Frank Depoix

Alhydrogel® adjuvant, ultrasonic dispersion and protein binding: a TEM and analytical study.

Aluminium-based vaccine adjuvants have been in use since the 1920s. Aluminium hydroxide (alum) that is the chemical basis of Alhydrogel, a widely used adjuvant, is a colloid that binds proteins to the particular surface for efficient presentation to the immune system during the vaccination process. Using conventional TEM and cryo-TEM we have shown that Alhydrogel can be finely dispersed by ultrasonication of the aqueous suspension. Clusters of ultrasonicated aluminium hydroxide micro-fibre crystals have been produced (∼ 10-100 nm), that are significantly smaller than those present the untreated Alhydrogel (∼ 2-12 μm). However, even prolonged ultrasonication did not produce a homogenous suspension of single aluminium hydroxide micro-fibres. The TEM images of unstained and negatively stained air-dried Alhydrogel are very similar to those obtained by cryo-electron microscopy. Visualization of protein on the surface of the finely dispersed Alhydrogel by TEM is facilitated by prior ultrasonication. Several examples are given, including some of medical relevance, using proteins of widely ranging molecular mass and oligomerization state. Even with the smaller mass proteins, their presence on the Alhydrogel surface can be readily defined by TEM. It has been found that low quantities of protein tend to cross-link and aggregate the small Alhydogel clusters, in a more pronounced manner than high protein concentrations. This indicates that complete saturation of the available Alhydrogel surface with protein may be achieved, with minimal cross-linkage, and future exploitation of this treatment of Alhydrogel is likely to be of immediate value for more efficient vaccine production.

10-Å CryoEM Structure and Molecular Model of the Myriapod (Scutigera) 6 × 6mer Hemocyanin: Understanding a Giant Oxygen Transport Protein

Oxygen transport in Myriapoda is maintained by a unique 6x6mer hemocyanin, that is, 36 subunits arranged as six hexamers (1x6mers). In the sluggish diplopod Spirostreptus, the 1x6mers seem to operate as almost or fully independent allosteric units (h approximately 1.3; P(50) approximately 5 torr), whereas in the swift centipede Scutigera, they intensively cooperate allosterically (h approximately 10; P(50) approximately 50 torr). Here, we show the chemomechanical basis of this differential behavior as deduced from hybrid 6x6mer structures, obtained by single-particle cryo-electron microscopy of the Scutigera 6x6mer (10.0 A resolution according to the 0.5 criterion) and docking of homology-modeled subunits from Scutigera and two diplopods, Spirostreptus and Polydesmus. The Scutigera 6x6mer hemocyanin is a trigonal antiprism assembled from six smaller trigonal antiprisms (1x6mers), thereby exhibiting D3 point group symmetry. It can be described as two staggered 3x6mers or three oblique 2x6mers. Topologically, the 6x6mer is subdivided into six subunit zones, thereby exhibiting a mantle (24 subunits) and a core (12 subunits). The six hexamers are linked by 21 bridges, subdivided into five types: two within each 3x6mer and three between both 3x6mers. The molecular models of the 6x6mer reveal intriguing amino acid appositions at these inter-hexamer interfaces. Besides opportunities for salt bridges, we found pairs of carboxylate residues for possible bridging via a Ca(2+) or Mg(2+) ion. Moreover, we detected histidine clusters, notably in Scutigera, allowing us to advance hypotheses as to how the hexamers are allosterically coupled in centipede hemocyanin and why they act more independently in diplopod hemocyanin.

The structure of gas-filled n-butyl-2-cyanoacrylate (BCA) polymer particles

Abstract The structure of gas-filled poly-[n-butyl-2-cyanoacrylate] (BCA) particles has been demonstrated by negative staining with uranyl acetate, platinum-carbon shadowing of air-dried material and thin sectioning of the aqueous suspension of BCA particles, embedded in water-soluble melamine resin. The polymer shell of the hollow particles possesses a globular outer surface and a smoother inner surface.

Pentafluorophenyl Ester-based Polymersomes as Nanosized Drug-Delivery Vehicles

In this work, activated ester chemistry is employed to synthesize biocompatible and readily functionalizable polymersomes. Via aminolysis of pentafluorophenyl methacrylate-based precursor polymers, an N-(2-hydroxypropyl) methacrylamide (HPMA)-analog hydrophilic block is obtained. The precursor polymers can be versatile functionalized by simple addition of suitable primary amines during aminolysis as demonstrated using a fluorescent dye. Vesicle formation is proven by cryoTEM and light scattering. High encapsulation efficiencies for hydrophilic cargo like siRNA are achieved using dual centrifugation and safe encapsulation is demonstrated by gel electrophoresis. In vitro studies reveal low cytotoxicity and no protein adsorption-induced aggregation in human blood serum occurs, making the vesicles interesting candidates as nanosized drug carriers.

A new automated plunger for cryopreparation of proteins in defined - even oxygen free - atmospheres

We study the structure and function of hemocyanins. They are giant extracellular oxygen carriers in the hemolymph of many molluscs and arthropods. Since some of these blue, copper-containing proteins show the highest cooperativity in nature (h = 10), one of our goals is to understand the chemomechanical interaction between the different substructures during allosteric oxygen binding.

8 Å cryo-EM structure of the giant hemoglobin from the planorbid snail Biomphalaria glabrata

Until 2006, snail red hemoglobin remained a phylogenetic enigma because it occurs quite isolated in a single gastropod family, the Planorbidae, whereas all other gastropods use blue hemocyanin as a respiratory protein (for recent cryo-EM of hemocyanin, see [1,2]). Moreover, sequence data on this snail hemoglobin were completely lacking. In 2006, our group published the complete cDNA and predicted amino acid sequence of two Biomphalaria glabrata hemoglobin polypeptides, termed BgHb1 and BgHb2 [3]. (Biomphalaria is intermediate host of the human parasite Schistosoma mansoni that causes Bilharziosis.) Resembling pearl-chains, both polypeptide subunits encompass 13 different, cysteine-free globin domains (∼17 kDa each; total subunit Mr ∼238 kDa). In addition, there is a small N-terminal non-globin “plug” domain (∼10 kDa) which contains three cysteines for subunit dimerization. Phylogenetic interpretation of the sequence data showed that Biomphalaria hemoglobin evolved from myoglobin which is present in the radula muscle of most gastropods. Possibly, this hemoglobin evolved to replace a less efficient hemocyanin which was detected in Biomphalaria hemolymph as a trace component [3].

Quaternary structure of recombinant human meprin β, a zinc peptidase of the astacin family

Meprins are astacin-type zinc peptidases distantly related to matrix metalloproteinases (MMPs) [1]. They are expressed in various epithelia, intestinal leukocytes and cancer cells. They cleave basement membrane proteins, cytokines and adhesion molecules, suggesting a role in epithelial differentiation, cell migration and immune reactions [2–4]. There is evidence for detachment of cells from the basal membrane caused by meprin alpha, while meprin beta probably plays a role in terminal differentiation and cell migration [5, 6].

Cryo TEM-based 3D reconstruction of the recombinant expressed human zinc peptidase Meprin β

Meprins are astacin-type zinc endopeptidases, which have been observed so far exclusively in vertebrates. Based on the structure of their catalytic domains these enzymes are distantly related to matrix metalloproteinases (MMPs) [1]. Typically, meprins are expressed in brush border membranes of intestine and kidney tubules, intestinal leukocytes, and certain cancer cells. This suggests a role in epithelial differentiation and cell migration [2]. For human meprin two subforms are described: Meprin α and meprin β [3].

Allosterism of Nautilus pompilius hemocyanin as deduced from 8 Å cryo-EM structures obtained under oxy and deoxy conditions

Hemocyanins are the blue copper-containing respiratory proteins of many molluscs. Nautilus pompilius hemocyanin (NpH) is a cylindrical decamer composed of ten copies of a 350 kDa polypeptide subunit, in turn consisting of seven O2-binding functional units (FUs, termed NpH-a to NpH-g). Ten copies of the subunit segment NpH-a to NpH-f form the cylinder wall (ca. 35 nm in diameter), whereas the ten copies of NpH-g build the internal collar. Recently we published a 9A cryo-EM structure and molecular model of NpH that solved the principal architecture of this protein [1]. Hemocyanins are highly allosteric, and the cooperativity of oxygen binding should be transferred between functional units by conformational changes. In this context, we try to monitor structural changes caused by the reversible oxygen binding process. Our approach is to prepare the specimens in their fully oxygenated and deoxygenated state, respectively, and to perform independent 3D reconstructions of both states. To achieve this, we developed an automated plunge-freeze apparatus capable of flash freezing the specimen in liquid ethane under controlled atmospheric conditions. Prior to fixation, the molecules are either incubated in an atmosphere with 25 % O2 + 75 % N2 (oxy-state), or in an atmosphere with 100 % N2 (deoxy-state). This led to two independent NpH datasets resulting in two 3D reconstructions with resolutions of 7.8 and 8.4 A (Figure 1), respectively. Their correlative analysis shows significant structural differences especially concerning the spatial orientation of certain FU types (Figure 2). On the basis of the two resulting molecular models it is now the task to interpret these differences in terms of allosteric interaction mechanisms, but also to reveal possible pitfalls.

9 Å cryo-EM structure and molecular model of a gastropod hemocyanin didecamer (KLH1) reveals the architecture of the asymmetric collar

Hemocyanins are blue copper proteins that transport oxygen in the hemolymph of many arthropods and molluscs. Molluscan hemocyanins are decamers, didecamers or multidecamers of a 350–400 kDa polypeptide subunit that is subdivided into seven or eight different functional units (FUs, each with a single copper active site). The quaternary structure is a semi-hollow cylinder consisting of a wall and a collar. Recently, we published a 9 A cryo-EM structure and molecular model of a cephalopod hemocyanin decamer (NpH, from Nautilus pompilius) that answered many hitherto unsolved questions concerning the quaternary structure of molluscan hemocyanin. Notably, it revealed the twisted pathway of the 10 subunit copies (each with seven FUs) and the various molecular interfaces between the 70 FUs [1]. Six FU types (termed NpH-a through NpH-f) constitute the cylinder wall, whereas the seventh type (NpH-g) forms the internal collar.