Ana Luísa M. de Carvalho

The structural and chemical diversity of β-glucans is reflected on the variety of essential biological roles tackled by these polysaccharides. This natural heterogeneity requires an elaborate assortment of enzymatic mechanisms to assemble, degrade or modify, as well as to extract their full biotechnological potential. Recent metagenomic efforts have provided an unprecedented growth in potential new biocatalysts, most of which remain unconfirmed or uncharacterized. Here we report the first biochemical and structural characterization of two bacterial β-glucanases from the recently created glycoside hydrolase family 157 (LaGH157 and BcGH157) and investigate their molecular basis for substrate hydrolysis. Structural analysis by X-ray crystallography revealed that GH157 enzymes belong to clan GH-A, possessing a (β/α)8-barrel fold catalytic domain, two β-sandwich accessory domains and two conserved catalytic glutamates residues, with relative positions compatible with a retaining mechanism of hydrolysis. Specificity screening and enzyme kinetics suggest that the enzymes prefer mixed-linkage glucans over β-1,3-glucans. Activity screening showed that both enzymes exhibit pH optimum at 6.5 and temperature optimum for LaGH157 and BcGH157 at 25 °C and 48 °C, respectively. Product analysis with HPAEC-PAD and LC-MS revealed that both enzymes are endo-1,3(4)-β-glucanases, capable of cleaving β-1,3 and β-1,4-linked glucoses, when preceded by a β-1,3 linkage. Moreover, BcGH157 needs a minimum of 4 subsites occupied for hydrolysis to occur, while LaGH157 only requires 3 subsites. Additionally, LaGH157 possesses exohydrolytic activity on β-1,3 and branching β-1,6 linkages. This unusual bifunctional endo-1,3(4)/exo-1,3–1,6 activity constitutes an expansion on our understanding of β-glucan deconstruction, with the potential to inspire future applications.

Many proteins naturally carry metal centers, with a large share of them being in the active sites of several enzymes. Paramagnetic effects are a powerful source of structural information and, therefore, if the native metal is paramagnetic, or it can be functionally substituted with a paramagnetic one, paramagnetic effects can be used to study the metal sites, as well as the overall structure of the protein. One notable example is cobalt(II) substitution for zinc(II) in carbonic anhydrase. In this manuscript we investigate the effects of sodium thiocyanate on the chemical environment of the metal ion of the human carbonic anhydrase II. The electron paramagnetic resonance (EPR) titration of the cobalt(II) protein with thiocyanate shows that the EPR spectrum changes from A-type to C-type on passing from 1:1 to 1:1000-fold ligand excess. This indicates the occurrence of a change in the electronic structure, which may reflect a sizable change in the metal coordination environment in turn caused by a modification of the frozen solvent glass. However, paramagnetic nuclear magnetic resonance (NMR) data indicate that the metal coordination cage remains unperturbed even in 1:1000-fold ligand excess. This result proves that the C-type EPR spectrum observed at large ligand concentration should be ascribed to the low temperature at which EPR measurements are performed, which impacts on the structure of the protein when it is destabilized by a high concentration of a chaotropic agent.

Reflectins are a family of intrinsically disordered proteins involved in cephalopod camouflage, making them an interesting source for bioinspired optical materials. Understanding reflectin assembly into higher-order structures by standard biophysical methods enables the rational design of new materials, but it is difficult due to their low solubility. To address this challenge, we aim to understand the molecular self-assembly mechanism of reflectin’s basic unit—the protopeptide sequence YMDMSGYQ—as a means to understand reflectin’s assembly phenomena. Protopeptide self-assembly was triggered by different environmental cues, yielding supramolecular hydrogels, and characterized by experimental and theoretical methods. Protopeptide films were also prepared to assess optical properties. Our results support the hypothesis for the protopeptide aggregation model at an atomistic level, led by hydrophilic and hydrophobic interactions mediated by tyrosine residues. Protopeptide-derived films were optically active, presenting diffuse reflectance in the visible region of the light spectrum. Hence, these results contribute to a better understanding of the protopeptide structural assembly, crucial for the design of peptide- and reflectin-based functional materials.

Silk fibroin is a biobased material with excellent biocompatibility and mechanical properties, but its use in bioelectronics is hampered by the difficult dissolution and low intrinsic conductivity. Some ionic liquids are known to dissolve fibroin but removed after fibroin processing. However, ionic liquids and fibroin can cooperatively give rise to functional materials, and there are untapped opportunities in this combination. The dissolution of fibroin, followed by gelation, in designer ionic liquids from the imidazolium chloride family with varied alkyl chain lengths (2–10 carbons) is shown here. The alkyl chain length of the anion has a large impact on fibroin secondary structure which adopts unconventional arrangements, yielding robust gels with distinct hierarchical organization. Furthermore, and due to their remarkable air-stability and ionic conductivity, fibroin ionogels are exploited as active electrical gas sensors in an electronic nose revealing the unravelled possibilities of fibroin in soft and flexible electronics.

Interactions of glycan-specific epitopes to human lectin receptors represent novel immune checkpoints for investigating cancer and infection diseases. By employing a multidisciplinary approach that combines isothermal titration calorimetry, NMR spectroscopy, molecular dynamics simulations, and X-ray crystallography, we disclosed the molecular determinants that govern the recognition of the tumour and pathogenic glycobiomarker LacdiNAc (GalNAc?1-4GlcNAc, LDN), including their comparison with the ubiquitous LacNAc epitope (Gal?1-4GlcNAc, LN), by two human immune-related lectins, galectin-3 (hGal-3) and the macrophage galactose C-type lectin (hMGL). A different mechanism of binding and interactions is observed for the hGal-3/LDN and hMGL/LDN complexes, which explains the remarkable difference in the binding specificity of LDN and LN by these two lectins. The new structural clues reported herein are fundamental for the chemical design of mimetics targeting hGal-3/hMGL recognition process.

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Protein crystallization still remains mostly an empirical science, as the production of crystals with the required quality for X-ray analysis is dependent on the intensive screening of the best protein crystallization and crystal’s derivatization conditions. Herein, this demanding step was addressed by the development of a high-throughput and low-budget microfluidic platform consisting of an ion exchange membrane (117 Nafion® membrane) sandwiched between a channel layer (stripping phase compartment) and a wells layer (feed phase compartment) forming 75 independent micro-contactors. This microfluidic device allows for a simultaneous and independent screening of multiple protein crystallization and crystal derivatization conditions, using Hen Egg White Lysozyme (HEWL) as the model protein and Hg2+ as the derivatizing agent. This microdevice offers well-regulated crystallization and subsequent crystal derivatization processes based on the controlled transport of water and ions provided by the 117 Nafion® membrane. Diffusion coefficients of water and the derivatizing agent (Hg2+) were evaluated, showing the positive influence of the protein drop volume on the number of crystals and crystal size. This microfluidic system allowed for crystals with good structural stability and high X-ray diffraction quality and, thus, it is regarded as an efficient tool that may contribute to the enhancement of the proteins’ crystals structural resolution.

The SOUL, or heme-binding protein HBP/SOUL, family represents a group of evolutionary conserved putative heme-binding proteins that contains a number of members in animal, plant andbacterial species. The structures of the murine form of HEBP1, or p22HBP, and the human form of HEBP2, or SOUL, have been determined in 2006 and 2011 respectively. In this work we discuss the structures of HEBP1 and HEBP2 in light of new X-ray data for heme bound murine HEBP1. The interaction between tetrapyrroles and HEBP1, initially proven to be hydrophobic in nature, was thought to also involve electrostatic interactions between heme propionate groups and positively charged amino acid side chains. However, the new X-ray structure, and results from murine HEBP1 variants and human HEBP1, confirm the hydrophobic nature of the heme-HEBP1 interaction, resulting in Kd values in the low nanomolar range, and rules out any electrostatic stabilization. Results from NMR relaxation time measurements for human HEBP1 describe a rigid globular protein with no change in motional regime upon heme binding. X-ray structures deposited in the PDB for human HEBP2 are very similar to each other and to the new heme-bound murine HEBP1 X-ray structure (backbone rmsd ca. 1 {\AA}). Results from a HSQC spectrum centred on the histidine side chain N$δ$-proton region for HEBP2 confirm that HEBP2 does not bind heme via H42 as no chemical shift differences were observed upon heme addition for backbone NH and N$δ$ protons. A survey of the functions attributed to HEBP1 and HEBP2 over the last 20 years span a wide range of cellular pathways. Interestingly, many of them are specific to higher eukaryotes, particularly mammals and a potential link between heme release under oxidative stress and human HEBP1 is also examined using recent data. However, at the present moment, trying to relate function to the involvement of heme or tetrapyrrole binding, specifically, makes little sense with our current biological knowledge and can only be applied to HEBP1, as HEBP2 does not interact with heme. We suggest that it may not be justified to call this very small family of proteins, heme-binding proteins. The family may be more correctly called “the SOUL family of proteins related to cellular fate” as, even though only HEBP1 binds heme tightly, both proteins may be involved in cell survival and/or proliferation.

One of the trends in downstream processing comprises the use of ?anything-but-chromatography? methods to overcome the current downfalls of standard packed-bed chromatography. Precipitation and magnetic separation are two techniques already proven to accomplish protein purification from complex media, yet never used in synergy. With the aim to capture antibodies directly from crude extracts, a new approach combining precipitation and magnetic separation was developed and named as affinity magnetic precipitation. A precipitation screening, based on the Hofmeister series, and a commercial precipitation kit were tested with affinity magnetic particles to assess the best condition for antibody capture from human serum plasma and clarified cell supernatant. The best conditions were obtained when using PEG3350 as precipitant at 4°C for 1h, reaching 80% purity and 50% recovery of polyclonal antibodies from plasma, and 99% purity with 97% recovery yield of anti-TNFα mAb from cell supernatants. These results show that the synergetic use of precipitation and magnetic separation can represent an alternative for the efficient capture of antibodies. This article is protected by copyright. All rights reserved

BackgroundHalf of human cancers harbour TP53 mutations that render p53 inactive as a tumor suppressor. As such, reactivation of mutant (mut)p53 through restoration of wild-type (wt)-like function represents one of the most promising therapeutic strategies in cancer treatment. Recently, we have reported the (S)-tryptophanol-derived oxazoloisoindolinone SLMP53-1 as a new reactivator of wt and mutp53 R280K with in vitro and in vivo p53-dependent antitumor activity. The present work aimed a mechanistic elucidation of mutp53 reactivation by SLMP53-1.
Methods and results
By cellular thermal shift assay (CETSA), it is shown that SLMP53-1 induces wt and mutp53 R280K thermal stabilization, which is indicative of intermolecular interactions with these proteins. Accordingly, in silico studies of wt and mutp53 R280K DNA-binding domain with SLMP53-1 unveiled that the compound binds at the interface of the p53 homodimer with the DNA minor groove. Additionally, using yeast and p53-null tumor cells ectopically expressing distinct highly prevalent mutp53, the ability of SLMP53-1 to reactivate multiple mutp53 is evidenced.
Conclusions
SLMP53-1 is a p53-activating agent with the ability to directly target wt and a set of hotspot mutp53.
General Significance
This work reinforces the encouraging application of SLMP53-1 in the personalized treatment of cancer patients harboring distinct p53 status.

Understanding the specific molecular interactions between proteins and β1,3-1,4-mixed-linked d-glucans is fundamental to harvest the full biological and biotechnological potential of these carbohydrates and of proteins that specifically recognize them. The family 11 carbohydrate-binding module from Clostridium thermocellum (CtCBM11) is known for its binding preference for β1,3-1,4-mixed-linked over β1,4-linked glucans. Despite the growing industrial interest of this protein for the biotransformation of lignocellulosic biomass, the molecular determinants of its ligand specificity are not well defined. In this report, a combined approach of methodologies was used to unravel, at a molecular level, the ligand recognition of CtCBM11. The analysis of the interaction by carbohydrate microarrays and NMR and the crystal structures of CtCBM11 bound to β1,3-1,4-linked glucose oligosaccharides showed that both the chain length and the position of the β1,3-linkage are important for recognition, and identified the tetrasaccharide Glcβ1,4Glcβ1,4Glcβ1,3Glc sequence as a minimum epitope required for binding. The structural data, along with site-directed mutagenesis and ITC studies, demonstrated the specificity of CtCBM11 for the twisted conformation of β1,3-1,4-mixed-linked glucans. This is mediated by a conformation–selection mechanism of the ligand in the binding cleft through CH-π stacking and a hydrogen bonding network, which is dependent not only on ligand chain length, but also on the presence of a β1,3-linkage at the reducing end and at specific positions along the β1,4-linked glucan chain. The understanding of the detailed mechanism by which CtCBM11 can distinguish between linear and mixed-linked β-glucans strengthens its exploitation for the design of new biomolecules with improved capabilities and applications in health and agriculture. Database Structural data are available in the Protein Data Bank under the accession codes 6R3M and 6R31.

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Overexpression of the Thomsen-Friedenreich (TF) antigen in cell membrane proteins occurs in 90% of adenocarcinomas. Additionally, the binding of the TF-antigen to human galectin-3 (Gal-3), also frequently overexpressed in malignancy, promotes cancer progression and metastasis. In this context, structures that interfere with this specific interaction display the potential to prevent cancer metastasis. Herein, a multidisciplinary approach, combining the optimized synthesis of a TF-antigen mimetic with NMR, X-ray crystallography methods and isothermal titration calorimetry assays has been employed to unravel the molecular structural details that govern the Gal-3/TF-mimetic interaction. The TF-mimetic presents a binding affinity for Gal-3 similar to the TF-natural antigen and retains the binding epitope and the bioactive conformation observed for the native antigen. Furthermore, from a thermodynamic perspective a decrease in the enthalpic contribution was observed for the Gal-3/TF-mimetic complex, however this behaviour is compensated by a favourable entropy gain. From a structural perspective, these results establish our TF-mimetic as a scaffold to design multivalent solutions to potentially interfere with Gal-3 aberrant interactions and likely be used to hamper Gal-3-mediated cancer cells adhesion and metastasis.

A new and never yet reported hetero arylidene-9(10H)-anthrone structure (4) was unexpectedly isolated on reaction of 1,2-dimethyl-3-ethylimidazolium iodide (2) and 9-anthracenecarboxaldehyde (3) under basic conditions. Its structure was unequivocally attributed by X-ray crystallography. No cytotoxicity in human healthy fibroblasts and in two different cancer cell lines was observed indicating its applicability in biological systems. Compound 4 interacts with CT-DNA by intercalation between the adjacent base pairs of DNA with a high binding affinity (Kb = 2.0(± 0.20) x 105 M-1) which is 10x higher than that described for doxorubicin (Kb = 3.2 (±0.23) × 104 M-1). Furthermore, compound 4 quenches the fluorescence emission of GelRed-CT-DNA system with a quenching constant (KSV) of 3.3(±0.3) x 103 M-1 calculated by the Stern-Volmer equation.

The functional and biological significance of the selected CASP12 targets are described by the authors of the structures. The crystallographers discuss the most interesting structural features of the target proteins and assess whether these features were correctly reproduced in the predictions submitted to the CASP12 experiment. This article is protected by copyright. All rights reserved.

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We show that a physical trigger, a non-ionizing infrared (IR) radiation at wavelengths strongly absorbed by liquid water, can be used to induce and kinetically control protein (periodic) self-assembly in solution. This phenomenon is explained by considering the effect of IR light on the structuring of protein interfacial water. Our results indicate that the IR radiation can promote enhanced mutual correlations of water molecules in the protein hydration shell. We report on the radiation-induced increase in both the strength and cooperativeness of H-bonds. The presence of a structured dipolar hydration layer can lead to attractive interactions between like-charged biomacromolecules in solution (and crystal nucleation events). Furthermore, our study suggests that enveloping the protein within a layer of structured solvent (an effect enhanced by IR light) can prevent the protein non-specific aggregation favoring periodic self-assembly. Recognizing the ability to affect protein-water interactions by means of IR radiation may have important implications for biological and bio-inspired systems.

Nucleation is a critical step determining the outcome of the entire crystallization process. Finding an effective nucleant for protein crystallization is of utmost importance for structural biology. The latter relies on good-quality crystals to solve the three-dimensional structures of macromolecules. In this study we show that crystalline barium sulfate (BaSO4) with an etched and/or ionic liquid (IL)-functionalized surface (1) can induce protein nucleation at concentrations well below the concentration needed to promote crystal growth under control conditions, (2) can shorten the nucleation time, (3) can increase the growth rate, and finally (4) may help to improve the protein crystal morphology. These effects were shown for lysozyme, RNase A, trypsin, proteinase K, myoglobin, and hemoglobin. Therefore, the use of BaSO4 particles enables us to reduce the amount of protein in crystallization trials and increases the chance of obtaining protein crystals of the desired quality. In the context of the underlying mechanism, it is shown that the protein-solid contact formation is governed by the interaction of the polar compartments of the biomacromolecule with the support. The tendency of a protein to concentrate near the solid surface is enhanced by both the hydrophobicity of the protein and that of the surface (tuned by the functionalizing IL). These mechanisms of interaction of biomacromolecules with inorganic hydrophilic solids correspond to the principles of amphiphilic IL-mineral interactions.

Glucans are polymers of D-glucose with differing linkages in linear or branched sequences. They are constituents of microbial and plant cell-walls and involved in important bio-recognition processes including immunomodulation, anti-cancer activities, pathogen virulence and plant cell-wall biodegradation. Translational possibilities for these activities in medicine and biotechnology are considerable. High-throughput micro-methods are needed to screen proteins for recognition of specific glucan sequences as a lead to structure-function studies and their exploitation. We describe construction of a glucome microarray, the first sequence-defined glycome-scale microarray, using a designer approach from targeted ligand-bearing glucans in conjunction with a novel high-sensitivity mass spectrometric sequencing method, as a screening tool to assign glucan recognition motifs. The glucome microarray comprises 153 oligosaccharide probes with high purity, representing major sequences in glucans. The negative-ion electrospray tandem mass spectrometry with collision-induced dissociation was used for complete linkage analysis of gluco-oligosaccharides in linear homo and hetero and branched sequences. The system is validated using antibodies and carbohydrate-binding modules known to target α- or β-glucans in different biological contexts, extending knowledge on their specificities, and applied to reveal new information on glucan recognition by two signalling molecules of the immune system against pathogens: Dectin-1 and DC-SIGN. The sequencing of the glucan oligosaccharides by the MS method and their interrogation on the microarrays provides detailed information on linkage, sequence and chain length requirements of glucan-recognizing proteins, and are a sensitive means of revealing unsuspected sequences in the polysaccharides.