The C terminus of the herpes simplex virus type 1 origin-binding protein, UL9ct, interacts directly with the viral single-stranded DNA-binding protein ICP8. We show that a 60-amino acid C-terminal deletion mutant of ICP8 (ICP8ΔC) also binds very strongly to UL9ct. Using small angle x-ray scattering, the low resolution solution structures of UL9ct alone, in complex with ICP8ΔC, and in complex with a 15-mer double-stranded DNA containing Box I of the origin of replication are described. Size exclusion chromatography, analytical ultracentrifugation, and electrophoretic mobility shift assays, backed up by isothermal titration calorimetry measurements, are used to show that the stoichiometry of the UL9ct-dsDNA_15-mer complex is 2:1 at micromolar protein concentrations. The reaction occurs in two steps with initial binding of UL9ct to DNA (K_d ∼ 6 nm) followed by a second binding event (K_d ∼ 0.8 nm). It is also shown that the stoichiometry of the ternary UL9ct-ICP8ΔC-dsDNA_15-mer complex is 2:1:1, at the concentrations used in the different assays. Electron microscopy indicates that the complex assembled on the extended origin, oriS, rather than Box I alone, is much larger. The results are consistent with a simple model whereby a conformational switch of the UL9 DNA-binding domain upon binding to Box I allows the recruitment of a UL9-ICP8 complex by interaction between the UL9 DNA-binding domains.

Background: Arabidopsis thalianatransthyretin-like (TTL) protein is a potential substrate in the brassinosteroid signalling cascade, having a role that moderates plant growth. Moreover, sequence homology revealed two sequence domains similar to 2-oxo-4-hydroxy-4-carboxy-5-ureidoimidazoline (OHCU) decarboxylase (N-terminal domain) and 5-hydroxyisourate (5-HIU) hydrolase (C-terminal domain). TTL is a member of the transthyretin-related protein family (TRP), which comprises a number of proteins with sequence homology to transthyretin (TTR) and the characteristic C-terminal sequence motif Tyr-Arg-Gly-Ser. TRPs are single domain proteins that form tetrameric structures with 5-HIU hydrolase activity. Experimental evidence is fundamental for knowing if TTL is a tetrameric protein, formed by the association of the 5-HIU hydrolase domains and, in this case, if the structural arrangement allows for OHCU decarboxylase activity. This work reports about the biochemical and functional characterization of TTL.Results: The TTL gene was cloned and the protein expressed and purified for biochemical and functional characterization. The results show that TTL is composed of four subunits, with a moderately elongated shape. We also found evidence for 5-HIU hydrolase and OHCU decarboxylase activitiesin vitro, in the full-length protein.Conclusions: TheArabidopsis thalianatransthyretin-like (TTL) protein is a tetrameric bifunctional enzyme, since it has 5-HIU hydrolase and OHCU decarboxylase activities, which were simultaneously observedin vitro.

Functional foods represent an emerging trend in the food industry. Fortifying foods with bioactive ingredients results in health benefits and reduces the risk of disease. Encapsulation techniques protect sensitive ingredients from degradation due to heat, light, moisture and other factors. Among encapsulating materials, milk whey proteins are particularly attractive due to their availability, GRAS status and remarkable ligand-binding ability. Whey protein was once considered a by-product in the dairy industry but is now seen as a promising resource given its natural role as a nutrient carrier. This work reviews the encapsulation systems that employ whey proteins in the food industry. The structural features of β-lactoglobulin (β-LG), the main protein constituent of milk whey, are presented in the context of its ligand-binding properties. Different types of encapsulation systems using whey proteins are discussed, focusing on the recent advances in stable formulations of bioactives using whey protein, alone or in hybrid systems. Whey proteins are a valuable asset capable of binding sensitive bioactive compounds such as vitamins, polyphenols and antioxidants and forming stable complexes that can be formulated as nanoparticles, nanofibrils, emulsions and other micro- and nanostructures. Developing scalable, solid and stable encapsulation systems is identified as a main challenge in the field.

Picornains 2A are cysteine proteases of picornaviruses, a virus family containing several human and animal pathogens. The pH dependencies of the alkylations of picornain 2A of rhinovirus type 2 with iodoacetamide and iodoacetate show two reactive thiol forms, namely the free thiolate ion at high pH and an imidazole assisted thiol group at low pH. Kinetic deuterium isotope effects do not support general base catalysis by the imidazole group, but rather the existence of a catalytically competent thiolate–imidazolium ion‐pair. The nature of the ion‐pair differs from that of papain, the paradigm of cysteine proteases. The ion‐pair is confined to the same, unusually narrow pH range in which the enzyme exhibits catalytic activity.