Fagopyrins from Buckwheat Flowers: Structural and Stereochemical Characterization Through Combined NMR/CD Spectroscopy and Theoretical Calculations.

Fagopyrins are phenantroperylenequinones present in the flowers of Fagopyrum esculentum (buckwheat) endowed with photodynamic activity. It has been reported that fagopyrin extracts actually contain a complex mixture of closely related compounds, differing only on the nature of the perylenequinone substituents. We report our systematic and detailed study on the chemical composition of fagopyrin extracts by a combination of preparative and analytical techniques. The combined use of 1H-NMR and CD spectroscopy was found to be particularly suited to fully characterize all stereochemical aspects of the extracted fagopyrins. For the first time nine isomers have been structurally characterized and their stereochemistry fully elucidated. The presence of two different heterocyclic ring substituents, two stereogenic centers and the inherent axial chirality of the aromatic system provides a complex stereochemical relationships among isomers, thus giving account of the high level of molecular multiplicity found in the extract.

A not-glycosylated isoform of γ-conglutin, a hexameric glycoprotein of Lupinus albus seed, participates in the oligomerization equilibrium.

γ-conglutin (γ-C) is a hexameric glycoprotein accumulated in lupin seeds and has long been considered as a storage protein. Recently, it has been investigated for its possible postprandial glycaemic regulating action in human nutrition and for its physiological role in plant defence. The quaternary structure of γ-C results from the assembly of six monomers in reversible pH-dependent association/dissociation equilibrium. Our working hypothesis was that the γ-C hexamer is made up of glycosylated subunits in association with not-glycosylated isoforms, that seem to have 'escaped' the correct glycosylation process in the Golgi. Here we describe the isolation of not-glycosylated γ-C monomers in native condition by two in tandem lectin-based affinity chromatography and the characterization of their oligomerization capacity. We report, for the first time, the observation that a plant multimeric protein may be formed by identical polypeptide chains that have undergone different post-translational modifications. All obtained considered, the results strongly suggest that the not-glycosylated isoform can also take part in the oligomerization equilibrium of the protein.

The structural bases for agonist diversity in an Arabidopsis thaliana glutamate receptor-like channel.

Arabidopsis thaliana glutamate receptor-like (GLR) channels are amino acid-gated ion channels involved in physiological processes including wound signaling, stomatal regulation, and pollen tube growth. Here, fluorescence microscopy and genetics were used to confirm the central role of GLR3.3 in the amino acid-elicited cytosolic Ca2+ increase in Arabidopsis seedling roots. To elucidate the binding properties of the receptor, we biochemically reconstituted the GLR3.3 ligand-binding domain (LBD) and analyzed its selectivity profile; our binding experiments revealed the LBD preference for l-Glu but also for sulfur-containing amino acids. Furthermore, we solved the crystal structures of the GLR3.3 LBD in complex with 4 different amino acid ligands, providing a rationale for how the LBD binding site evolved to accommodate diverse amino acids, thus laying the grounds for rational mutagenesis. Last, we inspected the structures of LBDs from nonplant species and generated homology models for other GLR isoforms. Our results establish that GLR3.3 is a receptor endowed with a unique amino acid ligand profile and provide a structural framework for engineering this and other GLR isoforms to investigate their physiology.

Apis mellifera RidA, a novel member of the canonical YigF/YER057c/UK114 imine deiminase superfamily of enzymes pre-empting metabolic damage.

The Reactive intermediate deiminase (Rid) protein family is a group of enzymes widely distributed in all Kingdoms of Life. RidA is one of the eight known Rid subfamilies, and its members act by preventing the accumulation of 2-aminoacrylate, a highly reactive enamine generated during the metabolism of some amino acids, by hydrolyzing the 2-iminopyruvate tautomer to pyruvate and ammonia. RidA members are homotrimers exhibiting a remarkable thermal stability. Recently, a novel subclass of RidA was identified in teleosts, which differs for stability and substrate specificity from the canonical RidA. In this study we structurally and functionally characterized RidA from Apis mellifera (AmRidA) as the first example of an invertebrate RidA to assess its belonging to the canonical RidA group, and to further correlate structural and functional features of this novel enzyme class. Circular dichroism revealed a spectrum typical of the RidA proteins and the high thermal stability. AmRidA exhibits the 2-imino acid hydrolase activity typical of RidA family members with a substrate specificity similar to that of the canonical RidA. The crystal structure confirmed the homotrimeric assembly and the presence of the typical structural features of RidA proteins, such as the proposed substrate recognition loop, and the ß-sheets ß1-ß9 and ß1-ß2. In conclusion, our data define AmRidA as a canonical member of the well-conserved RidA family and further clarify the diagnostic structural features of this class of enzymes.

Protein interactions in the biological assembly of iron-sulfur clusters in Escherichia coli: Molecular and mechanistic aspects of the earliest assembly steps.

This contribution focuses on the earliest steps of the assembly of FeS clusters and their insertion into acceptor apoproteins, that call for transient formation of a 2Fe2S cluster on a scaffold protein from sulfide and iron salts. For the sake of simplicity, this report is essentially limited to the Escherichia coli isc-encoded proteins and does not take into account agents that modulate the enzymatic synthesis of sulfide by protein in the same operon or the redox events associated with both sulfide generation and conversion of 2Fe2S structures in clusters of higher nuclearity. Therefore, the results discussed here are based on chemical reconstitution systems using inorganic sulfide, ferric salts, and excess thiols. This simplification offers the possibility to address some mechanistic issues related to the role of protein/protein interaction as for modulating: (a) the rate of cluster assembly on scaffold proteins; (b) the stability of the cluster on the scaffold protein; and (c) the rate of transfer to acceptor apoproteins as also influenced by the acceptor concentration. The emerging picture highlights the mechanistic versatility of the systems, that is discussed in terms of the capability of such an apparently simple combination of proteins to cope with various physiological situation. The hypothetical mechanism presented here may represent an additional way of modulating the rate and outcome of the overall process while avoiding potential toxicity issues.

Exploration of Lactiplantibacillus fabifermentans and Furfurilactobacillus rossiae as potential cocoa fermentation starters.

AIMS: To investigate the characteristics of two minority autochthonous LAB species, with particular regard to those properties that could be exploited in an improved cocoa fermentation process from a quality and safety point of view. METHODS AND RESULTS: Bacterial, yeast and mould strains characteristic of spontaneously fermented Dominican cocoa beans were isolated and identified by 16S or 26S rRNA gene sequencing. The potential of two autochthonous strains of LAB belonging to the species Lactiplantibacillus fabifermentans and Furfurilactibacillus rossiae were investigated. The two selected LAB strains were able to utilize glucose and fructose, produced mainly D-L lactic acid and had a good ability to resist to cocoa-related stress conditions such as low pH, high temperature and high osmotic pressure, as well as to grow in sterile cocoa pulp. The strains did not inhibit the growth of yeasts and acetic acid bacteria, that are essential to the cocoa fermentation process, and possessed a complex pool of peptidases especially active on hydrophobic amino acids. The strains also showed antifungal activity against mould species that can be found at the final stages of cocoa fermentation, as Aspergillus tamarii, A. nidulans, Lichtheimia ornata and Rhizomucor pusillus. CONCLUSIONS: The tested strains are good candidates for the design of starter cultures for a controlled cocoa fermentation process. SIGNIFICANCE AND IMPACT OF THE STUDY: This research showcases the potential of two alternative LAB species to the dominating Lactiplantibacillus plantarum and Limosilactibacillus fermentum as cocoa fermentation starters, with an interesting activity in improving the safety and quality of the process.

Cu(II) Binding Increases the Soluble Toxicity of Amyloidogenic Light Chains.

Light chain amyloidosis (AL) is caused by the aberrant overproduction of immunoglobulin light chains (LCs). The resulting abnormally high LC concentrations in blood lead to deposit formation in the heart and other target organs. Organ damage is caused not only by the accumulation of bulky amyloid deposits, but extensive clinical data indicate that circulating soluble LCs also exert cardiotoxic effects. The nematode C. elegans has been validated to recapitulate LC soluble toxicity in vivo, and in such a model a role for copper ions in increasing LC soluble toxicity has been reported. Here, we applied microscale thermophoresis, isothermal calorimetry and thermal melting to demonstrate the specific binding of Cu2+ to the variable domain of amyloidogenic H7 with a sub-micromolar affinity. Histidine residues present in the LC sequence are not involved in the binding, and yet their mutation to Ala reduces the soluble toxicity of H7. Copper ions bind to and destabilize the variable domains and induce a limited stabilization in this domain. In summary, the data reported here, elucidate the biochemical bases of the Cu2+-induced toxicity; moreover, they also show that copper binding is just one of the several biochemical traits contributing to LC soluble in vivo toxicity.

Beta-Lactoglobulin as a Model Food Protein: How to Promote, Prevent, and Exploit Its Unfolding Processes.

Bovine milk beta-lactoglobulin (BLG) is a small whey protein that is a common ingredient in many foods. Many of the properties of BLG relevant to the food industry are related to its unfolding processes induced by physical or chemical treatments. Unfolding occurs through a number of individual steps, generating transient intermediates through reversible and irreversible modifications. The rate of formation of these intermediates and of their further evolution into different structures often dictates the outcome of a given process. This report addresses the main structural features of the BLG unfolding intermediates under conditions that may facilitate or impair their formation in response to chemical or physical denaturing agents. In consideration of the short lifespan of the transient species generated upon unfolding, this review also discusses how various methodological approaches may be adapted in exploring the process-dependent structural modifications of BLG from a kinetic and/or a thermodynamic standpoint. Some of the conceptual and methodological approaches presented and discussed in this review can provide hints for improving the understanding of transient conformers formation by proteins present in other food systems, as well as when other physical or chemical denaturing agents are acting on proteins much different from BLG in complex food systems.

Impact of Thermal Treatment on the Starch-Protein Interplay in Red Lentils: Connecting Molecular Features and Rheological Properties.

Thermal treatments are widely applied to gluten-free (GF) flours to change their functionality. Despite the interest in using pulses in GF formulations, the effects of thermal treatment at the molecular level and their relationship with dough rheology have not been fully addressed. Raw and heat-treated red lentils were tested for starch and protein features. Interactions with water were assessed by thermogravimetric analysis and water-holding capacity. Finally, mixing properties were investigated. The thermal treatment of red lentils induced a structural modification of both starch and proteins. In the case of starch, such changes consequently affected the kinetics of gelatinization. Flour treatment increased the temperature required for gelatinization, and led to an increased viscosity during both gelatinization and retrogradation. Regarding proteins, heat treatment promoted the formation of aggregates, mainly stabilized by hydrophobic interactions between (partially) unfolded proteins. Overall, the structural modifications of starch and proteins enhanced the hydration properties of the dough, resulting in increased consistency during mixing.

Morpholino-based peptide oligomers: Synthesis and DNA binding properties.

The chemical structure of oligonucleotide analogues dictates the conformation of oligonucleotide analogue oligomers, their ability to hybridize complementary DNA and RNA, their stability to degradation and their pharmacokinetic properties. In a study aimed at investigating new analogues featuring a neutral backbone, we explored the ability of oligomers containing a morpholino-peptide backbone to bind oligonucleotides. Circular Dichroism studies revealed the ability of our oligomers to interact with DNA, molecular modelling studies revealed the interaction responsible for complex stabilization.

Gelsolin pathogenic Gly167Arg mutation promotes domain-swap dimerization of the protein.

AGel amyloidosis is a genetic degenerative disease characterized by the deposition of insoluble gelsolin protein aggregates in different tissues. Until recently, this disease was associated with two mutations of a single residue (Asp187 to Asn/Tyr) in the second domain of the protein. The general opinion is that pathogenic variants are not per se amyloidogenic but rather that the mutations trigger an aberrant proteolytic cascade, which results in the production of aggregation prone fragments. Here, we report the crystal structure of the second domain of gelsolin carrying the recently identified Gly167Arg mutation. This mutant dimerizes through a three-dimensional domain swapping mechanism, forming a tight but flexible assembly, which retains the structural topology of the monomer. To date, such dramatic conformational changes of this type have not been observed. Structural and biophysical characterizations reveal that the Gly167Arg mutation alone is responsible for the monomer to dimer transition and that, even in the context of the full-length protein, the pathogenic variant is prone to form dimers. These data suggest that, in addition to the well-known proteolytic-dependent mechanism, an alternative oligomerization pathway may participate in gelsolin misfolding and aggregation. We propose to integrate this alternative pathway into the current model of the disease that may also be relevant for other types of AGel amyloidosis, and other related diseases with similar underlying pathological mechanisms.

Influence of Free Fatty Acids on Lipid Membrane-Nisin Interaction.

The influence of free fatty acids (FFAs) on the nisin-membrane interaction was investigated through micro-DSC and fluorescence spectroscopy. A simple but informative model membrane was prepared (5.7 DMPC:3.8 DPPS:0.5 DOPC molar ratio) by considering the presence of different phospholipid headgroups in charge and size and different phospholipid tails in length and unsaturation level, allowing the discrimination of the combined interaction of nisin and FFAs with the single phospholipid constituents. The effects of six FFAs on membrane stability were evaluated, namely two saturated FFAs (palmitic acid and stearic acid), two monounsaturated FFAs (cis-unsaturated oleic acid and trans-unsaturated elaidic acid) and two cis-polyunsaturated FFAs (ω-6 linoleic acid and ω-3 docosahexaenoic acid). The results permitted assessment of a thermodynamic picture of such interactions which indicates that the peptide-membrane interaction does not overlook the presence of FFAs within the lipid bilayer since both FFAs and nisin are able to selectively promote thermodynamic phase separations as well as a general lipid reorganization within the host membrane. Furthermore, the magnitude of the effects may be different depending on the FFA chemical structure as well as the membrane lipid composition.

The structure of N184K amyloidogenic variant of gelsolin highlights the role of the H-bond network for protein stability and aggregation properties.

Mutations in the gelsolin protein are responsible for a rare conformational disease known as AGel amyloidosis. Four of these mutations are hosted by the second domain of the protein (G2): D187N/Y, G167R and N184K. The impact of the latter has been so far evaluated only by studies on the isolated G2. Here we report the characterization of full-length gelsolin carrying the N184K mutation and compare the findings with those obtained on the wild type and the other variants. The crystallographic structure of the N184K variant in the Ca2+-free conformation shows remarkable similarities with the wild type protein. Only minimal local rearrangements can be observed and the mutant is as efficient as the wild type in severing filamentous actin. However, the thermal stability of the pathological variant is compromised in the Ca2+-free conditions. These data suggest that the N to K substitution causes a local disruption of the H-bond network in the core of the G2 domain. Such a subtle rearrangement of the connections does not lead to significant conformational changes but severely affects the stability of the protein.

Glycosylation Tunes Neuroserpin Physiological and Pathological Properties.

Neuroserpin (NS) is a member of the serine protease inhibitors superfamily. Specific point mutations are responsible for its accumulation in the endoplasmic reticulum of neurons that leads to a pathological condition named familial encephalopathy with neuroserpin inclusion bodies (FENIB). Wild-type NS presents two N-glycosylation chains and does not form polymers in vivo, while non-glycosylated NS causes aberrant polymer accumulation in cell models. To date, all in vitro studies have been conducted on bacterially expressed NS, de facto neglecting the role of glycosylation in the biochemical properties of NS. Here, we report the expression and purification of human glycosylated NS (gNS) using a novel eukaryotic expression system, LEXSY. Our results confirm the correct N-glycosylation of wild-type gNS. The fold and stability of gNS are not altered compared to bacterially expressed NS, as demonstrated by the circular dichroism and intrinsic tryptophan fluorescence assays. Intriguingly, gNS displays a remarkably reduced polymerisation propensity compared to non-glycosylated NS, in keeping with what was previously observed for wild-type NS in vivo and in cell models. Thus, our results support the relevance of gNS as a new in vitro tool to study the molecular bases of FENIB.

Insights into the effects of N-glycosylation on the characteristics of the VC1 domain of the human receptor for advanced glycation end products (RAGE) secreted by Pichia pastoris.

Advanced glycation end products (AGEs) and advanced lipoxidation end products (ALEs), resulting from non-enzymatic modifications of proteins, are potentially harmful to human health. They directly act on proteins, affecting structure and function, or through receptor-mediated mechanisms. RAGE, a type I transmembrane glycoprotein, was identified as a receptor for AGEs. RAGE is involved in chronic inflammation, oxidative stress-based diseases and ageing. The majority of RAGE ligands bind to the VC1 domain. This domain was successfully expressed and secreted by Pichia pastoris. Out of two N-glycosylation sites, one (Asn25) was fully occupied while the other (Asn81) was under-glycosylated, generating two VC1 variants, named p36 and p34. Analysis of N-glycans and of their influence on VC1 properties were here investigated. The highly sensitive procainamide labeling method coupled to ES-MS was used for N-glycan profiling. N-glycans released from VC1 ranged from Man9GlcNAc2- to Man15GlcNAc2- with major Man10GlcNAc2- and Man11GlcNAc2- species for p36 and p34, respectively. Circular dichroism spectra indicated that VC1 maintains the same conformation also after removal of N-glycans. Thermal denaturation curves showed that the carbohydrate moiety has a small stabilizing effect on VC1 protein conformation. The removal of the glycan moiety did not affect the binding of VC1 to sugar-derived AGE- or malondialdehyde-derived ALE-human serum albumin. Given the crucial role of RAGE in human pathologies, the features of VC1 from P. pastoris will prove useful in designing strategies for the enrichment of AGEs/ALEs from plasma, urine or tissues, and in characterizing the nature of the interaction.

Cellulose nanofiber (CNF)-sakacin-A active material: production, characterization and application in storage trials of smoked salmon.

BACKGROUND: Sakacin-A due to its specific antimicrobial activity may represent a good candidate to develop active packaging solutions for food items supporting Listeria growth. In the present study a protein extract containing the bacteriocin sakacin-A, produced by Lactobacillus sakei Lb 706 in a low-cost culture medium containing deproteinized cheese whey, was adsorbed onto cellulose nanofibers (CNFs) to obtain an active material to be used as a mat (or a separator) in direct contact with foods. RESULTS: The applied fermentation conditions allowed 4.51 g L-1 of freeze-dried protein extract to be obtained, characterized by an antimicrobial activity of near 16 700 AU g-1 , that was used for the preparation of the active material by casting. The active material was then characterized by infrared spectra and thermogravimetric analyses. Antimicrobial trials were carried out in vitro using Listeria innocua as indicator strain; results were also confirmed in vivo, employing smoked salmon fillets intentionally inoculated with Listeria innocua: its final population was reduced to about 2.5-3 Log cycles after 28 days of storage at 6 °C in presence of sakacin-A, compared with negative control mats produced without the bacteriocin extract. CONCLUSION: This study demonstrates the possibility of producing an antimicrobial active material containing sakacin-A absorbed onto CNFs to decrease Listeria population in smoked salmon, a ready-to eat-food product. © 2019 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Imine Deaminase Activity and Conformational Stability of UK114, the Mammalian Member of the Rid Protein Family Active in Amino Acid Metabolism.

Reactive intermediate deaminase (Rid) protein family is a recently discovered group of enzymes that is conserved in all domains of life and is proposed to play a role in the detoxification of reactive enamines/imines. UK114, the mammalian member of RidA subfamily, was identified in the early 90s as a component of perchloric acid-soluble extracts from goat liver and exhibited immunomodulatory properties. Multiple activities were attributed to this protein, but its function is still unclear. This work addressed the question of whether UK114 is a Rid enzyme. Biochemical analyses demonstrated that UK114 hydrolyzes α-imino acids generated by l- or d-amino acid oxidases with a preference for those deriving from Ala > Leu = l-Met > l-Gln, whereas it was poorly active on l-Phe and l-His. Circular Dichroism (CD) analyses of UK114 conformational stability highlighted its remarkable resistance to thermal unfolding, even at high urea concentrations. The half-life of heat inactivation at 95 °C, measured from CD and activity data, was about 3.5 h. The unusual conformational stability of UK114 could be relevant in the frame of a future evaluation of its immunogenic properties. In conclusion, mammalian UK114 proteins are RidA enzymes that may play an important role in metabolism homeostasis also in these organisms.

Bacterial Production, Characterization and Protein Modeling of a Novel Monofuctional Isoform of FAD Synthase in Humans: An Emergency Protein?

FAD synthase (FADS, EC 2.7.7.2) is the last essential enzyme involved in the pathway of biosynthesis of Flavin cofactors starting from Riboflavin (Rf). Alternative splicing of the human FLAD1 gene generates different isoforms of the enzyme FAD synthase. Besides the well characterized isoform 1 and 2, other FADS isoforms with different catalytic domains have been detected, which are splice variants. We report the characterization of one of these novel isoforms, a 320 amino acid protein, consisting of the sole C-terminal 3'-phosphoadenosine 5'-phosphosulfate (PAPS) reductase domain (named FADS6). This isoform has been previously detected in Riboflavin-Responsive (RR-MADD) and Non-responsive Multiple Acyl-CoA Dehydrogenase Deficiency (MADD) patients with frameshift mutations of FLAD1 gene. To functionally characterize the hFADS6, it has been over-expressed in Escherichia coli and purified with a yield of 25 mg·L-1 of cell culture. The protein has a monomeric form, it binds FAD and is able to catalyze FAD synthesis (kcat about 2.8 min-1), as well as FAD pyrophosphorolysis in a strictly Mg2+-dependent manner. The synthesis of FAD is inhibited by HgCl2. The enzyme lacks the ability to hydrolyze FAD. It behaves similarly to PAPS. Combining threading and ab-initio strategy a 3D structural model for such isoform has been built. The relevance to human physio-pathology of this FADS isoform is discussed.

Structural changes in emulsion-bound bovine beta-lactoglobulin affect its proteolysis and immunoreactivity.

Adsorption on the surface of sub-micrometric oil droplets resulted in significant changes in the tertiary structure of bovine beta-lactoglobulin (BLG), a whey protein broadly used as a food ingredient and a major food allergen. The adsorbed protein had increased sensitivity to trypsin, and increased immunoreactivity towards specific monoclonal antibodies. In spite of the extensive tryptic breakdown of emulsion-bound BLG, some sequence stretches in BLG became trypsin-insensitive upon absorption of the protein on the fat droplets. As a consequence - at contrast with free BLG - proteolysis of emulsion-bound BLG did not decrease the immunoreactivity of the protein, and some of the large peptides generated by trypsinolysis of emulsion-bound BLG were still recognizable by specific monoclonal antibodies. Structural changes occurring in emulsion-bound BLG and their consequences are discussed in comparison with those occurring when the tertiary structure of BLG is modified by lipophilic salts, by urea, or upon interaction with solid hydrophobic surfaces. Such a comparison highlights the relevance of situation-specific structural modifications, that in turn may affect physiologically relevant features of the protein.

Sakacin-A antimicrobial packaging for decreasing Listeria contamination in thin-cut meat: preliminary assessment.

BACKGROUND: Minimally processed ready-to-eat products are considered a high-risk food because of the possibility of contamination with pathogenic bacteria, including Listeria monocytogenes from the animal reservoir, and the minimal processing they undergo. In this study, a sakacin-A anti-Listeria active package was developed and tested on thin-cut veal meat slices (carpaccio). RESULTS: Enriched food-grade sakacin-A was obtained from a cell-free supernatant of a Lactobacillus sakei culture and applied (0.63 mg cm-2 ) onto the surface of polyethylene-coated paper sheets to obtain an active antimicrobial package. The coating retained antimicrobial features, indicating that the process did not affect sakacin-A functionality, as evidenced in tests carried out in vitro. Thin-cut veal meat slices inoculated with Listeria innocua (a surrogate of pathogenic L. monocytogenes) were laid on active paper sheets. After 48 h incubation at 4 °C, the Listeria population was found to be 1.5 log units lower with respect to controls (3.05 vs 4.46 log colony-forming units (CFU) g-1 ). CONCLUSION: This study demonstrates the possibility of using an antimicrobial coating containing sakacin-A to inhibit or decrease the Listeria population in ready-to-eat products, thus lowering the risk of food-related diseases. © 2016 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.