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Iota toxin, a type of A-B toxin produced by Clostridium perfringens, comprises an enzymatic component (Ia) and a membrane-binding component (Ib). The translocation of Ia to the target cell via the pore formed by Ib allows it to function as an ADP-ribosyltransferase that inhibits actin polymerization in the host cell. The structure of Ia-bound Ib-pore has been determined using cryo-electron microscopy (cryo-EM), thereby elucidating the mechanism of the initial Ia translocation; however, open questions regarding Ia translocation still exist. In this chapter, we describe a new method of preparing Ia-bound Ib-pore complex samples for structural analysis at high resolution using cryo-EM. This method is different from previously reported methods for other A-B toxins. Consequently, it produces Ib-pore with two different states with short and long membrane-spanning β-barrel stem. We expect that this method will be useful in functional and structural studies of iota toxin and other binary toxins.Pore forming proteins (PFPs) undergo dramatic conformational changes to punch holes in the target membrane. These PFPs have the ability to self-assemble, by way of oligomerization, and have the capacity to transform from a water soluble state (commonly referred to as fluid phase) to a membrane adhered form. Accordingly, PFPs are metastable, that is they are inert until the right conditions cause the release of potential energy stored in the conformational fold leading to a vast structural rearrangement into a membrane-inserted oligomeric form. However, the metastable state of PFPs poses a problem of leading to aggregation and precipitation in conditions typically required for structural biology techniques. Here, we discuss the protein chemistry of the MACPF protein complement component 9 (C9). Selleckchem Amcenestrant C9 is part of a larger complex assembly known as the membrane attack complex (MAC) that has been studied extensively for its ability to form pores in bacteria. An unusual artifact of human C9 is the ability to form a soluble oligomeric state of the channel portion of the MAC, called polyC9. PolyC9 formation does not require the presence of membranes or other complement factors. It is only in recent years that structural studies of the MAC have become successful owing to improved recombinant DNA expression systems and the improvement of high-resolution techniques (both X-ray crystallography and single particle cryo-EM). We discuss the expression and purification of recombinant C9, crystallization of the soluble monomeric form of C9 and the preparation of the oligomeric polyC9.A common form of cellular attack by pathogenic bacteria is to secrete pore-forming toxins (PFTs). Capable of forming transmembrane pores in various biological membranes, PFTs have also been identified in a diverse range of other organisms such as sea anemones, earthworms and even mushrooms and trees. The mechanism of pore formation by PFTs is associated with substantial conformational changes in going from the water-soluble to transmembrane states of the protein. The determination of the crystal structures for numerous PFTs has shed much light on our understanding of these proteins. Other than elucidating the atomic structural details of PFTs and the conformational changes that must occur for pore formation, crystal structures have revealed structural homology that has led to the discovery of new PFTs and new PFT families. Here we review some key crystallographic results together with complimentary approaches for studying PFTs. We discuss how these studies have impacted our understanding of PFT function and guided research into biotechnical applications.Exopolysaccharide (EPS) secretion by Sclerotium rolfsii ATCC 201126 in submerged cultures, already identified as high-osmolarity responsive, was assessed by reducing C-source without compromising EPS yields. A designed medium with 80 g sucrose L-1 (MOPT80) was tested at 3 L-bioreactor scale at different temperature, agitation, aeration and pH (uncontrolled vs. controlled) values. Optimal operative conditions (200 rpm, 28 °C, 0.5 vvm and initial pH -pHi- 4.5) were validated, as well as the possibility to work at pHi 5.5 to reduce biomass production. Purified EPSs produced in MOPT80 at optimal and other valid operative conditions exhibited refined grade ( less then 1 % proteins and ash, 3-4 % reducing sugars, 87-99 % total sugars). EPS purity, MW and rheological parameters led to discourage pH controlled at 4.5. Relatively constant MW (6-8 × 106 Da) and outstanding viscosifying ability were found. Polyphasic EPS analysis (titre, purity, macromolecular features and rheological fitness) would support to properly select production conditions.The development of effective and environment-friendly corrosion inhibitors is of great significance for the protection of mild steel in hydrochloric acid media. Accordingly, a natural polysaccharide mixture inhibitor composed of chondroitin sulfate derived from pig cartilage (CS-PC) and sodium alginate (SA) is developed here, and the synergistic effect of the two polysaccharides towards adsorption on mild steel in 1 M HCl is studied. The inhibition performance has been studied using weight loss test, electrochemical investigations, SEM, SECM and UV methods. The results indicate that the mixtures of CS-PC and SA strongly inhibit the corrosion of mild steel compared to individual inhibitors (i.e., 95.18 % versus 72.78 %), and show a synergistic inhibition effect. The structure-activity relationship between the molecular structure of the CS-PC + SA mixture and its corrosion inhibition performance has been discussed by using the quantum chemistry calculation and molecular dynamics simulations. It is believed that these results have certain guiding significance for the rational design of efficient corrosion inhibitor.Highly efficient shielding materials with an excellent electromagnetic wave absorption have gained increased attention. A new design was used to provide cellulose paper with a high electromagnetic shielding effectiveness (EMI SE) and improve the absorption performance by constructing an asymmetry sandwich structure that consisted of a dense nickel coating, Fe3O4 nanoparticles and a porous nickel layer. This unique structure caused a "multiple reflection-absorb-reflection" process when the electromagnetic waves penetrated the sample. The EMI absorption (SEA) and total SE (SET) increased with Fe3O4 absorption time increasing at 8.2-12.4 GHz, which was attributed to the synergistic effect between porous nickel layer and Fe3O4 nanoparticles. The SEA and SET of the sample with a thickness of 0.195 mm can achieved 18.57 and 41.88 dB, respectively. The design was conducive to improving the magnetic and corrosion resistance properties. This study provided a novel path to obtain a low cost and lightweight electromagnetic shielding material that can reduce secondary radiation.
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