Whey protein mouth drying influenced by thermal denaturation

Whey proteins are becoming an increasingly popular functional food ingredient. There are, however, sensory properties associated with whey protein beverages that may hinder the consumption of quantities sufficient to gain the desired nutritional benefits. One such property is mouth drying. The influence of protein structure on the mouthfeel properties of milk proteins has been previously reported. This paper investigates the effect of thermal denaturation of whey proteins on physicochemical properties (viscosity, particle size, zeta-potential, pH), and relates this to the observed sensory properties measured by qualitative descriptive analysis and sequential profiling. Mouthcoating, drying and chalky attributes built up over repeated consumption, with higher intensities for samples subjected to longer heating times (p < 0.05). Viscosity, pH, and zeta-potential were found to be similar for all samples, however particle size increased with longer heating times. As the pH of all samples was close to neutral, this implies that neither the precipitation of whey proteins at low pH, nor their acidity, as reported in previous literature, can be the drying mechanisms in this case. The increase in mouth drying with increased heating time suggests that protein denaturation is a contributing factor and a possible mucoadhesive mechanism is discussed.

The adrenal secretory serine protease AsP is a short secretory isoform of the transmembrane airway trypsin-like protease

To further elucidate the role of proteases capable of cleaving N-terminal proopiomelanocortin (N-POMC)-derived peptides, we have cloned two cDNAs encoding isoforms of the airway trypsin-like protease (AT) from mouse (MAT) and rat ( RAT), respectively. The open reading frames comprise 417 amino acids (aa) and 279 aa. The mouse AT gene was located at chromosome 5E1 and contains 10 exons. The longer isoform, which we designated MAT1 and RAT1, has a simple type II transmembrane protein structure, consisting of a short cytoplasmic domain, a transmembrane domain, a SEA (63-kDa sea urchin sperm protein, enteropeptidase, agrin) module, and a serine protease domain. The human homolog of MAT1 and RAT1 is the human AT ( HAT). The shorter isoform, designated MAT2 and RAT2, which contains an alternative N terminus, was formerly described in the rat as adrenal secretory serine protease (AsP) and has been shown to be involved in the processing of N-POMC-derived peptides. In contrast to the long isoform, neither MAT2 and RAT2 ( AsP) contain a transmembrane domain nor a SEA domain but an N-terminal signal peptide to direct the enzyme to the secretory pathway. The C terminus, covering the catalytic triad, is identical in both isoforms. Immunohistochemically, MAT/RAT was predominantly expressed in tissues of the upper gastrointestinal and the respiratory tract - but also in the adrenal gland. Moreover, isoform-specific RT-PCR and quantitative PCR analysis revealed a complex expression pattern of the two isoforms with differences between mice and rats. These findings indicate a multifunctional role of these proteases beyond adrenal proliferation.

Fermentation of heated gluten systems by gut microflora

The Maillard reaction causes changes to protein structure and occurs in foods mainly during thermal treatment. Melanoidins, the final products of the Maillard reaction, may enter the gastrointestinal tract, which is populated by different species of bacteria. In this study, melanoidins were prepared from gluten and glucose. Their effect on the growth of faecal bacteria was determined in culture with genotype and phenotype probes to identify the different species involved. Analysis of peptic and tryptic digests showed that low molecular mass products are formed from the degradation of melanoidins. Results showed a change in the growth of bacteria. This in vitro study demonstrated that melanoidins, prepared from gluten and glucose, affect the growth of the gut microflora.

Proteomic analysis of arginine adducts on glyoxal-modified ribonuclease

Accumulation of advanced glycation end-products (AGEs) on proteins is associated with the development of diabetic complications. Although the overall extent of modification of protein by AGEs is limited, localization of these modifications at a few critical sites might have a significant effect on protein structure and function. In the present study, we describe the sites of modification of RNase by glyoxal under physiological conditions. Arg(39) and Arg(85), which are closest to the active site of the enzyme, were identified as the primary sites of formation of the glyoxal-derived dihydroxyimidazolidine and hydroimidazolone adducts. Lower amounts of modification were detected at Arg(10), while Arg(33) appeared to be unmodified. We conclude that dihydroxyimidazolidine adducts are the primary products of modification of protein by glyoxal, that Arg(39) and Arg(85) are the primary sites of modification of RNase by glyoxal, and that modification of arginine residues during Maillard reactions of proteins is a highly selective process.

The use of proteomics to identify novel therapeutic targets for the treatment of disease

The completion of the Human Genome Project has revealed a multitude of potential avenues for the identification of therapeutic targets. Extensive sequence information enables the identification of novel genes but does not facilitate a thorough understanding of how changes in gene expression control the molecular mechanisms underlying the development and regulation of a cell or the progression of disease. Proteomics encompasses the study of proteins expressed by a population of cells, and evaluates changes in protein expression, post-translational modifications, protein interactions, protein structure and splice variants, all of which are imperative for a complete understanding of protein function within the cell. From the outset, proteomics has been used to compare the protein profiles of cells in healthy and diseased states and as such can be used to identify proteins associated with disease development and progression. These candidate proteins might provide novel targets for new therapeutic agents or aid the development of assays for disease biomarkers. This review provides an overview of the current proteomic techniques available and focuses on their application in the search for novel therapeutic targets for the treatment of disease.

Effect of high-hydrostatic pressure and pH treatments on the emulsification properties of gum arabic

This study investigated the emulsification properties of the native gums and those treated at high pressure (800 MPa) both at their “natural” pH (4.49 and 4.58, respectively) and under “acidic and basic” pH (2.8 and 8.0) conditions. The emulsification behaviour of KLTA gum was found to be superior to that of the GCA gum. High pressure and pH treatment changed the emulsification properties of both gums. The acidic amino acids in gum arabic were shown to play an important role in their emulsification behaviour, and mechanisms of emulsification for the two gums were suggested to be different. The highly “branched” nature of the carbohydrate in GCA gum was also thought to be responsible for the “spreading” of droplet size distributions observed. Coomassie brilliant blue binding was used to indicate conformational changes in protein structure and Ellman’s assay was used to estimate any changes in levels of free thiols present.

The DISOPRED server for the prediction of protein disorder

Dynamically disordered regions appear to be relatively abundant in eukaryotic proteomes. The DISOPRED server allows users to submit a protein sequence, and returns a probability estimate of each residue in the sequence being disordered. The results are sent in both plain text and graphical formats, and the server can also supply predictions of secondary structure to provide further structural information.

Rapid protein domain assignment from amino acid sequence using predicted secondary structure

The elucidation of the domain content of a given protein sequence in the absence of determined structure or significant sequence homology to known domains is an important problem in structural biology. Here we address how successfully the delineation of continuous domains can be accomplished in the absence of sequence homology using simple baseline methods, an existing prediction algorithm (Domain Guess by Size), and a newly developed method (DomSSEA). The study was undertaken with a view to measuring the usefulness of these prediction methods in terms of their application to fully automatic domain assignment. Thus, the sensitivity of each domain assignment method was measured by calculating the number of correctly assigned top scoring predictions. We have implemented a new continuous domain identification method using the alignment of predicted secondary structures of target sequences against observed secondary structures of chains with known domain boundaries as assigned by Class Architecture Topology Homology (CATH). Taking top predictions only, the success rate of the method in correctly assigning domain number to the representative chain set is 73.3%. The top prediction for domain number and location of domain boundaries was correct for 24% of the multidomain set (±20 residues). These results have been put into context in relation to the results obtained from the other prediction methods assessed

Intrinsic disorder prediction from the analysis of multiple protein fold recognition models

Motivation: Intrinsic protein disorder is functionally implicated in numerous biological roles and is, therefore, ubiquitous in proteins from all three kingdoms of life. Determining the disordered regions in proteins presents a challenge for experimental methods and so recently there has been much focus on the development of improved predictive methods. In this article, a novel technique for disorder prediction, called DISOclust, is described, which is based on the analysis of multiple protein fold recognition models. The DISOclust method is rigorously benchmarked against the top.ve methods from the CASP7 experiment. In addition, the optimal consensus of the tested methods is determined and the added value from each method is quantified. Results: The DISOclust method is shown to add the most value to a simple consensus of methods, even in the absence of target sequence homology to known structures. A simple consensus of methods that includes DISOclust can significantly outperform all of the previous individual methods tested.

Retention time prediction and protein identification

Proteins are commonly identified through enzymatic digestion and generation of short sequence tags or fingerprints of peptide masses by mass spectrometry. Separation methods, such as liquid chromatography and electrophoresis, are often used to fractionate complex protein or peptide mixtures and these separations also provide information on the different species, such as molecular weight and isoelectric point from electrophoresis and hydrophobicity in reversed-phase chromatography. These are also properties that can be predicted from amino acid sequences derived from genomic sequences and used in protein identification. This chapter reviews recently introduced methods based on retention time prediction to extract information from chromatographic separations and the applications to protein identification in organisms with small and large genomes. Novel data on retention time prediction of posttranslationally modified peptides is also presented.

Nuclear magnetic resonance structure of the N-terminal domain of nonstructural protein 3 from the severe acute respiratory syndrome coronavirus

This paper describes the structure determination of nsp3a, the N-terminal domain of the severe acute respiratory syndrome coronavirus (SARS-CoV) nonstructural protein 3. nsp3a exhibits a ubiquitin-like globular fold of residues 1 to 112 and a flexibly extended glutamic acid-rich domain of residues 113 to 183. In addition to the four beta-strands and two alpha-helices that are common to ubiquitin-like folds, the globular domain of nsp3a contains two short helices representing a feature that has not previously been observed in these proteins. Nuclear magnetic resonance chemical shift perturbations showed that these unique structural elements are involved in interactions with single-stranded RNA. Structural similarities with proteins involved in various cell-signaling pathways indicate possible roles of nsp3a in viral infection and persistence.

The measurement of maternal plasma corticotropin-releasing factor (CRF) and CRF-binding protein improves the early prediction of preeclampsia

In the present study we measured maternal plasma concentrations of two placental neurohormones, corticotropin-releasing factor (CRF) and CRF-binding protein (CRF-BP), in 58 at-risk pregnant women consecutively enrolled between 28 and 29 wk of pregnancy to evaluate whether their evaluation may predict third trimester-onset preeclampsia ( PE). The statistical significance was assessed by t test. The cut-off points for defining altered CRF and CRF-BP levels for prediction of PE were chosen by receiving operator characteristics curve analysis, and the probability of developing PE was calculated for several combinations of hormone testing results. CRF and CRF-BP levels were significantly ( both P < 0.0001) higher and lower, respectively, in the patients (n = 20) who later developed PE than in those who did not present PE at follow-up. CRF at the cut-off 425.95 pmol/liter achieved a sensitivity of 94.8% and a specificity of 96.9%, whereas CRF-BP at the cut-off 125.8 nmol/liter combined a sensitivity of 92.5% and a specificity of 82.5% as single markers for prediction of PE. The probability of PE was 34.5% in the whole study population, 93.75% when both CRF and CRF-BP levels were changed, and 0% if both hormone markers were unaltered. The measurement of CRF and CRF-BP levels may add significant prognostic information for predicting PE in at-risk pregnant women.

The structure of echovirus type 12 bound to a two-domain fragment of its cellular attachment protein decay-accelerating factor (CD 55)

Echovirus type 12 (EV12), an enterovirus of the Picornaviridae family, uses the complement regulator, decay-accelerating factor (DAF, CD55) as a cellular receptor. We have calculated a three-dimensional reconstruction of EV12 bound to a fragment of DAF, consisting of short consensus repeat domains 3 and 4, from cryo-negative stain electron microscopy data (EMD #1057). This shows that, as for an earlier reconstruction of the related echovirus type 7 bound to DAF, attachment is not within the viral canyon but occurs close to the two-fold symmetry axes. Despite this general similarity, our reconstruction reveals a receptor interaction that is quite different from that observed for EV7. Fitting of the crystallographic co-ordinates for DAF34 and EV11 into the reconstruction shows a close agreement between the crystal structure of the receptor fragment and the density for the virus-bound receptor, allowing unambiguous positioning of the receptor with respect to the virion (PDB #1UPN). Our finding that the mode of virus-receptor interaction in EV12 is distinct from that seen for EV7 raises interesting questions regarding the evolution and biological significance of the DAF-binding phenotype in these viruses.

Automatic prediction of functional site regions in low-resolution protein structures

World-wide structural genomics initiatives are rapidly accumulating structures for which limited functional information is available. Additionally, state-of-the art structural prediction programs are now capable of generating at least low resolution structural models of target proteins. Accurate detection and classification of functional sites within both solved and modelled protein structures therefore represents an important challenge. We present a fully automatic site detection method, FuncSite, that uses neural network classifiers to predict the location and type of functionally important sites in protein structures. The method is designed primarily to require only backbone residue positions without the need for specific side-chain atoms to be present. In order to highlight effective site detection in low resolution structural models FuncSite was used to screen model proteins generated using mGenTHREADER on a set of newly released structures. We found effective metal site detection even for moderate quality protein models illustrating the robustness of the method.