Protein structural modifications induced by physical processing

This contribution focuses on the molecular determinants and the biotechnologically relevant consequences of structural modification of protein ensuing from: 1) physical treatments that alter the structure of solvent water (such as temperature of high pressure); 2) shear forces (as used, for example, in food processing); 3) the interaction of proteins with hydrophobic surfaces (either in liquid or in solid phases). For the sake of simplicity, most of the data to be presented and discussed relate to bovine betalactoglobulin (BLG), that offers a number of distinctive advantages for this type of studies: i) its structure is known in extreme detail; ii) it contains a number of \u201creporter\u201d residues that facilitate analysis of individual unfolding steps; iii) it is a food allergen with both sequential and conformational epitopes, for which selected monoclonal antibodies are available. The presentation will take off by considering temperature stability of the protein structure, with a focus on how unfolding can be controlled by the presence/absence of ligands and cosolutes, and on how temperature-swollen conformers of BLG may be used for selective and stable binding of hydrophobes (including bioactives and species of pharmaceutical interest) or to generate non-immunoreactive species upon selective proteolytic breakdown breakdown of sequential epitopes. Further, focus will shift on mechanical stress, as used in generating BLG conformers trapped at the interface between polar and non-polar liquid phases, as happens when BLG is used as a stabilizing agent in emulsions. The effects of these conformational changes on the protein structure will be discussed also in terms of altered proteolytic patterns and of changes in immunoreactivity. Finally, recent data on the unfolding of BLG upon contact with the hydrophobic surface of polystyrene nanoparticles (NPs, of various size) will be presented and discussed. Molecular dynamic (MD) studies highlighted that this \u201ccontact\u201d unfolding is extensive and extremely fast. Structural features of the NP-bound BLG have been assessed by monitoring the reactivity and the spectroscopic features of \u201creporter\u201d residues in the protein structure, as well as the immunoreactivity of NP-bound BLG and its sensitivity to proteolysis. Results confirm what predicted by MD studies, and provide also evidence of the relevance of \u201cgeometric effects\u201d (related to the NP size) and of \u201cmolecular crowding\u201d effects (related to the BLG/NP mass ratio)

Affinity and selectivity of plant proteins for red wine components relevant to color and aroma traits

The effects of fining with various plant proteins were assessed on Aglianico red wine, using both the young wine and wine aged for twelve and twenty-four months, and including wine unfined or fined with gelatin as controls. Color traits and fining efficiency were considered, along with the content of various types of phenolics and of aroma-related compounds of either varietal or fermentative origin. All agents had comparable fining efficiency, although with distinct kinetics, and had similar effects on wine color. Individual plant proteins and enzymatic hydrolyzates differed in their ability to interact with some anthocyanins, with specific proanthocyanidins complexes, and with some aroma components of fermentative origin. Changes in varietal aroma components upon fining were very limited or absent. Effects of all the fining agents tested in this study on the anthocyanidin components were most noticeable in young red wine, and decreased markedly with increasing wine ageing

Studies on the mechanism of catalysis of iron-sulfur cluster transfer from IscU[2Fe2S] by HscA/HscB chaperones

The HscA/HscB chaperone/cochaperone system accelerates transfer of iron-sulfur clusters from the FeS-scaffold protein IscU (IscU(2)[2Fe2S], holo-IscU) to acceptor proteins in an ATP-dependent manner. We have employed visible region circular dichroism (CD) measurements to monitor chaperone-catalyzed cluster transfer from holo-IscU to apoferredoxin and to investigate chaperone-induced changes in properties of the IscU(2)[2Fe2S] cluster. HscA-mediated acceleration of [2Fe2S] cluster transfer exhibited an absolute requirement for both HscB and ATP. A mutant form of HscA lacking ATPase activity, HscA(T212V), was unable to accelerate cluster transfer, suggesting that ATP hydrolysis and conformational changes accompanying the ATP (T-state) to ADP (R-state) transition in the HscA chaperone are required for catalysis. Addition of HscA and HscB to IscU(2)[2Fe2S] did not affect the properties of the [2Fe2S] cluster, but subsequent addition of ATP was found to cause a transient change of the visible region CD spectrum, indicating distortion of the IscU-bound cluster. The dependence of the rate of decay of the observed CD change on ATP concentration and the lack of an effect of the HscA(T212V) mutant were consistent with conformational changes in the cluster coupled to ATP hydrolysis by HscA. Experiments carried out under conditions with limiting concentrations of HscA, HscB, and ATP further showed that formation of a 1:1:1 HscA-HscB-IscU(2)[2Fe2S] complex and a single ATP hydrolysis step are sufficient to elicit the full effect of the chaperones on the [2Fe2S] cluster. These results suggest that acceleration of iron-sulfur cluster transfer involves a structural change in the IscU(2)[2Fe2S] complex during the T --> R transition of HscA accompanying ATP hydrolysis

Grape skin phenolics as inhibitors of mammalian α-glucosidase and α-amylase – effect of food matrix and processing on efficacy

Inhibition of mammalian α-amylase and α-glucosidase was studied for white grape skin samples recovered from wineries and found to be higher than that of the drug acarbose

Structuring and texturing gluten‑free pasta: egg albumen or whey proteins?

The effects of adding egg albumen or whey proteins to pasta made from parboiled rice flour (PR) were investigated. Pasta quality was evaluated in terms of color, furosine content, and cooking properties (water absorption, cooking loss, and consistency at the optimal cooking time). The surface heterogeneity of the cooked and uncooked materials was studied, and some starch properties (pasting properties and starch susceptibility to \u3b1-amylase hydrolysis) were assessed, along with the features of the protein network as determined by conditional solubility studies and with ultrastructural features of the cooked products. Egg albumen improved pasta appearance and gave a product with low cooking loss, firmer, and nutritionally more valuable than the other ones. In albumen-enriched pasta, small starch granules appear homogeneously surrounded by a protein network. In the uncooked product, the protein network is stabilized mostly by hydrophobic interactions, but additional disulfide interprotein bonds form upon cooking. Thus, addition of 15 % liquid albumen to PR results in significant improvement of the textural and structural features of rice-based gluten-free pasta

Characterization of Whole Grain Pasta: Integrating Physical, Chemical, Molecular, and Instrumental Sensory Approaches

The consumption of whole-grain foodincluding pastahas been increasing steadily. In the case of whole-grain pasta, given the many different producers, it seems important to have some objective parameters to define its overall quality. In this study, commercial whole-grain pasta samples representative of the Italian market have been characterized from both molecular and electronic-senses (electronic nose and electronic tongue) standpoint in order to provide a survey of the properties of different commercial samples. Only 1 pasta product showed very low levels of heat damage markers (furosine and pyrraline), suggesting that this sample underwent to low temperature dry treatment. In all samples, the furosine content was directly correlated to protein structural indices, since protein structure compactness increased with increasing levels of heat damage markers. Electronic senses were able to discriminate among pasta samples according to the intensity of heat treatment during the drying step. Pasta sample with low furosine content was discriminated by umami taste and by sensors responding to aliphatic and inorganic compounds. Data obtained with this multidisciplinary approach are meant to provide hints for identifying useful indices for pasta quality. Practical ApplicationAs observed for semolina pasta, objective parameters based on heat-damage were best suited to define the overall quality of wholegrain pasta, almost independently of compositional differences among commercial samples. Drying treatments of different intensity also had an impact on instrumental sensory traits that may provide a reliable alternative to analytical determination of chemical markers of heat damage in all cases where there is a need for avoiding time-consuming procedures

Probiotics action on gliadin sequences relevant to gluten sensitivity

The Celiac disease in genetically predisposed individuals is mainly induced by specific repetitive sequences in gliadins (PQPYP). This autoimmune disease stems from the interaction between toxic sequences and lamina propria cells, that is relevant also to other forms of gluten sensitivity. Specific endo-esoprolinase were isolated from lactic acid bacteria, suggesting possible practical applications. The ability of some probiotics at removing "toxic" celiac sequences was investigated, at first by assessing the presence and level of endo- and eso-prolinase activity in some of the most popular probiotic bacteria. Significant activities were detected in Lactobacillus and Bifidum species, as well as in the probiotic Escherichia coli Niessle 1917. On the basis of prolinase data, we investigated by mass spectroscopy the removal of "toxic" sequences in gliadin. A complete disappearance of these sequences was observed only with Escherichia coli Niessle 1917. Among the Bifidus and Lactobacillus species, only B. bifidum MIMBb23SG and L. acidophilus LA5 showed a significant decrease in the "toxic" sequences. All together, this study suggests a potential use of lactic bacteria to lower gluten response in sensitive individuals, including celiacs and gluten-sensitive

Structural consequences of the interaction of puroindolines with gluten proteins

The effect of puroindolines (PINs) on structural characteristics of wheat proteins was investigated in Triticum turgidum ssp. durum (cv. Svevo) and Triticum aestivum (cv. Alpowa) and in their respective derivatives in which PIN genes were expressed (Soft Svevo) or the distal end of the short arm of chromosome 5D was deleted and PINs were not expressed (Hard Alpowa). The presence of PINs decreased the amount of cold-SDS extractable proteins and the accessibility of protein thiols to specific reagents, but resulted in facilitated solvation of gluten proteins, as detected by tryptophan fluorescence measurements carried out on minimally mixed flour/water mixtures. We propose that PINs and gluten proteins are interacting in the grain or flour prior to mixing. Hydrophobic interactions between PINs and some of the gluten proteins modify the pattern of interactions among gluten proteins, thus providing an additional mechanistic rationale for the effects of PINs on kernel hardness

Stabilization of the “open” conformer of apoIscU on the surface of polystyrene nanobeads accelerates assembly of a 2Fe2S structure

The scaffold protein IscU is involved in the assembly/transfer of FeS clusters. IscU exists in both open and closed conformation. The clusterless open conformation of IscU adheres to the hydrophobic surface of polystyrene nanobeads, as observed for other proteins. Increased accessibility of the ligand cysteines in bound IscU facilitates assembly of a 2Fe2S cluster, and the cluster-bearing structured form of IscU does not interact with the nanobeads, thus ensuring turnover. The dependence of accelerated cluster assembly on the nanobeads concentration pointed to steric and crowding effects as for promoting cluster formation, and confirms the requirement for structural flexibility of IscU

Stabilization of the "open" conformer of apoIscU on the surface of polystyrene nanobeads accelerates assembly of a 2Fe2S structure

AbstractThe scaffold protein IscU is involved in the assembly/transfer of FeS clusters. IscU exists in both open and closed conformation. The clusterless open conformation of IscU adheres to the hydrophobic surface of polystyrene nanobeads, as observed for other proteins. Increased accessibility of the ligand cysteines in bound IscU facilitates assembly of a 2Fe2S cluster, and the cluster-bearing structured form of IscU does not interact with the nanobeads, thus ensuring turnover. The dependence of accelerated cluster assembly on the nanobeads concentration pointed to steric and crowding effects as for promoting cluster formation, and confirms the requirement for structural flexibility of IscU