed by fluorescence spectroscopy studies. The maximum dipyridamole loadings within PMPC(30)-b-PDPA(30) (R(H) = 14.0 nm; W = 4.8 nm; R(c) = 9.2 nm) and PMPC(30)-b-PDPA(60) (R(H) = 27.1 nm; W = 11.0 nm; R(c) = 16.1 nm) micelles were 7 and 12% w/w(p), respectively. This preferential solubilization of DIP into micelles formed by copolymer chains having longer core-forming blocks (i.e., possessing larger core volumes) reflects the larger partition coefficient (K(V)) of DIP between the aqueous phase and PMPC(30)-b-PDPA(60) micelles (K(V) = 5.7 x 10(4)) compared to PMPC(30)-b-PDPA(30) micelles (K(V) = 1.1 x 10(4)). This enhanced ability of PMPC(30)-b-PDPA(60) aggregates to entrap/stabilize small hydrophobic molecules also produces slower release kinetics. Rapid release can be triggered by lowering the pH to induce micellar dissociation.

Dipyridamole↗

PubMed2005

Structural determinants for plant annexin-membrane interactions.

The interactions of two plant annexins, annexin 24(Ca32) from Capsicum annuum and annexin Gh1 from Gossypium hirsutum, with phospholipid membranes have been characterized using liposome-based assays and adsorption to monolayers. These two plant annexins show a preference for phosphatidylserine-containing membranes and display a membrane binding behavior with a half-maximum calcium concentration in the sub-millimolar range. Surprisingly, the two plant annexins also display calcium-independent membrane binding at levels of 10-20% at neutral pH. This binding is regulated by three conserved surface-exposed residues on the convex side of the proteins that play a pivotal role in membrane binding. Due to quantitative differences in the membrane binding behavior of N-terminally His-tagged and wild-type annexin 24(Ca32), we conclude that the N-terminal domain of plant annexins plays an important role, reminiscent of the findings in their mammalian counterparts. Experiments elucidating plant annexin-mediated membrane aggregation and fusion, as well as the effect of these proteins on membrane surface hydrophobicity, agree with findings from the membrane binding experiments. Results from electron microscopy reveal elongated rodlike assemblies of plant annexins in the membrane-bound state. It is possible that these structures consist of protein molecules directly interacting with the membrane surface and molecules that are membrane-associated but not in direct contact with the phospholipids. The rodlike structures would also agree with the complex data from intrinsic protein fluorescence. The tubular lipid extensions suggest a role in the membrane cytoskeleton scaffolding or exocytotic processes. Overall, this study demonstrates the importance of subtle changes in an otherwise conserved annexin fold where these two plant annexins possess distinct modalities compared to mammalian and other nonplant annexins.

Animals↗

PubMed2005

The basic framework of VE-cadherin junctions revealed by cryo-EM.

Artificial adherens junctions were reconstituted in vitro by assembly of cadherin fragments at the surfaces of liposomes. The architecture of the adherens junctions was revealed by cryo-electron microscopy (cryo-EM). The formation of these artificial adherens junctions was shown to result from the two-dimensional (2D) self-assembly of cadherin fragments at membrane surfaces. The molecular architecture of the junctions was resolved by combining information from several cryo-EM views. This study concludes to the 2D ordered nature of the cadherin assembly and shows that the minimal information required to build up an adherens junction is contained within the extracellular moiety of cadherin molecules.

Adherens Junctions↗

PubMed2005

Trimeric structure of OprN and OprM efflux proteins from Pseudomonas aeruginosa, by 2D electron crystallography.

OprM and OprN belong to the outer membrane factor family of multidrug efflux proteins from Pseudomonas aeruginosa, a bacterium responsible of nosocomial infections. We report here the two-dimensional (2D) crystallization of OprN and OprM into lipid bilayers and the determination of their 2D projected structure by cryo-electron crystallography, at 1 and 1.4 nm, respectively. Both proteins present a dense ring of protein density, of approximately 7 nm diameter. An additional thin peripheral ring is resolved in OprN structure. Both proteins are assembled as trimers. The results presented here indicate a high structural homology between OprN (and OprM) and TolC, a multidrug efflux protein from Escherichia coli.

Bacterial Outer Membrane Proteins↗

PubMed2005

The formation of supported lipid bilayers on silica nanoparticles revealed by cryoelectron microscopy.

The controlled fabrication of biocompatible devices made of lipid bilayers deposited onto flat solid supports presents interest as models of cell membranes as well as for their biotechnological applications. We report here on the formation of supported lipid bilayers on silica nanoparticles (nanoSLBs). The successive steps of the adsorption of lipid vesicles on nanoparticles and the formation of nanoSLBs are revealed in detail by cryotransmission electron microscopy (cryo-EM). The formation of nanoSLBs was achieved for liposomes with positive, neutral, and low net negative charge, while liposomes with a high net negative charge adsorbed to silica nanoparticles but did not rupture. The nanoSLBs were found to follow faithfully the surface contours of the particles, information yet unavailable for SLB formation on planar solid substrates.

Coated Materials, Biocompatible↗

PubMed2004

Mechanism of formation of multilayered 2D crystals of the enzyme IIC-mannitol transporter.

We have recently reported the crystallization by reconstitution into lipid bilayer structures of Enzyme IIC(mtl), the transmembrane C-domain of the mannitol transporter from E. coli. The projected structure was determined to a resolution of 0.5 nm [J. Mol. Biol. 287 5 (1999) 845]. However, further investigation proved that these crystals were multilamellar stacks instead of 2D crystals, and therefore were unsuitable for three-dimensional structural analysis by electron crystallography. Understanding the crystallogenesis of these crystals could reveal the mechanism of formation of multilayers. In the present study, cryo-electron microscopy (cryo-EM) and turbidimetry are used to study the successive steps of reconstitution of Enzyme IIC(mtl) into phospholipid-containing structures and its crystallization under different conditions. Our experimental approach enabled us to distinguish the separate steps of reconstitution and crystallization. The salt concentration especially influenced the nature of the vesicles, either half open unilamellar or aggregated multilamellar, formed during reconstitution of Enzyme IIC(mtl). The presence of DOPE and DOPC and the temperature influenced the type of lipid structures that were formed during the crystallization phase of Enzyme IIC(mtl). Cryo-EM showed that protein crystallization is closely associated with the formation of isotropic lipid (cubic) phases. We believe that DOPE is responsible for the formation of these lipid cubic phases, and that crystallization is driven by exclusion of protein from these phases and its concentration into the lamellar phases. This mechanism is inextricably associated with the formation of multilayers.

Cholic Acids↗

PubMed2004

QCM-D on mica for parallel QCM-D-AFM studies.

Quartz crystal microbalance with dissipation monitoring (QCM-D) has developed into a recognized method to study adsorption processes in liquid, such as the formation of supported lipid bilayers and protein adsorption. However, the large intrinsic roughness of currently used gold-coated or silica-coated QCM-D sensors limits parallel structural characterization by atomic force microscopy (AFM). We present a method for coating QCM-D sensors with thin mica sheets operating in liquid with high stability and sensitivity. We define criteria to objectively assess the reliability of the QCM-D measurements and demonstrate that the mica-coated sensors can be used to follow the formation of supported lipid membranes and subsequent protein adsorption. This method allows combining QCM-D and AFM investigations on identical supports, providing detailed physicochemical and structural characterization of model membranes.

Aluminum Silicates↗

PubMed2003

Pathways of lipid vesicle deposition on solid surfaces: a combined QCM-D and AFM study.

Supported lipid bilayers (SLBs) are popular models of cell membranes with potential biotechnological applications, yet the mechanism of SLB formation is only partially understood. In this study, the adsorption and subsequent conformational changes of sonicated unilamellar vesicles on silica supports were investigated by quartz crystal microbalance with dissipation monitoring and atomic force microscopy, using mixtures of zwitterionic, negatively charged, and positively charged lipids, both in the presence and in the absence of Ca(2+) ions. Four different pathways of vesicle deposition could be distinguished. Depending on their charge, vesicles i). did not adsorb; ii). formed a stable vesicular layer; or iii). decomposed into an SLB after adsorption at high critical coverage or iv). at low coverage. Calcium was shown to enhance the tendency of SLB formation for negatively charged and zwitterionic vesicles. The role of vesicle-support, interbilayer, and intrabilayer interactions in the formation of SLBs is discussed.

Adsorption↗

PubMed2003

Preparation of flat carbon support films.

Wrinkling of carbon support films is known to limit the resolution of electron microscopy images of protein two-dimensional crystals. The origin of carbon wrinkling during preparation of the support films was investigated by reflected light microscopy. We observed that carbon films go through several states during their preparation. While dried carbon films have a tendency to be wrinkled, a flat state is observed transiently before complete drying. This state could be stabilized by the addition of sugars or tannic acid to the embedding medium. An alternative method for preparing flat carbon films was developed, in which a sandwich is formed by two symmetrical carbon films positioned on both sides of a grid. The formation of sandwiched carbon films was facilitated by the use of grids with thin bars. The carbon sandwich films were flat, stable, and easily and reproducibly prepared.

Carbohydrates↗

PubMed2002

Self-assembly of the hydrophobin SC3 proceeds via two structural intermediates.

Hydrophobins self assemble into amphipathic films at hydrophobic-hydrophilic interfaces. These proteins are involved in a broad range of processes in fungal development. We have studied the conformational changes that accompany the self-assembly of the hydrophobin SC3 with polarization-modulation infrared reflection absorption spectroscopy, attenuated total reflection Fourier transform infrared spectroscopy, and circular dichroism, and related them to changes in morphology as observed by electron microcopy. Three states of SC3 have been spectroscopically identified previously as follows: the monomeric state, the alpha-helical state that is formed upon binding to a hydrophobic solid, and the beta-sheet state, which is formed at the air-water interface. Here, we show that the formation of the beta-sheet state of SC3 proceeds via two intermediates. The first intermediate has an infrared spectrum indistinguishable from that of the alpha-helical state of SC3. The second intermediate is rich in beta-sheet structure and has a featureless appearance under the electron microscope. The end state has the same secondary structure, but is characterized by the familiar 10-nm-wide rodlets.

Bacterial Proteins↗