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ABSTRACT: Curcumin is a bioactive polyphenolic compound extracted from turmeric with known anti-inflammatory properties, and its hydrophobic nature restricts its solubility and its bioaccessibility. Solubility may be improved upon binding of curcumin to native or treatment-modified casein micelles. The present work demonstrated that high hydrostatic pressure treatment of skim milk increases the binding of curcumin to caseins. The association of curcumin to casein micelles was assessed using fluorescence spectroscopy, either directly or by tryptophan quenching. The amount of curcumin associated with the milk proteins increased in pressure-treated milk, and a further improvement in the amount of bound curcumin was observed upon pressure treatment of a milk/curcumin mixture. However, in this case, some of the curcumin dissociated during storage, contrarily to what was observed for untreated milk. From a molecular standpoint, the data presented here indicate that structural modifications induced by high-pressure treatment and known to affect the structure of milk proteins result in a rearrangement of the amino acid residues in close proximity to the protein-associated curcumin.
Journal of Dairy Research 04/2013; · 1.34 Impact Factor
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ABSTRACT: Structural changes of starch and proteins in rice pasta were investigated as a function of raw-materials and pasta-making conditions, and their impact on cooking behaviour and glycaemic index was assessed. Rice pasta was prepared from untreated or parboiled rice flour by conventional extrusion or by extrusion-cooking. Starch structure was studied by assessing starch accessibility to specific enzymes (α-amylase and pullulanase), and by evaluating the molecular properties of fragments from enzymatic action. Protein solubility in presence/absence of chaotropes and accessibility of protein cysteine thiols allowed to evaluate the intensity and nature of inter-protein interactions. Parboiling stiffens the protein network in rice flour and makes starch more accessible to hydrolysis. Pasta-making induced further changes in the starch structure, that were most evident in pasta made from untreated rice and were mainly related to the amylopectin fraction. Thus, the interplay among structural modifications on starch and/or proteins affects the features of products.
Carbohydrate polymers. 02/2013; 92(2):1865-72.
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ABSTRACT: Presence and stability of a protein network was evaluated by fluorescence spectroscopy, by protein solubility studies, and by assessing the accessibility of protein thiols in samples of commercial Italian semolina pasta made in industrial plants using different processes. The pasting properties of starch in each sample were evaluated by means of a viscoamylograph. Magnetic resonance imaging (MRI) was used to evaluate water distribution and water mobility in dry pasta, and at various cooking times. The molecular information derived from these studies was related to sensory indices, indicating that protein reticulation was dependent on the process conditions, which affected water penetration, distribution, and mobility during cooking. Products with a crosswise gradient of water mobility once cooked had the best sensory scores at optimal cooking time, whereas products with a less compact protein network performed better when slightly overcooked.
Food Chemistry 11/2012; 135(2):348-55. · 3.65 Impact Factor
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Food Chemistry 08/2012; 133:1011-1016. · 3.65 Impact Factor
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ABSTRACT: Sakacin A was purified to homogeneity through simple chromatographic procedures from cultures of Lactobacillus sakei DSMZ 6333 grown on a low-cost medium. The highly purified protein dissipated both transmembrane potential (ΔΨ) and transmembrane pH gradient (ΔpH) in Listeria cells in a very intense, rapid, and energy-dependent fashion. On a slower timescale, purified sakacin A also showed a lytic activity toward isolated cell walls of Listeria. Mass spectrometry was used to analyze the products of sakacin A action on cell walls, evidencing that sakacin A acts on various types of bonds within peptoglycans.
FEMS Microbiology Letters 07/2012; 334(2):143-9. · 2.04 Impact Factor
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ABSTRACT: a b s t r a c t Gluten-free pasta represents a challenge for food technologists and nutritionists since gluten-free materials used in conventional formulations have poor functional and nutritional properties. A novel extrusion-cooking process was set up to improve the textural characteristics of rice-based pasta, and to enrich it with amaranth. Mineral and fiber content, and protein digestibility were improved by amaranth enrichment. Extrusion-cooking of a 75/25 mixture of rice flour and amaranth prior to pasta-making gave the best results as for the textural characteristics of the final product. The firmness of cooked pasta increased due to the extrusion-cooking process, that also decreased protein solubility in the amaranth-enriched pasta. The content in accessible thiols also decreased in amaranth-enriched pastas, indicating that amaranth proteins may be involved in forming disulphide bonds during the pasta-making process. Our results suggest that starch in rice flour interacts best with amaranth proteins when starch gelati-nization occurs simultaneously to protein denaturation in the extrusion-cooking process.
LWT - Food Science and Technology. 07/2012; 47(2-ISSN 0023-6438):421.
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ABSTRACT: Fluorescence spectroscopy was used to acquire information on the structural changes of proteins at the oil/water interface in emulsions prepared by using soy protein isolate, glycinin, and β-conglycinin rich fractions. Spectral changes occurring from differences in the exposure of tryptophan residues to the solvent were evaluated with respect to spectra of native, urea-denatured, and heat treated proteins. The fluorescence emission maxima of the emulsions showed a red shift with respect to those of native proteins, indicating that the tryptophan residues moved toward a more hydrophilic environment after adsorption at the interface. The heat-induced irreversible transitions were investigated using microcalorimetry. Fluorescence spectroscopy studies indicated that while the protein in solution underwent irreversible structural changes with heating at 75 and 95°C for 15 min, the interface-adsorbed proteins showed very little temperature-induced rearrangements. The smallest structural changes were observed in soy protein isolate, probably because of the higher extent of protein-protein interactions in this material, as compared to the β-conglycinin and to the glycinin fractions. This work brings new evidence of structural changes of soy proteins upon adsorption at the oil water interface, and provides some insights on the possible protein exchange events that may occur between adsorbed and unadsorbed proteins in the presence of oil droplets.
Colloids and surfaces. B, Biointerfaces 05/2012; 93:41-8. · 2.60 Impact Factor
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ABSTRACT: A study was made on the molecular and structural changes occurring in the protein and starch components of sorghum flour when
fermented to prepare typical non-malted Sudanese foods. Protein solubility and SDS-PAGE studies indicated that water-soluble
proteins are the main target of hydrolysis during fermentation. Proteolysis products are taken up for bacterial growth. Kafirins
are among the proteins left intact by proteolytic events in the fermentation step. Upon cooking in boiling water kafirins
are converted into protein aggregates almost insoluble even in the presence of 8M urea and of disulphide-reducing agents.
Viscoamylographic and microstructural studies indicate that fermentation leads to the release of starch granules from very
compact structures in the original sorghum flour, in which proteolysis-sensitive, water-soluble proteins form an essential
part of the outermost shell of large structures, where starch granules are embedded into a kafirin-rich protein matrix that
is not affected by proteolytic events during fermentation.
European Food Research and Technology 04/2012; 222(5):559-564. · 1.57 Impact Factor
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ABSTRACT: Maize is used as an alternative to wheat to elaborate food stuffs for celiac patients in a gluten-free diet.However, some maize prolamins (zeins) contain amino acid sequences that resemble the wheat gluten immunodominant peptides and their integrity after gastrointestinal proteolysisis unknown. In this study, the celiac IgA-immunoreactivity to zeins from raw or nixtamalized grains, before and after peptic/tryptic digestion was evaluated and their possible immunogenicity was investigated by in silico methods.IgA from some celiac patients with HLA-DQ2 or DQ8 haplotypes recognized two alpha-zeins even after peptic/ tryptic proteolysis. However, digestion affected zeins after denaturation, reduction, and alkylation, used for identification of prolamins as alpha-zein A20 and A30 by MS/MS sequencing. An in silico analysis indicated that other zeins contain similar sequences, or sequences that may bind even better to the HLA-DQ2/DQ8 molecules compared to the already identified ones. Results concur to indicate that relative abundance of these zeins, along with factors affecting their resistance to proteolysis, may be of paramount clinical relevance, and the use of maize in the formulation and preparation of gluten-free foods must be reevaluated in some cases of celiac disease.
Materiae Vegetabiles 02/2012; 67(1):24, 30. · 2.51 Impact Factor
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ABSTRACT: We report on specific magneto-capturing followed by Multidimensional Protein Identification Technology (MudPIT) for the analysis of surface-exposed proteins of intact cells of the bacterial opportunistic pathogen Pseudomonas aeruginosa. The magneto-separation of cell envelope fragments from the soluble cytoplasmic fraction allowed the MudPIT identification of the captured and neighboring proteins. Remarkably, we identified 63 proteins captured directly by nanoparticles and 67 proteins embedded in the cell envelope fragments. For a high number of proteins, our analysis strongly indicates either surface exposure or localization in an envelope district. The localization of most identified proteins was only predicted or totally unknown. This novel approach greatly improves the sensitivity and specificity of the previous methods, such as surface shaving with proteases that was also tested on P. aeruginosa. The magneto-capture procedure is simple, safe, and rapid, and appears to be well-suited for envelope studies in highly pathogenic bacteria.
PLoS ONE 01/2012; 7(11):e51062. · 4.09 Impact Factor
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ABSTRACT: The scaffold protein IscU and molecular chaperones HscA and HscB play central roles in biological assembly of iron-sulfur clusters and maturation of iron-sulfur proteins. However, the structure of IscU-FeS complexes and the molecular mechanism whereby the chaperones facilitate cluster transfer to acceptor proteins are not well understood. We have prepared amino acid substitution mutants of Escherichia coli IscU in which potential ligands to the FeS cluster (Cys-37, Cys-63, His-105, and Cys-106) were individually replaced with alanine. The properties of the IscU-FeS complexes formed were investigated by measuring both their ability to transfer preformed FeS clusters to apo-ferredoxin and the activity of the IscU proteins in catalyzing cluster assembly on apo-ferredoxin using inorganic iron with inorganic sulfide or with IscS and cysteine as a sulfur source. The ability of the HscA/HscB chaperone system to accelerate ATP-dependent cluster transfer from each IscU substitution mutant to apo-ferredoxin was also determined. All of the mutants formed FeS complexes with a stoichiometry similar to the wild-type holo-protein, i.e., IscU(2)[2Fe2S], raising the possibility that different cluster ligation states may occur during iron-sulfur protein maturation. Spectroscopic properties of the mutants and the kinetics of transfer of performed IscU-FeS clusters to apo-ferredoxin indicate that the most stable form of holo-IscU involves iron coordination by Cys-63 and Cys-106. Results of studies on the ability of mutants to catalyze formation of holo-ferredoxin using iron and different sulfur sources were consistent with proposed roles for Cys-63 and Cys-106 in FeS cluster binding and also indicated an essential role for Cys-106 in sulfide transfer to IscU from IscS. Measurements of the ability of the chaperones HscA and HscB to facilitate cluster transfer from holo-IscU to apo-ferredoxin showed that only IscU(H105A) behaved similarly to wild-type IscU in exhibiting ATP-dependent stimulation of cluster transfer. IscU(C63A) and IscU(C106A) displayed elevated rates of cluster transfer in the ±ATP whereas IscU(C37A) exhibited low rates of cluster transfer ±ATP. In interpreting these findings, we propose that IscU(2)[2Fe2S] is able undergo structural isomerization to yield conformers having different cysteine residues bound to the cluster. On the basis of the crystal structure of HscA complexed with an IscU-derived peptide, we propose that the chaperone binds and stabilizes an isomer of IscU(2)[2Fe2S] in which the cluster is bound by cysteine residues 37 and 63 and that the [2Fe2S] cluster, being held less tightly than that coordinated by Cys-63 and Cys-106 in free IscU(2)[2Fe2S], is more readily transferred to acceptor proteins such as apo-ferredoxin.
Biochemistry 11/2011; 50(44):9641-50. · 3.42 Impact Factor
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ABSTRACT: A soluble form of human FAD synthase (isoform 2; hFADS2) was produced and purified to homogeneity as a recombinant His-tagged protein. The enzyme binds 1 mole of the FAD product very tightly, although noncovalently. Complete release of FAD from the 'as isolated' protein requires extensive denaturation. A 75 : 25 mixture of apo/holoprotein could be prepared by treatment with mild chaotropes, allowing estimatation of the contribution made by bound FAD to the protein stability and evaluatation of whether structural rearrangements may be required for FAD release. Under turnover conditions, the enzyme catalyzes FAD assembly from ATP and FMN and, at a much lower rate, the pyrophosphorolytic hydrolysis of FAD. Several mechanistic features of both reactions were investigated in detail, along with their dependence on environmental conditions (pH, temperature, dependence on metals). Our data indicate that FAD release may represent the rate-limiting step of the whole catalytic cycle and that the process leading to FAD synthesis, and delivery to client apoproteins may be tightly controlled.
FEBS Journal 09/2011; 278(22):4434-49. · 3.79 Impact Factor
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ABSTRACT: To improve our understanding of the interaction of food allergens with cells of the immune system, the endocytosis by human monocytes of bovine β-lactoglobulin (BLG) and ovomucoid (OM)--two major food allergens--and human serum albumin (HSA) was studied.
BLG was covalently conjugated to dextran-coated magnetic nanoparticles (MNPs) without affecting its structure and immunoreactivity. BLG-conjugated MNPs were taken up by human monocytes much more efficiently than non-conjugated MNPs, allowing easy magnetic separation of cells that had adsorbed the allergen. BLG, OM, and HSA were conjugated to MNPs also labeled with a fluorescent probe. The uptake of these materials by human monocytes was monitored through flow cytometry, and compared with fluorescent MNPs and the free fluorescently labeled proteins, confirming higher uptake of the BLG-conjugated MNPs versus non-conjugated MNPs. OM but not HSA conjugation to particles enhanced uptake of the MNPs. Confocal microscopy provided direct evidence of the actual internalization of BLG-MNP conjugates into the cytoplasm.
These results contribute to the current understanding of the interaction between food allergens and antigen-presenting cells, and demonstrate that the BLG is readily endocytosed by monocytes both as the single protein and as a conjugate.
Molecular Nutrition & Food Research 09/2011; 55(11):1708-16. · 4.30 Impact Factor
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Stavros Azinas,
Matteo Colombo,
Alberto Barbiroli,
Carlo Santambrogio,
Sofia Giorgetti,
Sara Raimondi, Francesco Bonomi,
Rita Grandori,
Vittorio Bellotti,
Stefano Ricagno,
Martino Bolognesi
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ABSTRACT: Proteins hosting main β-sheets adopt specific strategies to avoid intermolecular interactions leading to aggregation and amyloid deposition. Human beta-2 microglobulin (β2m) displays a typical immunoglobulin fold and is known to be amyloidogenic in vivo. Upon severe kidney deficiency, β2m accumulates in the bloodstream, triggering, over the years, pathological deposition of large amyloid aggregates in joints and bones. A β-bulge observed on the edge D β-strand of some β2m crystal structures has been suggested to be crucial in protecting the protein from amyloid aggregation. Conversely, a straight D-strand, observed in different crystal structures of monomeric β2m, could promote amyloid aggregation. More recently, the different conformations observed for the β2m D-strand have been interpreted as the result of intrinsic flexibility, rather than being assigned to a functional protective role against aggregation. To shed light on such contrasting picture, the mutation Asp53→Pro was engineered in β2m, aiming to impair the formation of a regular/straight D-strand. Such a mutant was characterized structurally and biophysically by CD, X-ray crystallography and MS, in addition to an assessment of its amyloid aggregation trends in vitro. The results reported in the present study highlight the conformational plasticity of the edge D-strand, and show that even perturbing the D-strand structure through a Pro residue has only marginal effects on protecting β2m from amyloid aggregation in vitro.
FEBS Journal 07/2011; 278(13):2349-58. · 3.79 Impact Factor
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Stavros Azinas,
Matteo Colombo,
Alberto Barbiroli,
Carlo Santambrogio,
Sofia Giorgetti,
Sara Raimondi, Francesco Bonomi,
Rita Grandori,
Vittorio Bellotti,
Stefano Ricagno,
Martino Bolognesi
FEBS Journal 05/2011; 278(13):2349 - 2358. · 3.79 Impact Factor
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ABSTRACT: Fatty acids are the natural ligands associated with the bovine milk lipocalin, β-lactoglobulin (BLG), and were identified by means of mass spectrometry. The naturally bound ligands were found to contribute to the stability of the proteins toward denaturation by both temperature and chaotropes. To assess the nature of the structural regions involved in this stabilization, the thermodynamic and kinetic aspects of the stability of various structural regions of the proteins were studied in the presence of bound palmitate, which is the most abundant natural ligand. Binding of a single palmitate molecule was found to affect not only the stability of the calyx region, where palmitate is bound, but also that of the region at the hydrophobic interface between the barrel itself and the long helix in the protein structure, where the thiol group of Cys121 is buried. This region is known to be essential for the stability of the BLG dimer and is relevant to the generation of "reactive monomers" that are involved in covalent and noncovalent polymerization of BLG and in the formation of covalent adducts with other milk proteins.
Journal of Agricultural and Food Chemistry 05/2011; 59(10):5729-37. · 2.82 Impact Factor
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ABSTRACT: Beta-2 microglobulin (β2m) is the light chain of Class I major histocompatibility complex (MHC-I) complex. β2m is an intrinsically amyloidogenic protein capable of forming amyloid fibrils in vitro and in vivo. β2m displays the typical immunoglobulin-like fold with a disulphide bridge (Cys25-Cys80) cross-linking the two β-sheets. Engineering of the loop comprised between β-strands D and E has shown that mutations in this region affect protein structure, fold stability, folding kinetics and amyloid aggregation properties. Such overall effects have been related to the DE loop backbone structure, which presents a strained conformation in the wild-type (wt) protein, and a type I β-turn in the W60G mutant. Here, we report a biophysical and structural characterization of the K58P-W60G β2m mutant, where a Pro residue has been introduced in the type I β-turn i + 1 position. The K58P-W60G mutant shows improved chemical and temperature stability and faster folding relative to wt β2m. The crystal structure (1.25 Å resolution) shows that the Cys25-Cys80 disulphide bridge is unexpectedly severed, in agreement with electrospray ionization-mass spectrometry (ESI-MS) spectra that indicate that a fraction of the purified protein lacks the internal disulphide bond. These observations suggest a stabilizing role for Pro58, and stress a crucial role for the DE loop in determining β2m biophysical properties.
Journal of biochemistry 03/2011; 150(1):39-47. · 1.95 Impact Factor
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ABSTRACT: Human neuroserpin (hNS) is a serine protease inhibitor that belongs to the serpin superfamily and is expressed in nervous tissues. The serpin fold is generally characterized by a long exposed loop, termed the reactive center loop, that acts as bait for the target protease. Intramolecular insertion of the reactive center loop into the main serpin β-sheet leads to the serpin latent form. As with other known serpins, hNS pathological mutants have been shown to accumulate as polymers composed of quasi-native protein molecules. Although hNS polymerization has been intensely studied, a general agreement about serpin polymer organization is still lacking. Here we report a biophysical characterization of native hNS that is shown to undergo two distinct conformational transitions, at 55°C and 85°C, both leading to distinct latent and polymeric species. The latent and polymer hNS forms obtained at 45°C and 85°C differ in their chemical and thermal stabilities; furthermore, the hNS polymers also differ in size and morphology. Finally, the 85°C polymer shows a higher content of intermolecular β-sheet interactions than the 45°C polymer. Together, these results suggest a more complex conformational scenario than was previously envisioned, and, in a general context, may help reconcile the current contrasting views on serpin polymerization.
Biophysical Journal 11/2010; 99(10):3402-11. · 3.65 Impact Factor
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ABSTRACT: Plant proteins are being used as a replacement for animal proteins in wine fining. The surface hydrophobicity of plant proteins in four commercial preparations differing for their origin and processing was assessed by using a fluorescent hydrophobic probe in wine-like media. Displacement of the probe by addition of wine phenolics was measured as a way to compare and predict to some extent the efficiency of these proteins in wine fining. It was found that the binding of polyphenols was much more specific than that of the hydrophobic probe. Further analysis of the polyphenol pattern in protein-treated wine-like solutions pointed out two relevant facts: (1) proteins may interfere with the chemistry of the interactions between polyphenols and other wine components; (2) individual protein preparation having different surface hydrophobicities also have different specificities in binding different polymeric forms of the polyphenols and in their substitution products. These findings are related to the possible carry-over of transition metals and may be worth exploring for custom tailoring the fining process. Whether the practical application of the latter finding will call for production and/or screening of plant-derived proteins with features appropriate to this task remains to be investigated. However, the approaches presented in this study may be used for large-scale screening of protein suitability for fining application under laboratory conditions, providing guidelines for their use in actual winemaking applications.
Journal of Agricultural and Food Chemistry 10/2010; 58(22):11969-76. · 2.82 Impact Factor
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ABSTRACT: Addition of iron salts to chaotrope-denatured aporubredoxin (apoRd) leads to nearly quantitative recovery of its single Fe(SCys)(4) site and native protein structure without significant dilution of the chaotrope. This "high-chaotrope" approach was used to examine iron binding and protein folding events using stopped-flow UV-vis absorption and CD spectroscopies. With a 100-fold molar excess of ferrous iron over denatured apoRd maintained in 5 M urea, the folded holoFe(III)Rd structure was recovered in >90% yield with a t(1/2) of <10 ms. More modest excesses of iron also gave nearly quantitative holoRd formation in 5 M urea but with chronological resolution of iron binding and protein folding events. The results indicate structural recovery in 5 M urea consists of the minimal sequence: (1) binding of ferrous iron to the unfolded apoRd, (2) rapid formation of a near-native ferrous Fe(SCys)(4) site within a protein having no detectable secondary structure, and (3) recovery of the ferrous Fe(SCys)(4) site chiral environment nearly concomitantly with (4) recovery of the native protein secondary structure. The rate of step 2 (and, by inference, step 1) was not saturated even at a 100-fold molar excess of iron. Analogous results obtained for Cys --> Ser iron ligand variants support formation of an unfolded-Fe(SCys)(3) complex between steps 1 and 2, which we propose is the key nucleation event that pulls together distal regions of the protein chain. These results show that folding of chaotrope-denatured apoRd is iron-nucleated and driven by extraordinarily rapid formation of the Fe(SCys)(4) site from an essentially random coil apoprotein. This high-chaotrope, multispectroscopy approach could clarify folding pathways of other [M(SCys)(3)]- or [M(SCys)(4)]-containing proteins.
Biochemistry 08/2010; 49(31):6627-34. · 3.42 Impact Factor
