Influence of Cortisol on the Fibril Formation Kinetics of Aß42 Peptide: A Multi-Technical Approach.

Amyloid-ß peptide (Aß) aggregates are known to be correlated with pathological neurodegenerative diseases. The fibril formation process of such peptides in solution is influenced by several factors, such as the ionic strength of the buffer, concentration, pH, and presence of other molecules, just to mention a few. In this paper, we report a detailed analysis of in vitro Aß42 fibril formation in the presence of cortisol at different relative concentrations. The thioflavin T fluorescence assay allowed us to monitor the fibril formation kinetics, while a morphological characterization of the aggregates was obtained by atomic force microscopy. Moreover, infrared absorption spectroscopy was exploited to investigate the secondary structure changes along the fibril formation path. Molecular dynamics calculations allowed us to understand the intermolecular interactions with cortisol. The combined results demonstrated the influence of cortisol on the fibril formation process: indeed, at cortisol-Aß42 concentration ratio (ρ) close to 0.1 a faster organization of Aß42 fragments into fibrils is promoted, while for ρ = 1 the formation of fibrils is completely inhibited.

Legume Proteins and Peptides as Compounds in Nutraceuticals: A Structural Basis for Dietary Health Effects.

In the current climate of food security, quality aspects of legume crops have primary market economic and health impact. Legume proteins and peptides have been discovered to have a role far beyond supplying amino acids for growth and maintenance of body tissues. Several proteins (enzymatic inhibitors, lectins, storage globulins) and peptides derived from them (lunasin, hydrophobic peptides) have shown anticarcinogenic, hypocholesterolemic, glucose-lowering, antioxidant, antimicrobial, and immunostimulant properties. Further understanding of how structural features of legume proteins affect in vivo digestion and production of bioactive sequences represents a key step in the valorization of nutraceutical potentiality of legume proteins and peptides derived from them. In this work, the relationship between structure and bioavailability of protein and peptides are reviewed and discussed.

Normal mode calculation and infrared spectroscopy of proteins in water solution: Relationship between amide I transition dipole strength and secondary structure.

Dipole Strength (DS) of the amides has gained a renewed interest in chemical physics since it provides an important tool to disclose the on-site vibrational energy distributions. Apart from earlier experimental efforts on polypeptides, little is still known about DS in complex proteins. We accurately measured the Fourier Transform Infrared absorption spectra of nine proteins in water solution obtaining their Molar Extinction Coefficient in the amide I and II spectral region. Our results show that the amide I DS value depends on the protein secondary structure, being that of the α-rich and unstructured proteins lower by a factor of 2 than that of the ß-rich proteins. The average DS for amino acids in α and ß secondary structures confirms this finding. Normal Mode calculation and Molecular Dynamics were performed and used as tools for data analysis and interpretation. The present outcomes corroborate the hypothesis that antiparallel ß-sheet environment is more prone to delocalize the on-site CO stretching vibration through coupling mechanisms between carbonyl groups, whereas α-helix structures are energetically less stable to permit vibrational mode delocalization.

The Puzzling Problem of Cardiolipin Membrane-Cytochrome c Interactions: A Combined Infrared and Fluorescence Study.

The interaction of cytochrome c (cyt c) with natural and synthetic membranes is known to be a complex phenomenon, involving both protein and lipid conformational changes. In this paper, we combined infrared and fluorescence spectroscopy to study the structural transformation occurring to the lipid network of cardiolipin-containing large unilamellar vesicles (LUVs). The data, collected at increasing protein/lipid ratio, demonstrate the existence of a multi-phase process, which is characterized by: (i) the interaction of cyt c with the lipid polar heads; (ii) the lipid anchorage of the protein on the membrane surface; and (iii) a long-distance order/disorder transition of the cardiolipin acyl chains. Such effects have been quantitatively interpreted introducing specific order parameters and discussed in the frame of the models on cyt c activity reported in literature.

Cytochrome c aggregation: A dataset at and far from the isoelectric point.

We present SEM, ThT fluorescence and circular dichroism (CD) data of amyloidogenic aggregates of cytochrome c (cyt c).This protein is of outmost relevance in many biochemical processes, such as respiratory chain in mitochondria and cells apoptosis. The present data focus on polymorphism of the protein aggregates obtained at the isoelectric point (IP) and by changing the environmental pH above and below the IP, the protein concentration and the base. The SEM images provide evidence for a large variety of structures, depending on the pH and on protein concentration: mature amyloid fibrils and overstructured platelets are distinguishable in the aggregates below IP, and relatively high cyt c concentration, whereas inhomogeneous amyloid formations are observed above it. At pH 10, i.e. close to IP, only characteristic protein particulates at the micrometric scale are observed. SEM and Fluorescence data have been acquired in dried drops of protein solution, prepared in different bases: TRIS-HCl, at the different pH values, or NaOH (pH 13). Along with this, at relatively low cyt c concentration compact layered structures are visible below the IP, though still made of a thin fibrils reticulate, whereas above the IP, also at low cyt c concentration, granulates structures are present, merging into compact layer, alongside with platelets and mature fibers. These areas are characterized by diffuse ThT-fluorescence and typical fibrils. The loss of the predominant alpha helix secondary structure was verified by CD spectra. Besides the intrinsic scientific relevance, this data collection provides a set of images useful for spectroscopists to discriminate among different morphologic protein formations and suggests pathways for the achievement of different kinds of cytochrome c aggregates. These data are add-ons of the paper published in the International Journal of Biomacromolecules, 138 (2019) 106-115, https://doi.org/10.1016/j.ijbiomac.2019.07.060.

Achieving cytochrome c fibril/aggregate control towards micro-platelets and micro-fibers by tuning pH and protein concentration: A combined morphological and spectroscopic analysis.

Cytochrome c is a metalloprotein with primary physiological functions in the respiratory chain and in the regulation of cell death signals. Investigating the mechanisms leading to cytochrome c fibril formation is of primary importance for understanding its misfunctioning and, in a wider perspective, for its technologic applications in the field of bio-nanoscience. In this work, we analyzed the morphology and the spectroscopic properties of cytochrome c aggregates, combining the outcomes from electron microscopy, fluorescence, infrared and Raman spectroscopies and making use of statistical tools for the data analysis. The morphology scenario is quite complex, as it points out the presence of aggregates in the shape of platelets as well as fibers at micrometric scale. By infrared and Raman spectroscopy we analyzed the secondary and tertiary structures of unordered aggregates and fibrils, drawing a pathway for their formation at the timescales from tents to hundreds of minutes. Dependence of the fibrillation route on environmental pH, above and below the isoelectric point, and on protein concentration has also been explored. We found that it is possible to direct the process towards the formation of superstructures with different morphologies and different sizes along with fibrils, after destabilization of the native fold and the formation of ß-sheet rich structures. A different mechanism characterizes aggregate/fibril elongation of cyt c in Tris-HCl, in comparison with NaOH environment.

Hydrogen-Deuterium exchange kinetics in ß-lactoglobulin (-)-epicatechin complexes studied by FTIR spectroscopy.

Hydrogen-Deuterium exchange kinetics of ß-lactoglobulin and ß-lactoglobulin (-)-epicatechin solutions has been investigated through the analysis of the amide I absorption band at 1650cm-1 in the FTIR spectrum. H-D substitution in NH amides and residues of the protein results in a slight red-shift and in intensity changes of the amide I components: either these effects have been inspected in the framework of the Principal Components Analysis methods. The present analysis allowed to unveil three H-D kinetics at the timescale of the oligomeric fluctuations of the protein. A fast mechanism (lifetime from 5 to 10min) can be ascribed to the dynamics of protein oligomers and aggregates at the scale of the quaternary structure variations, and it is not observed in the complexes ß-lactoglobulin (-)-epicatechin. The other slowest kinetics, whose lifetimes are in the range 1-10h, are here associated to dynamics of high-molecular weight complexes that hamper the proton exchange. The role of (-)-epicatechin as an enhancer of the formation of stable high-molecular weight aggregates from ß-lactoglobulin is also discussed by comparison of the lifetimes at different protein concentrations.

Epicatechin-induced conformational changes in ß-lactoglobulin B monitored by FT-IR spectroscopy.

ABSTRACT: The interaction between whey carrier protein ß-lactoglobulin B and (-)-epicatechin, a major dietary flavonoid with a wide range of health-promoting biological activities, was investigated by Fourier transform infrared spectroscopy in physiological conditions. Amide I spectra of epicatechin - ß-lactoglobulin complexes, in D2O buffer solutions, pD= 6.8, at molar ratios from 0.5:1 to 15:1, were measured by using a cell device specifically created. Changes in secondary structure elements at increasing epicatechin concentrations were quantified. Two different trends were observed for the intensities of ß-sheet, random coil, and side chain contributions. At molar ratios ≤2 the ß-exposed strand contributions (1625 cm(-1)) increased at the expence of the ß-antiparallel sheet band (1637 cm(-1)). At molar ratios >2 the intensities of both ß structures slightly decreased. The same behaviour was observed for the side chain contributions (band around 1610 ÷ 1620 cm(-1)). In addition, a conformational transition to a slightly opened structure, followed by aggregate formation at the highest molar ratios, were revealed. The results suggest that binding of epicatechin to ß-lactoglobulin in physiological conditions occurs at the surface of the protein molecule, resulting in protein dissociation at molar ratios ≤2 with minor changes in secondary structure. This finding provides further evidence for the possibility of successful use of the protein as a carrier of flavonoids, epicatechin included.

Bioactive molecules and antioxidant activity in durum wheat grains and related millstream fractions.

The present study was performed to evaluate the distribution of phytochemicals in wheat grains and millstreams and the contribution of these compounds to the antioxidant activity. Bioactive molecules, although related to genotype, were highly affected by the milling procedure applied. Carotenoids were unevenly distributed in the kernel and lutein resulted to be the main carotenoid in all samples considered. The WSB method was confirmed to be a useful tool to detect yellow color intensity of semolina but it was not suitable to get information on the true content of carotenoids of the sample. Water-methanol soluble components showed antioxidant activity which decreased from the outer to the inner parts of the kernel. Chloroform-methanol soluble compounds showed generally negligible or negative antioxidant activity due to the inadequate amount of antioxidants unable to counteract oxidative processes already in progress.

7S globulins from Phaseolus vulgaris L.: Impact of structural aspects on the nutritional quality.

7S globulins were extracted from common bean (Phaseolus vulgaris L.) seeds and characterized. SDS-PAGE showed major bands corresponding to the phaseolin subunits (43-53 kDa). An amino acid analysis indicated that, in spite of the limited amounts of sulphur amino acids and tryptophan, the globulins contained very high levels of essential amino acids. The protein solubility profiles of native and denatured (120 degrees C for 20 min) 7S globulins in water and in 0.5 M NaCl showed that NaCl had a limited effect on increasing the solubility of either the native or denatured proteins. The in vivo small intestinal digestibility of the 7S globulins was 90%, this being decreased to 86% after a thermal treatment. Fourier transform infrared spectroscopy revealed a high content of beta-sheet and beta-turn structures, together with a contribution at 1687 cm(-1) that was assigned to intramolecular beta-sheets. These features are diagnostic of a high propensity to irreversible aggregation that may be related to an adverse effect on the protein quality.

Absorption of quercetin and rutin in rat small intestine.

BACKGROUND/AIMS: Dietary antioxidant flavonoids, especially flavonols, are ubiquitous constituents of plant foods with potential health-promoting effects. However, the actual bioavailability of these compounds in vivo, especially in the prevalent glycosidic form, remains a controversial point in making an assessment of their biological importance. Thus, absorption of quercetin and rutin in the small intestine has been determined. METHODS: The bioavailability of quercetin and rutin (quercetin-3-rutinoside) was assessed in vivo, with single-meal experiments with rats and by an in vitro method with ligated loops of rat small intestine. The amount of quercetin or rutin in the plasma of rats or in the lumen of each intestinal segment was assayed by maximum absorption in the UV/VIS optical spectrum as was the amount of compound that had crossed the gut wall into the incubation buffer. In addition, uptake of [14C]quercetin was tested in vitro. RESULTS: Absorption of both quercetin and rutin from the small intestine of rat was evident. However, rutin appeared to be absorbed more slowly than quercetin. Experiments with [14C]quercetin showed that only 1.5% quercetin crossed the gut wall in vitro and more than half of the total quercetin was bound to the small intestinal tissue. CONCLUSIONS: Both quercetin and rutin can attach to and traverse the small intestine of the rat. Binding of flavonoids to the intestinal wall components may however greatly limit their absorption from the small intestine.

Modulation of antioxidant compounds in organic vs conventional fruit (peach, Prunus persica L., and pear, Pyrus communis L.).

Despite the increasing interest in organic products, knowledge about how different levels of fertilization affect nutritionally relevant components is still limited. The concentration of polyphenols and the activity of polyphenoloxidase (PPO), together with the content in ascorbic acid, citric acid, and alpha- and gamma-tocopherol, were assayed in conventional and organic peach (Prunus persica L., cv. Regina bianca) and pear (Pyrus communis L., cv. Williams). 2-Thiobarbituric acid reactive substances and the tocopherolquinone/alpha-tocopherol ratio were used as markers of oxidative damage in fruits. A parallel increase in polyphenol content and PPO activity of organic peach and pear as compared with the corresponding conventional samples was found. Ascorbic and citric acids were higher in organic than conventional peaches, whereas alpha-tocopherol was increased in organic pear. The concentration of oxidation products in organic samples of both fruits was comparable to that of the corresponding conventional ones. These data provide evidence that an improvement in the antioxidant defense system of the plant occurred as a consequence of the organic cultivation practice. This is likely to exert protection against damage of fruit when grown in the absence of pesticides.