Daniel Lévy

Detergent removal by non-polar polystyrene beads

Abstract Detergent removal from lipid-protein-detergent micellar solutions is the most successful strategy for reconstitution of integral membrane proteins into proteoliposomes or into two-dimensional crystals. This review establishes the potential of polystyrene beads as a simple alternative to other conventional detergent removing strategies such as dialysis, gel chromatography and dilution. Kinetics and equilibrium aspects of removal of different detergents by hydrophobic adsorption onto polystyrene beads have been systematically investigated. A mechanism of adsorption onto polystyrene beads is proposed and provide useful information about the use of these beads in reconstitution experiments. The usefulness of this detergent removal strategy to produce quasi-ideal proteoliposomes is evaluated by considering the morphology and the size of the reconstituted vesicles, the homogeneity in size and protein distribution, the final protein orientation and the permeability of resulting proteoliposomes. Finally, the advantages of detergent removal by polystyrene beads as an alternative to conventional dialysis in two-dimensional crystallization trials are evaluated through review of recent structural reconstitution studies.

Structure of the Dimeric PufX-containing Core Complex of Rhodobacter blasticus by in Situ Atomic Force Microscopy

We have studied photosynthetic membranes of wild type Rhodobacter blasticus, a closely related strain to the well studied Rhodobacter sphaeroides, using atomic force microscopy. High-resolution atomic force microscopy topographs of both cytoplasmic and periplasmic surfaces of LH2 and RC-LH1-PufX (RC, reaction center) complexes were acquired in situ. The LH2 is a nonameric ring inserted into the membrane with the 9-fold axis perpendicular to the plane. The core complex is an S-shaped dimer composed of two RCs, each encircled by 13 LH1 α/β-heterodimers, and two PufXs. The LH1 assembly is an open ellipse with a topography-free gap of ∼25 Å. The two PufXs, one of each core, are located at the dimer center. Based on our data, we propose a model of the core complex, which provides explanation for the PufX-induced dimerization of the Rhodobacter core complex. The QB site is located facing a ∼25-Å wide gap within LH1, explaining the PufX-favored quinone passage in and out of the core complex.

Use of detergents in two-dimensional crystallization of membrane proteins.

Structure determination at high resolution is actually a difficult challenge for membrane proteins and the number of membrane proteins that have been crystallized is still small and far behind that of soluble proteins. Because of their amphiphilic character, membrane proteins need to be isolated, purified and crystallized in detergent solutions. This makes it difficult to grow the well-ordered three-dimensional crystals that are required for high resolution structure analysis by X-ray crystallography. In this difficult context, growing crystals confined to two dimensions (2D crystals) and their structural analysis by electron crystallography has opened a new way to solve the structure of membrane proteins. However, 2D crystallization is one of the major bottlenecks in the structural studies of membrane proteins. Advances in our understanding of the interaction between proteins, lipids and detergents as well as development and improvement of new strategies will facilitate the success rate of 2D crystallization. This review deals with the various available strategies for obtaining 2D crystals from detergent-solubilized intrinsic membrane proteins. It gives an overview of the methods that have been applied and gives details and suggestions of the physical processes leading to the formation of the ordered arrays which may be of help for getting more proteins crystallized in a form suitable for high resolution structural analysis by electron crystallography.

AFM Characterization of Tilt and Intrinsic Flexibility of Rhodobacter sphaeroides Light Harvesting Complex 2 (LH2)

Atomic force microscopy (AFM) has developed into a powerful tool to investigate membrane protein surfaces in a close-to-native environment. Here we report on the surface topography of Rhodobacter sphaeroides light harvesting complex 2 (LH2) reconstituted into two-dimensional crystals. These photosynthetic trans-membrane proteins formed cylindrical oligomeric complexes, which inserted tilted into the lipid membrane. This peculiar packing of an integral membrane protein allowed us to determine oligomerization and tilt of the LH2 complexes, but also protrusion height and intrinsic flexibility of their individual subunits. Furthermore the surface contouring reliability and limits of the atomic force microscopy could be studied. The two-dimensional crystals examined had sizes of up to 5 microm and, as revealed by a 10 A cryo electron microscopy projection map, p22(1)2(1) crystal symmetry. The unit cell had dimensions of a = b = 150 A and gamma = 90 degrees, and housed four nonameric complexes, two pointing up and two pointing down. AFM topographs of these 2D crystals had a lateral resolution of 10 A. Further, the high vertical resolution of approximately 1 A, allowed the protrusion height of the cylindrical LH2 complexes over the membrane to be determined. This was maximally 13.1 A on one side and 3.8 A on the other. Interestingly, the protrusion height varied across the LH2 complexes, showing the complexes to be inserted with a 6.2 degree tilt with respect to the membrane plane. A detailed analysis of the individual subunits showed the intrinsic flexibility of the membrane protruding peptide stretches to be equal and independent of their protrusion height. Furthermore, our analysis of membrane proteins within this peculiar packing confirmed the high vertical resolution of the atomic force microscopy on biological samples, and led us to conclude that the image acquisition function was equally accurate for contouring protrusions with heights up to approximately 15 A.

Two-dimensional crystallization of membrane proteins: the lipid layer strategy.

Due to the difficulty to crystallize membrane proteins, there is a considerable interest to intensify research topics aimed at developing new methods of crystallization. In this context, the lipid layer crystallization at the air/water interface, used so far for soluble proteins, has been recently adapted successfully to produce two‐dimensional (2D) crystals of membrane proteins, amenable to structural analysis by electron crystallography. Besides to represent a new alternative strategy, this approach gains the advantage to decrease significantly the amount of material needed in incubation trials, thus opening the field of crystallization to those membrane proteins difficult to surexpress and/or purify. The systematic studies that have been performed on different classes of membrane proteins are reviewed and the physico‐chemical processes that lead to the production of 2D crystals are addressed. The different drawbacks, advantages and perspectives of this new strategy for providing structural information on membrane proteins are discussed.

Smectic polymer micellar aggregates with temperature-controlled morphologies

The morphological control of polymer micellar aggregates is a very important issue in applications such as drug delivery and material science. We report the temperature-controlled formation of nanotubes, nanofibers, ellipsoidal and faceted vesicles, and spherical aggregates by nanoprecipitation of amphiphilic diblock copolymers in dioxane/water mixture. The copolymers used are composed of a cholesterol-based smectic liquid crystal core-forming block and a PEG hydrophilic block. The morphology of the micellar self-assemblies was studied by transmission electron microscopy (TEM), cryo-electron microscopy (cryo-TEM) and atomic force microscopy (AFM). In all these aggregates smectic organization is clearly present in the hydrophobic cores. We propose a smectic “liquid crystallization”-driven self-assembly process for the formation of nanofibers and nanotubes on the basis of small angle X-ray scattering (SAXS) studies during the nanoprecipitation. The temperature dependence of the morphology (from T = 5–55 °C) is explained by the free energy consideration. The different aggregates finally dispersed in water after the removal of dioxane are thermally stable at temperature ≤55 °C and can be preserved for years at room temperature without structural change.

Apolipoprotein E Regulates Amyloid Formation within Endosomes of Pigment Cells

Accumulation of toxic amyloid oligomers is a key feature in the pathogenesis of amyloid-related diseases. Formation of mature amyloid fibrils is one defense mechanism to neutralize toxic prefibrillar oligomers. This mechanism is notably influenced by apolipoprotein E variants. Cells that produce mature amyloid fibrils to serve physiological functions must exploit specific mechanisms to avoid potential accumulation of toxic species. Pigment cells have tuned their endosomes to maximize the formation of functional amyloid from the protein PMEL. Here, we show that ApoE is associated with intraluminal vesicles (ILV) within endosomes and remain associated with ILVs when they are secreted as exosomes. ApoE functions in the ESCRT-independent sorting mechanism of PMEL onto ILVs and regulates the endosomal formation of PMEL amyloid fibrils in vitro and in vivo. This process secures the physiological formation of amyloid fibrils by exploiting ILVs as amyloid nucleating platforms.

Optimized Purification of a Heterodimeric ABC Transporter in a Highly Stable Form Amenable to 2-D Crystallization

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.

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

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.

Two-dimensional structures of the Shiga toxin B-subunit and of a chimera bound to the glycolipid receptor Gb3.

The B-subunit of Shiga toxin has been demonstrated as a powerful vector for carrying attached peptides into cells for intracellular transport studies and for medical research. We have investigated the structure of the B-subunit and of a chimera bearing a peptide extension, bound to the membranous lipidic receptor, the globotriaosylceramide (Gb3). Two-dimensional crystals of both B-subunits have been obtained by the lipid layer method and projection maps have been calculated at 8.5A resolution from ice-embedded samples. The B-subunits as the chimera are organized in a pentameric form similar to the X-ray structure of the B-subunit not bound to Gb3. A difference map of both proteins has been calculated in which no density could be attributed to the peptide extension. Cross-correlations with projections of the B-subunit X-ray structure revealed that pentamers in the 2D crystals were oriented with their binding sites pointing to the lipid layer. Thus, it is likely that the peptide extension was disordered and confined to the surface of the pentamer opposite to the Gb3 binding sites. This location confirms the hypothesis that addition of peptide extension to the C-terminus conserves the ability of the modified B-subunit to bind the membranous receptor Gb3.