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Caseins are a diverse family of intrinsically disordered proteins present in the milks of all mammals. A property common to two cow paralogues, α

Monitoring SARS-CoV-2 in wastewater is a valuable approach to track COVID-19 transmission. Designing wastewater surveillance (WWS) with representative sampling sites and quantifiable results requires knowledge of the sewerage system and virus fate and transport. We developed a multi-level WWS system to track COVID-19 in Atlanta using an adaptive nested sampling strategy. From March 2021 to April 2022, 868 wastewater samples were collected from influent lines to wastewater treatment facilities and upstream community manholes. Variations in SARS-CoV-2 concentrations in influent line samples preceded similar variations in numbers of reported COVID-19 cases in the corresponding catchment areas. Community sites under nested sampling represented mutually-exclusive catchment areas. Community sites with high SARS-CoV-2 detection rates in wastewater covered high COVID-19 incidence areas, and adaptive sampling enabled identification and tracing of COVID-19 hotspots. This study demonstrates how a well-designed WWS provides actionable information including early warning of surges in cases and identification of disease hotspots.

Bovine milk alpha(S2)-casein, an intrinsically disordered protein, readily forms amyloid fibrils in vitro and is implicated in the formation of amyloid fibril deposits in mammary tissue. Its two cysteine residues participate in the formation of either intra- or intermolecular disulphide bonds, generating monomer and dimer species. X-ray solution scattering measurements indicated that both forms of the protein adopt large, spherical oligomers at 20 degrees C. Upon incubation at 37 degrees C, the disulphide-linked dimer showed a significantly greater propensity to form amyloid fibrils than its monomeric counterpart. Thioflavin T fluorescence, circular dichroism and infrared spectra were consistent with one or both of the dimer isomers (in a parallel or antiparallel arrangement) being predisposed toward an ordered, amyloid-like structure. Limited proteolysis experiments indicated that the region from Ala(81) to Lys(113) is incorporated into the fibril core, implying that this region, which is predicted by several algorithms to be amyloidogenic, initiates fibril formation of alpha(S2)-casein. The partial conservation of the cysteine motif and the frequent occurrence of disulphide-linked dimers in mammalian milks despite the associated risk of mammary amyloidosis, suggest that the dimeric conformation of alpha(S2)-casein is a functional, yet amyloidogenic, structure. Refereed/Peer-reviewed

Caseins (αS1, αS2, β and κ) are the main protein fraction of bovine milk. Together with nanoclusters of amorphous calcium phosphate (CaP) and divalent cations, they combine to form a polydisperse distribution of particles called casein micelles. A casein micelle model is proposed which is consistent with the way in which intrinsically disordered proteins interact through predominantly polar, short, linear, motifs. Using the model, an expression is derived for the size distribution of casein micelles formed when caseins bind to the CaP nanoclusters and the complexes further associate with each other and the remaining mixture of free caseins. The result is a refined coat-core model in which the core is formed mainly by the nanocluster complexes and the coat is formed exclusively by the free caseins. Example calculations of the size distribution and surface composition of an average bovine milk are compared with experiment. The average size, size distribution and surface composition of the micelles is shown to depend on the affinity of the nanocluster complexes for each other in competition with their affinity for free caseins, and on the concentrations of free caseins, calcium ions and other salts in the continuous phase.

Bovine milk α

Biofluids that contain stable calcium phosphate nanoclusters sequestered by phosphopeptides make it possible for soft and hard tissues to co-exist in the same organism with relative ease. The stability diagram of a solution of nanocluster complexes shows how the minimum concentration of phosphopeptide needed for stability increases with pH. In the stable region, amorphous calcium phosphate cannot precipitate. Nevertheless, if the solution is brought into contact with hydroxyapatite, the crystalline phase will grow at the expense of the nanocluster complexes. The physico-chemical principles governing the formation, composition, size, structure, and stability of the complexes are described. Examples are given of complexes formed by casein, osteopontin, and recombinant phosphopeptides. Application of these principles and properties to blood serum, milk, urine, and resting saliva is described to show that under physiological conditions they are in the stable region of their stability diagram and so cannot cause soft tissue calcification. Stimulated saliva, however, is in the metastable region, consistent with its role in tooth remineralization. Destabilization of biofluids, with consequential ill-effects, can occur when there is a failure of homeostasis, such as an increase in pH without a balancing increase in the concentration of sequestering phosphopeptides.

Increasing dietary calcium has been suggested to have a range of health benefits, such as reducing the risk of osteoporosis and hypertension. However, producing calcium-fortified products is challenging due to the destabilizing effect caused by added calcium. We provide new data on the effect of adding either calcium gluconate or calcium lactate at up to 50 mM on the partition of salts and the structure and solubility of micellar calcium phosphate (MCP). The empirical chemical formula of the MCP in milk with added calcium was Ca(HPO4)0.6(PO4)0.267, similar to that previously reported for the MCP in native bovine casein micelles. Ion equilibria calculations showed that the solubility of the MCP was decreased as measured by an increase in negative logarithm of the solubility constant (pKS) from 6.8 to 7.3 ± 0.1 and 7.5 ± 0.1 for milk with added calcium gluconate and calcium lactate, respectively. No substantial change in the amorphous structure of the MCP was observed by either X-ray powder diffraction or infrared spectroscopy of dried casein micelles as a result of added calcium. The conclusion is that the added calcium caused an increase in the concentration of the MCP and decreased its solubility without changing its amorphous structure or chemical composition.

The white appearance of skim milk is due to strong light scattering by colloidal particles called casein micelles. Bovine casein micelles comprise expressed proteins from four casein genes together with significant fractions of the total calcium, inorganic phosphate, magnesium and citrate ions in the milk. Thus, the milk salts are partitioned between the casein micelles, where they are mostly in the form of nanoclusters of an amorphous calcium phosphate sequestered by caseins through their phosphorylated residues, with the remainder in the continuous phase. Previously, a salt partition calculation was made assuming that the nanoclusters are sequestered only by short, highly phosphorylated casein sequences, sometimes called phosphate centres. Three of the four caseins have a proportion of their phosphorylated residues in either one or two phosphate centres and these were proposed to react with the nanoclusters equally and independently. An improved model of the partition of caseins and salts in milk is described in which all the phosphorylated residues in competent caseins act together to bind to and sequester the nanoclusters. The new model has been applied to results from a recent study of variation in salt and casein composition in the milk of individual cows. Compared to the previous model, it provides better agreement with experiment of the partition of caseins between free and bound states and equally good results for the partition of milk salts. In addition, new calculations are presented for the charge on individual caseins in their bound and free states.

The influence of transglutaminase (TG) treatment on the changes in the physico-chemical properties of reconstituted skim milk (10 wt%) with different amounts of added ethylenediaminetetraacetic acid (EDTA) (0–50 mM) was studied. The partition of salts (Ca, Mg, K, Na and inorganic phosphate) was determined experimentally by atomic absorption and emission spectroscopy, colorimetry and 31P-nuclear magnetic resonance spectroscopy, and calculated with a model of the salt partition in milk based on equilibrium thermodynamics. Hydrodynamic radius, skim milk viscosity and casein micelle medium resolution substructure were measured by dynamic light scattering, rheology, and small-angle X-ray scattering (SAXS), respectively. TG treatment of milk did not influence the partition of salts upon the addition of EDTA as TG crosslinking is unlikely to disrupt the phosphate centre motifs on the short exon-encoded amino acid sequences on caseins. However, TG treatment prevented full dissociation of casein micelles when >15 mM EDTA was added, and induced higher viscosity increase than TG-untreated milk upon addition of EDTA. In accordance with the particle sizing results, SAXS demonstrated that TG treatment reduced the extent of EDTA-induced micellar disruption, as compared to milk without TG treatment.

Caseins are a group of closely related intrinsically disordered proteins (IDPs), best known for their occurrence in milk as stable, polydisperse, roughly spherical, amorphous particles, typically containing thousands of protein chains and hundreds of nanoclusters of calcium phosphate. The particles are called casein micelles though their structure bears no resemblance to detergent micelles. Caseins have an open and flexible conformation with a preponderance of poly-l-proline II secondary structure and hence cannot be described as hydrophobic proteins. Individually, and in combination, they associate through polar and non-polar interactions to form polydisperse fuzzy complexes (including the native casein micelle) while retaining their hydrated and flexible conformation to a large degree. Like many other IDPs, caseins are prone to form cytotoxic amyloid fibrils. However, they are also highly effective molecular chaperones so that a mixture of different caseins can form fuzzy complexes that are often self-limiting in size and, within which, amyloid fibril formation is suppressed. The remarkable ability of caseins to sequester nanoclusters of calcium phosphate in stable complexes is due to their flexible conformation and multiply-phosphorylated short sequences. These features combine to form a dense peptide shell around the calcium phosphate making the core-shell complex thermodynamically stable, even at high calcium and phosphate concentrations. Thus, the casein micelle provides a readily digested, high calcium food for the neonate. It also preserves the functional properties of caseins as IDPs and protects the mammary gland against amyloid formation and pathological calcification, dysfunctional processes that would reduce the future reproductive success of the mother.

Milk caseins and dental amelogenins are intrinsically disordered proteins (IDPs) that associate with themselves and others. Paradoxically, they are also described as hydrophobic proteins, which is difficult to reconcile with a solvent-exposed conformation. We attempt to resolve this paradox. We show that caseins and amelogenins are not hydrophobic proteins but they are more hydrophobic than most IDPs. Remarkably, uncharged residues from different regions of these mature proteins have a nearly constant average hydropathy but these regions exhibit different charged residue frequencies. A novel sequence analysis method was developed to identify hydrophobic and order-promoting regions that would favor conformational collapse. We found that such regions were uncommon; most hydrophobic and order-promoting residues were adjacent to hydrophilic or disorder-promoting residues. A further reason why caseins and amelogenins do not collapse is their high proportion of disorder-promoting proline residues. We conclude that in these proteins the hydrophobic effect is not large enough to cause conformational collapse but it can contribute, along with polar interactions, to protein-protein interactions. This behaviour is similar to the interaction of the disordered N-terminal region of small heat-shock proteins with either themselves during oligomer formation or other, unfolding, proteins during chaperone action.

Primary structures of caseins from 20 species, including two monotremes and two marsupials, have been compared. Sequences of the mature proteins are very divergent, whereas variation in amino acid composition is mostly restricted to a range of disorder-promoting residues. The number and size of clusters of phosphorylation sites in the caseins is variable, blurring the boundaries between them. Casein polar tract sequences were found in all caseins, though of variable lengths, and are chiefly responsible for weak and dynamic interactions among the tangled web of peptide chains in the matrix of casein micelles. The interactions take the predominant form of backbone-to-backbone contacts rather than the sequence-specific side chain interactions of the hydrophobic effect. It is suggested that the dynamic casein micelle matrix be represented by an ensemble of interchanging structures with different types and degrees of inhomogeneity, influenced by solvent quality and other environmental factors.

Caseins are a group of closely related intrinsically disordered proteins (IDPs), best known for their occurrence in milk as stable, polydisperse, roughly spherical, amorphous particles, typically containing thousands of protein chains and hundreds of nanoclusters of calcium phosphate. The particles are called casein micelles though their structure bears no resemblance to detergent micelles. Caseins have an open and flexible conformation with a preponderance of poly-l-proline II secondary structure and hence cannot be described as hydrophobic proteins. Individually, and in combination, they associate through polar and non-polar interactions to form polydisperse fuzzy complexes (including the native casein micelle) while retaining their hydrated and flexible conformation to a large degree. Like many other IDPs, caseins are prone to form cytotoxic amyloid fibrils. However, they are also highly effective molecular chaperones so that a mixture of different caseins can form fuzzy complexes that are often self-limiting in size and, within which, amyloid fibril formation is suppressed. The remarkable ability of caseins to sequester nanoclusters of calcium phosphate in stable complexes is due to their flexible conformation and multiply-phosphorylated short sequences. These features combine to form a dense peptide shell around the calcium phosphate making the core-shell complex thermodynamically stable, even at high calcium and phosphate concentrations. Thus, the casein micelle provides a readily digested, high calcium food for the neonate. It also preserves the functional properties of caseins as IDPs and protects the mammary gland against amyloid formation and pathological calcification, dysfunctional processes that would reduce the future reproductive success of the mother.

Caseins were among the first proteins to be recognised as functional but unfolded. Many others are now known, providing better models of casein behaviour than either detergents or folded proteins. Caseins are members of a paralogous group of unfolded phosphoproteins, some of which share the ability to sequester amorphous calcium phosphate through phosphate centres. Non-covalent interactions of caseins can be through Pro- and Gln-rich sequences. Similar sequences in other unfolded proteins can also form open and highly hydrated structures such as gels, mucus and slimes. Many unfolded proteins, including κ- and αS2-caseins, can form amyloid fibrils under physiological conditions. The sequence-specific interactions that lead to fibrils can be reduced or eliminated by low specificity interactions among a mixture of caseins to yield, instead, amorphous aggregates. The size of amorphous whole casein aggregates is limited by the C-terminal half of κ-casein whose sequence resembles that of a soluble mucin.

Molecular chaperone proteins perform a diversity of roles inside and outside the cell. One of the most important is the stabilization of misfolding proteins to prevent their aggregation, a process that is potentially detrimental to cell viability. Diseases such as Alzheimer's, Parkinson's, and cataract are characterized by the accumulation of protein aggregates. In vivo, many proteins are metastable and therefore under mild destabilizing conditions have an inherent tendency to misfold, aggregate, and hence lose functionality. As a result, protein levels are tightly regulated inside and outside the cell. Protein homeostasis, or proteostasis, describes the network of biological pathways that ensures the proteome remains folded and functional. Proteostasis is a major factor in maintaining cell, tissue, and organismal viability. We have extensively investigated the structure and function of intra- and extracellular molecular chaperones that operate in an ATP-independent manner to stabilize proteins and prevent their misfolding and subsequent aggregation into amorphous particles or highly ordered amyloid fibrils. These types of chaperones are therefore crucial in maintaining proteostasis under normal and stress (e.g., elevated temperature) conditions. Despite their lack of sequence similarity, they exhibit many common features, i.e., extensive structural disorder, dynamism, malleability, heterogeneity, oligomerization, and similar mechanisms of chaperone action. In this Account, we concentrate on the chaperone roles of α-crystallins and caseins, the predominant proteins in the eye lens and milk, respectively. Intracellularly, the principal ATP-independent chaperones are the small heat-shock proteins (sHsps). In vivo, sHsps are the first line of defense in preventing intracellular protein aggregation. The lens proteins αA- and αB-crystallin are sHsps. They play a crucial role in maintaining solubility of the crystallins (including themselves) with age and hence in lens proteostasis and, ultimately, lens transparency. As there is little metabolic activity and no protein turnover in the lens, crystallins are very long lived proteins. Lens proteostasis is therefore very different to that in normal, metabolically active cells. Crystallins undergo extensive post-translational modification (PTM), including deamidation, racemization, phosphorylation, and truncation, which can alter their stability. Despite this, the lens remains transparent for tens of years, implying that lens proteostasis is intimately integrated with crystallin PTMs. Many PTMs do not significantly alter crystallin stability, solubility, and functionality, which thereby facilitates lens transparency. In the long term, however, extensive accumulation of crystallin PTMs leads to large-scale crystallin aggregation, lens opacification, and cataract formation. Extracellularly, various ATP-independent molecular chaperones exist that exhibit sHsp-like structural and functional features. For example, caseins, the major milk proteins, exhibit chaperone ability by inhibiting the amorphous and amyloid fibrillar aggregation of a diversity of destabilized proteins. Caseins maintain proteostasis within milk by preventing deleterious casein amyloid fibril formation via incorporation of thousands of individual caseins into an amorphous structure known as the casein micelle. Hundreds of nanoclusters of calcium phosphate are sequestered within each casein micelle through interactions with short, highly phosphorylated casein sequences. This results in a stable biofluid that contains a high concentration of potentially amyloidogenic caseins and concentrations of calcium and phosphate that can be far in excess of the solubility of calcium phosphate. Casein micelle formation therefore performs vital roles in neonatal nutrition and calcium homeostasis in the mammary gland.

International audience; The last decade established that the dynamic properties of the phosphoproteome are central to function and its modulation. The temporal dimension of phosphorylation effects remains nonetheless poorly understood, particularly for intrinsically disordered proteins. Osteopontin, selected for this study due to its key role in biomineralization, is expressed in many species and tissues to play a range of distinct roles. A notable property of highly phosphorylated isoforms of osteopontin is their ability to sequester nanoclusters of calcium phosphate to form a core-shell structure, in a fluid that is supersaturated but stable. In Biology, this process enables soft and hard tissues to coexist in the same organism with relative ease. Here, we extend our understanding of the effect of phosphorylation on a disordered protein, the recombinant human-like osteopontin rOPN. The solution structures of the phosphorylated and unphosphorylated rOPN were investigated by small-angle x-ray scattering and no significant changes were detected on the radius of gyration or maximum interatomic distance. The picosecond-to-nanosecond dynamics of the hydrated powders of the two rOPN forms were further compared by elastic and quasi-elastic incoherent neutron scattering. Phosphorylation was found to block some nanosecond side-chain motions while increasing the flexibility of other side chains on the faster timescale. Phosphorylation can thus selectively change the dynamic behavior of even a highly disordered protein such as osteopontin. Through such an effect on rOPN, phosphorylation can direct allosteric mechanisms, interactions with substrates, cofactors and, in this case, amorphous or crystalline biominerals

No abstract available.

A typical casein micelle contains thousands of casein molecules, most of which form thermodynamically stable complexes with nanoclusters of amorphous calcium phosphate. Like many other unfolded proteins, caseins have an actual or potential tendency to assemble into toxic amyloid fibrils, particularly at the high concentrations found in milk. Fibrils do not form in milk because an alternative aggregation pathway is followed that results in formation of the casein micelle. As a result of forming micelles, nutritious milk can be secreted and stored without causing either pathological calcification or amyloidosis of the mother's mammary tissue. The ability to sequester nanoclusters of amorphous calcium phosphate in a stable complex is not unique to caseins. It has been demonstrated using a number of noncasein secreted phosphoproteins and may be of general physiological importance in preventing calcification of other biofluids and soft tissues. Thus, competent noncasein phosphoproteins have similar patterns of phosphorylation and the same type of flexible, unfolded conformation as caseins. The ability to suppress amyloid fibril formation by forming an alternative amorphous aggregate is also not unique to caseins and underlies the action of molecular chaperones such as the small heat-shock proteins. The open structure of the protein matrix of casein micelles is fragile and easily perturbed by changes in its environment. Perturbations can cause the polypeptide chains to segregate into regions of greater and lesser density. As a result, the reliable determination of the native structure of casein micelles continues to be extremely challenging. The biological functions of caseins, such as their chaperone activity, are determined by their composition and flexible conformation and by how the casein polypeptide chains interact with each other. These same properties determine how caseins behave in the manufacture of many dairy products and how they can be used as functional ingredients in other foods.

In the native bovine casein micelle the calcium sensitive caseins (α(S1)-, α(S2)- and β-casein) sequester amorphous calcium phosphate in nanometer-sized clusters, whereas the calcium-insensitive κ-casein limits the growth of the micelle. In this paper, we further investigate the self-association of κ- and β-casein, which are two of the key proteins that control the substructure of the milk casein micelle, using neutron and light scattering techniques and cryogenic transmission electron microscopy. Results demonstrate that κ-casein can, apart from the known self-assembly, form amyloid-like fibrils already at temperatures of 25 °C when subject to agitation. This extended aggregation behavior of κ-casein is inhibited by β-casein, as reported by others. These findings have implications for the structure and stability of casein micelles. The neutron scattering data was used to gain information on the self-assembly structure of κ-casein. β-Casein shows similar self-association behavior as κ-casein, but unlike κ-casein, the self-association exhibits temperature dependence within the studied temperatures (6 and 25 °C). Here, we will discuss our extended study of the known self-assembly of casein in the context of the fibrillation of κ-casein.

In an early challenge to an aspect of Darwin’s theory of natural selection, Jackson Mivart contended that milk could not have evolved ‘from a scarcely nutritious fluid from an accidentally hypertrophied cutaneous gland’. The evolutionary change from a gland secretion to milk involves an increase in calcium and protein concentrations by up to 100- and 1000-fold, respectively. Even so, the challenge, we suggest, is not just a problem of scale. An increase in the concentrations of calcium and phosphate brings an increased risk of calcification of the secretory gland because calcium phosphate is highly insoluble. In addition, two of the four constituent milk casein proteins (κ and αS2) aggregate to produce toxic amyloid fibrils. It is proposed that both problems were solved through the cosecretion of ancestral β- and κ-caseins to form a stable amorphous aggregate of both proteins with sequestered amorphous calcium phosphate, that is, a primordial casein micelle. Evolutionarily, a gradual increase in the concentration of casein micelles could therefore produce progressively more nutritious fluids for the neonate without endangering the reproductive potential of the mother.

Neutron reflectometry was used to study the co-adsorption of calcium phosphate nanoclusters (CPN) and beta-casein at hydrophobized and hydrophilic silica-water interfaces. The structural characteristics of the adsorbed layer were determined from neutron reflectivity curves analysed with multi-layer optical models. We used a highly specific proteolytic enzyme, endoproteinase Asp-N in conjunction with a single neutron contrast to verify the model of the protein layer structure. The results showed that the calcium phosphate nanoclusters profoundly affected the rate of adsorption and structure of the interface compared to the adsorption of beta-casein alone and for the hydrophobic interface the effects depended on the point at which the nanoclusters were added. It is proposed that the nanoclusters become surface active because whole beta-casein molecules can replace one or more of the hydrophilic peptides in the shell of the nanoclusters. (C) 2010 Elsevier Ltd. All rights reserved. (Less)

The sequestration of calcium phosphate by unfolded proteins is fundamental to the stabilization of biofluids supersaturated with respect to hydroxyapatite, such as milk, blood or urine. The unfolded state of osteopontin (OPN) is thought to be a prerequisite for this activity, which leads to the formation of core–shell calcium phosphate nanoclusters. We report on the structures and dynamics of a native OPN peptide from bovine milk, studied by neutron spectroscopy and small-angle X-ray and neutron scattering. The effects of sequestration are quantified on the nanosecond– ångström resolution by elastic incoherent neutron scattering. The molecular fluctuations of the free phosphopeptide are in agreement with a highly flexible protein. An increased resilience to diffusive motions of OPN is corroborated by molecular fluctuations similar to those observed for globular proteins, yet retaining conformational flexibilities. The results bring insight into the modulation of the activity of OPN and phosphopeptides with a role in the control of biomineralization. The quantification of such effects provides an important handle for the future design of new peptides based on the dynamics–activity relationship.

There are abundant examples of nanoclusters and inorganic microcrystals in biology. Their study under physiologically relevant conditions remains challenging due to their heterogeneity, instability, and the requirements of sample preparation. Advantages of using neutron diffraction and contrast matching to characterize biomaterials are highlighted in this article. We have applied these and complementary techniques to search for nanocrystals within clusters of calcium phosphate sequestered by bovine phosphopeptides, derived from osteopontin or casein. The neutron diffraction patterns show broad features that could be consistent with hexagonal hydroxyapatite crystallites smaller than 18.9 A. Such nanocrystallites are, however, undetected by the complementary X-ray and FTIR data, collected on the same samples. The absence of a distinct diffraction pattern from the nanoclusters supports the generally accepted amorphous calcium phosphate structure of the mineral core.

The ability to discriminate successfully among cultures of all species of the Listeria genus by infrared spectroscopy in combination with canonical variate analysis was confirmed. The robustness of the method was demonstrated by showing that the separation of L. monocytogenes and L. grayi was hardly affected by variations in broth medium, incubation temperature, incubation time and cell washing procedure. Discrimination among 24 strains of L. monocytogenes according to serotype allowed two groups to be recognised, one comprising serotypes 4 and 4b and the other containing serotypes 1, 1/2b and 1/2c. When strain variation was included in the species discrimination model, the classification of all the L. monocytogenes strains was virtually 100% correct.

Caseins belong to a larger group of secreted calcium phosphate-binding phosphoproteins that have a natively unfolded conformation. Nearly all members of the group are involved in aspects of calcium phosphate biology and nearly all have recognition sites for phosphorylation by the Golgi protein kinase. In the caseins these are often close together in the primary structure, forming the so-called phosphate centres. Certain highly phosphorylated phosphopeptides derived from the calcium-sensitive caseins will combine with amorphous calcium phosphate to form defined chemical complexes called calcium phosphate nanoclusters. Both the substructure of casein micelles and the partition of salts in milk can be explained quantitatively by the ability of the calcium-sensitive caseins to sequester calcium phosphate and form nanocluster-like structures. A simple stability rule for milk can be derived by applying equilibrium thermodynamics to the process of calcium phosphate sequestration. In principle, the stability rule can be extended to problems of instability encountered in milk-processing operations and to the formulation of other types of high calcium foods.

β -Lactoglobulin ( β -LG) is the major whey protein of ruminant species and is also present in the milks of many, but not all, other species. Its amino-acid sequence and 3-dimensional structure show that it is a lipocalin, a widely diverse family, most of which bind small hydrophobic ligands and thus may act as specific transporters, as does serum retinol binding protein. Bovine β -LG binds a wide range of ligands, but this may not be the reason for its presence in milk. In reviewing the structure and physicochemical properties of the protein, we present the structures of the ligands cholesterol (at a resolution of 2.0A, R=0.221; R free = 0.295) and vitamin D 2 (at a resolution of 2.4A, R=0.212; R free = 0.297) each bound to the central binding cavity of bovine β -LG at pH 7.3. Neither ligand is fully visible in the electron density maps, and the less well-ordered regions are the polar end groups at the mouth of the binding site. In a separate experiment, a mercury ion was bound to the free Cys121 (at a resolution of 2.2A, R=0.218; R free = 0.288) in a way that transmitted a small structural change through Asp137 via Arg148 to the dimer interface. It is not clear if the known dissociation that arises from the reaction of β -LG with HgCl 2 results from this perturbation. In reviewing the structural studies that reveal the ligand binding sites for long-chain fatty acids, retinoids, and steroids, only the central location, common to all lipocalins so far examined, is occupied under the conditions used. We find that there is no crystallographic evidence of another ligand binding site in our crystals grown in approximately 1.3 M citrate, although low ionic strength studies in solution indicate the possible presence of at least one other low affinity site. The apparent ability of the binding site to accommodate a wide range of ligands may point to a possible physiological function. However, by considering the lipocalin family in general, and the species distribution of β -LG in particular, some speculation as to the physiological function can be made. β -Lactoglobulin has been reported as being implicated, inter alia, in hydrophobic ligand transport and uptake, enzyme regulation, and the neonatal acquisition of passive immunity. However, these functions do not appear to be consistent between species. Sequence comparisons among members of the lipocalin family reveal that glycodelin, found in the human endometrium during early pregnancy, is the most closely related to β -LG. Although the function of glycodelin is also unknown, it appears to have effects on the immune system and/or to be involved in differentiation. It is proposed that β -LG, over-expressed in the lactating mammary gland of many, but not all, species, is primarily an important source of amino acids for the offspring of those animals that produce it, but that this function arose by gene duplication from the physiologically essential glycodelin. The other functions that have been associated with β -LG in the neonate are, therefore, fortuitous.

Neutron reflectometry was used to investigate effects of calcium ions on the interfacial behavior of beta-casein at the silicon oxide-aqueous solution interface. The structural characteristics of the adsorbed layer were determined from reflectivity curves fitted to three- and two-layer optical models. The results showed that the presence of divalent calcium ions decreased the specific electrostatic adsorption affinity of the protein to silica compared with the calcium-free buffer system studied in an earlier work. In addition, it speeded up the adsorption suggesting that the slow kinetics seen in the calcium-free system are related to conformational adjustments of the beta-casein structure driven by the maximization of the number of positive charges on the polypeptide interacting with negative surface charges. In the calcium-free system, a dense inner layer resulted from this process, with cationic segments firmly bound to the negative surface, whereas in the presence of calcium, a less dense inner layer was formed. The difference in binding is also mirrored by the effects on the interfacial layer of a specific proteolytic enzyme, i.e., endoproteinase Asp-N. In the calcium-free case, an inner dense layer remained at the surface after the proteolytic cleavage of the polypeptide, whereas virtually nothing was left after enzymatic action in the presence of calcium ions.

This paper describes the possible usefulness of knowledge of the three-dimensional structure of milk proteins to dairy scientists. After a brief introduction of the available methodology and the structures of milk proteins that are already available, the structure of bovine β-lactoglobulin is used to illustrate the possible applications of the structure to understanding the problems to which the protein contributes during milk processing.

The casein milk proteins and the brain proteins alpha-synuclein and tau have been described as natively unfolded with random coil structures, which, in the case of alpha-synuclein and tau, have a propensity to form the fibrils found in a number of neurodegenerative diseases. New insight into the structures of these proteins has been provided by a Raman optical activity study, supplemented with differential scanning calorimetry, of bovine beta- and kappa-casein, recombinant human alpha-, beta- and gamma-synuclein, together with the A30P and A53T mutants of alpha-synuclein associated with familial cases of Parkinson's disease, and recombinant human tau 46 together with the tau 46 P301L mutant associated with inherited frontotemporal dementia. The Raman optical activity spectra of all these proteins are very similar, being dominated by a strong positive band centred at approximately 1318 cm(-1) that may be due to the poly(l-proline) II (PPII) helical conformation. There are no Raman optical activity bands characteristic of extended secondary structure, although some unassociated beta strand may be present. Differential scanning calorimetry revealed no thermal transitions for these proteins in the range 15-110 degrees C, suggesting that the structures are loose and noncooperative. As it is extended, flexible, lacks intrachain hydrogen bonds and is hydrated in aqueous solution, PPII helix may impart a rheomorphic (flowing shape) character to the structure of these proteins that could be essential for their native function but which may, in the case of alpha-synuclein and tau, result in a propensity for pathological fibril formation due to particular residue properties.

Evidence is provided from studies on natural and artificial biofluids that the sequestration of amorphous calcium phosphate by peptides or proteins to form nanocluster complexes is of general importance in the control of physiological calcification. A naturally occurring mixture of osteopontin peptides was shown, by light and neutron scattering, to form calcium phosphate nanoclusters with a core–shell structure. In blood serum and stimulated saliva, an invariant calcium phosphate ion activity product was found which corresponds closely in form and magnitude to the ion activity product observed in solutions of these osteopontin nanoclusters. This suggests that types of nanocluster complexes are present in these biofluids as well as in milk. Precipitation of amorphous calcium phosphate from artificial blood serum, urine and saliva was determined as a function of pH and the concentration of osteopontin or casein phosphopeptides. The position of the boundary between stability and precipitation was found to agree quantitatively with the theory of nanocluster formation. Artificial biofluids were prepared that closely matched their natural counterparts in calcium and phosphate concentrations, pH, saturation, ionic strength and osmolality. Such fluids, stabilised by a low concentration of sequestering phosphopeptides, were found to be highly stable and may have a number of beneficial applications in medicine.

We have investigated the scattering properties of mixtures of aggregated-whey-protein colloid (AWC) particles and an exocellular polysaccharide (EPS) from a lactic acid bacterium. Exceeding a certain concentration leads to a phase separation, which can be explained by an effective depletion attraction between the AWCs due to non-adsorbing EPS. The measured scattering intensity of a AWC/EPS mixture shows that the structure factor of the AWC particles increases at low wave vectors with increasing EPS concentration. The measurements can be described qualitatively in terms of the structure factor, calculated using integral theory including an expression for the depletion interaction potential.

We have produced β-lactoglobulin gene constructs in the pGAPZαA vector for over expression of recombinant wild-type and a putative monomeric mutant protein in Pichia pastoris. Levels of expression of at least 150 mg L ' of the wild-type were achieved in shake flask culture. Ion exchange chromatography showed that the expressed recombinant wild-type β-Lg eluted as a heterogeneous mixture of species, some of which may be owing to small variations in the N-terminus and some to ligand binding. The putative monomeric β-lactoglobulin construct gave an expressed protein that was recognized by anti bovine β-lactoglobulin antibody, but we do not know yet if it forms monomers rather than dimers.

The adsorption of proteins to interfaces is a vital and complex process for the formation and stabilization of multiphase food systems (emulsions and foams). The process of protein adsorption is generally understood only at the phenomenological level, as the complexity of protein unfolding during adsorption is very difficult to predict and model. By comparing proteins with very similar structures, it is possible to attribute observed changes in adsorption behaviour. The A and B genetic variants of β-lactoglobulin (β-lg) differ by only two amino acids (Asp-64, Val-118 in A, and Gly-64, Ala-118 in B), thus making them ideal candidates for this type of comparison. In this study we monitored the surface behaviour of β-lg A and B, measuring the surface tension and surface dilational modulus of adsorbed protein, and the compression behaviour of spread protein films. At pH 7, variant B lowered the surface tension and increased the surface dilational modulus more rapidly than variant A. Raising the pH to 7.8 should increase the level of dissociation into monomers. Indeed, this was confirmed by the rate of adsorption, which increased in both cases. Also, the surface tension of both variants was much lower than at pH7. Variant B was less sensitive to the change of pH than A. Regardless of pH, after 3 h adsorption the difference between the variants in surface tension or surface dilational modulus was negligible. The differences in surface behaviour between the variants are discussed in terms of interactions between monomers at and with the interface, and the dimer: monomer equilibrium in the bulk solution.

Profound differences in the rate of aggregation and denaturation among β-lactoglobulin samples from different sources can be reduced or even eliminated by dialysis of the samples first against a phosphate buffer. Visual evidence and thermograms indicated a reduced rate of aggregation in a phosphate buffered salt solution compared to the salt solution alone. The same effect was not achieved by another divalent tetrahedral ion, sulphate, nor by imidazole-buffered NaCl. Replacement of the background electrolyte by Na acetate had no discernible effect on the thermogram in the phosphate buffer. Phosphate is particularly good at buffering the protein solution at neutral pH at elevated temperature so the main effect is to maintain the net protein charge. In the imidazole and unbuffered media, the protein can move towards its isoelectric point and although the protein is more stable, aggregation is faster. It is speculated that there might also be a specific interaction of the phosphate anion with a cation-rich region of the β-lactoglobulin surface which could also inhibit aggregation at elevated temperatures.

Casein micelles become mutually attractive when an exocellular polysaccharide produced by Lactococcus lactis subsp. cremoris NIZO B40 (hereafter called EPS) is added to skim milk. The attraction can be explained as a depletion interaction between the casein micelles induced by the nonadsorbing EPS. We used three scattering techniques (small-angle neutron scattering, turbidity measurements, and dynamic light scattering) to measure the attraction. In order to connect the theory of depletion interaction with experiment, we calculated structure factors of hard spheres interacting by a depletion pair potential. Theoretical predictions and all the experiments showed that casein micelles became more attractive upon increasing the EPS concentration.

Experiments were designed to manipulate only one of the factors known to be important in maintaining the integrity of casein micelles while holding all of the other factors constant. Casein micelles were dissociated either by reducing the content of colloidal calcium phosphate or by adding κ -casein at a constant free calcium ion concentration, pH, and ionic strength and, in the latter case, also at a constant concentration of colloidal calcium phosphate. Size fractionation of casein micelles and partially dissociated casein micelles was achieved by gel filtration chromatography using an eluent that had an ionic strength and concentrations of free calcium and magnesium ions comparable with those of milk and was saturated with respect to colloidal calcium phosphate. Dissociation by selective reduction of colloidal calcium phosphate produced particles the average size of which changed during dialysis, but at no time was there an accumulation of the putative submicelles. Similarly, the addition of κ -casein, even in great excess, did not completely reverse the suggested process of association by submicelles. Surprisingly, in an attempt to speed the dissociation of casein micelles by urea addition, it was observed that the micelles could form a temperature-dependant precipitate at 3.5 to 4.0 M . None of the submicellar models of casein micelle structure adequately describe these controlled dissociation experiments.

Three samples each of the pure A and B genetic variants together with three samples of the mixed A/B variants of β-lactoglobulin were studied by differential scanning microcalorimetry. All the samples were characterised with respect to whey protein composition and shown to be almost pure β-lactoglobulin. Other analyses showed only minor amounts of other contaminants but variable degrees of lactolation ranging from

Previous CD measurements of changes in the conformation of beta-lactoglobulin at neutral pH as a function of temperature indicated the formation of a molten globule state above approx. 70 degrees C. New CD measurements are reported at temperatures up to 80 degrees C with an instrument on the Daresbury synchrotron radiation source which gives spectra of good signal-to-noise ratio down to 170 nm. IR spectra were recorded up to 94.8 degrees C with a ZnSe circle cell and a single simplified model of the substructure of the amide I' band was used to give the fractional contents of beta-sheet structure unambiguously and independently of the CD spectroscopy. The results of both techniques, however, were in agreement in showing a progressive loss of beta-sheet structure with increasing temperature, beginning below the denaturation temperature. Nevertheless, the CD spectroscopy showed a fairly abrupt loss of virtually all the helical conformation at approx. 65 degrees C. Comparison of the present results with other studies on the molten globule formed at acid pH in the lipocalin family suggests that above 65 degrees C a partly unfolded state is formed, possibly by destabilization of the intermolecular beta-strand I and the loss of the main helix, but it is not a classical molten globule transition.

Mineralized collagen composites are of interest because they have the potential to provide a bone-like scaffold that stimulates the natural processes of resorption and remodeling. Working toward this goal, our group has previously shown that the nanostructure of bone can be reproduced using a polymer-induced liquid-precursor (PILP) process, which enables intrafibrillar mineralization of collagen with hydroxyapatite (HA) to be achieved. This prior work used polyaspartic acid (pASP), a simple mimic for acidic non-collagenous proteins (NCPs), to generate nanodroplets/nanoparticles of an amorphous mineral precursor which can infiltrate the interstices of type-I collagen fibrils. In this study we show that osteopontin (OPN) can similarly serve as a process-directing agent for the intrafibrillar mineralization of collagen, even though OPN is generally considered a mineralization inhibitor. We also found that inclusion of OPN in the mineralization process promotes the interaction of mouse marrow-derived osteoclasts with PILP-remineralized bone that was previously demineralized, as measured by actin ring formation. While osteoclast activation occurred when pASP was used as the process-directing agent, using OPN resulted in a dramatic effect on osteoclast activation, presumably because of the inherent arginine-glycine-aspartate acid (RGD) ligands of OPN. By capitalizing on the multifunctionality of OPN, these studies may lead the way to producing biomimetic bone substitutes with the capability of tailorable bioresorption rates.

Previous work on the thermal denaturation of beta-lactoglobulin at about neutral pH and concentrations generally above 50 mg/ml has shown that the temperature of the maximum in the thermogram increases only slightly with concentration. Likewise, there is little if any concentration dependence at acid pH over a wide concentration range. However, so far as we are aware, no work has been described on the thermal denaturation of beta-lactoglobulin in the physiological range of protein concentration and pH appropriate to milk. We report measurements at pH 6.75 and 8.05 in the concentration range 2-120 mg/ml and show that below about 50 mg/ml the position of the maximum becomes strongly dependent on concentration, passing through a minimum near 25 mg/ml and increasing towards the lowest concentrations where measurements were practicable. Moreover, the narrow, well defined and nearly symmetrical thermal transition observed at high protein concentrations contrasts with a broader and more asymmetric curve at lower concentrations. An explanation for the behaviour seen at the lower protein concentrations is suggested, based on the temperature- and concentration-dependent dissociation of the beta-lactoglobulin dimer and an associated conformational transition. The position of the maximum in the thermogram has a marked dependence on the rate of heating down to the lowest rate investigated of 10 degrees C per hour, showing the importance of slow kinetic effects in the denaturation of this protein.

Unfolded phosphopolypeptides that contain one or more multiply-phosphorylated short sequences can sequester amorphous calcium phosphate to form stable complexes of constant average size and chemical composition. . A biofluid containing such complexes is supersaturated with respect to the bone and tooth mineral, hydroxyapatite but is undersaturated with respect to the amorphous precursor phase. Thus, soft tissues permeated by the biofluid should not experience ectopic calcification and hard tissues should remain mineralised. Sequestration by caseins allows high concentrations of calcium and phosphate to be attained in milk while osteopontin, fetuin and other phosphopolypeptides may act in a similar way in blood, other biofluids, soft and hard tissues.

A brief overview is given of the methods of determining and predicting secondary structure in proteins. The secondary structures of the milk serum proteins, lactoferrin, alpha-lactalbumin, lysozyme, and beta-lactoglobulin, as determined by x-ray crystallography, are compared with the results of a joint prediction method. This comparison evaluates critically the degree of success achieved and helps define what can reasonably be expected from a prediction in the absence of a known structure. The value of supplementary information from spectroscopic methods and the use of templates and sequence information from related proteins in improving the confidence of predictions are illustrated. One point that emerges is the general overprediction of helix content by the joint prediction method such that, for an all-beta protein such as beta-lactoglobulin, the method of Garnier, Osguthorpe, and Robson, applied with the correctly selected decision constants, provides a somewhat better approach. Secondary structure of the caseins can be predicted with less confidence than for globular proteins, and the results should be interpreted as evidence of a propensity to form transient secondary structures of the indicated type in view of the generally open and flexible conformation of caseins in solution.

Caseins are members of a class of proteins with extremely open and flexible conformations. Here, we consider what features of their sequences are important in maintaining such a structure. Primary structures of the αS1-, β- and κ-caseins from species including the cow, sheep, rat, mouse, rabbit and guinea pig were aligned both by a variety of automatic multiple alignment procedures, and manually, to identify conserved features. Fully conserved residues in the mature proteins were unusually rare and involved mainly residues that have high mutation rates in conventional alignment scoring schemes. Autocorrelation of sequences using residue mutation rate scores as measures of similarity revealed that around the PQNI conserved sequence of β-caseins there appear to be repeated sequences similar to Pro-rich domain-linking peptides found in a number of other proteins. Other cryptic repeats were found in αS1-caseins. Predicted secondary structures were calculated and it is argued that apart from the regions around the centres of phosphorylation, the caseins are essentially of the all-β-strand type. However, condensation into β-sheets is inhibited by certain of the conserved features of the primary structure, allowing the proteins to maintain an open and mobile (rheomorphic) conformation.

Phosphoproteins and phosphopeptides were expressed by E. coli to give yields of 30–200 mg of purified protein per litre with an average degree of phosphorylation at multiple sites of 61–83%. The method employed two compatible cohabiting plasmids having low and high copy number, expressing a protein kinase and, more abundantly, the substrate (poly)peptide, respectively. It was used to phosphorylate recombinant β-casein or osteopontin at multiple casein kinase-2 sites. Two constructs were designed to produce shorter peptides containing one or more clusters of phosphorylation sites resembling the phosphate centres of caseins. In the first, a 53-residue 6-His tagged phosphopeptide was expressed at a 5-fold higher molar yield. The second had multiple tandem repeats of a tryptic phosphopeptide sequence to give a similar increase in efficiency. Each recombinant phosphopeptide was purified (30–100 mg) and small-angle X-ray scattering measurements showed that they, like certain casein and osteopontin phosphopeptides, sequester amorphous calcium phosphate to form calcium phosphate nanoclusters. In principle, the method can provide novel phosphopeptides for the control of biocalcification or be adapted for use with other kinases and cognate proteins or peptides to study the effect of specific phosphorylations on protein structure. Moreover, the insertion of a phosphate centre sequence, possibly with a linker peptide, may allow thermodynamically stable, biocompatible nanoparticles to be made from virtually any sequence.