Protein-rich Domains Research Articles

Fabrication and characterization of glycerin-plasticized soy protein amyloid fibril scaffolds by unidirectional freeze casting method

Soy protein amyloid fibrils (SAFs) plasticized with glycerin were prepared into aligned porous scaffolds through the unidirectional freeze casting method. The impact of the amount of glycerin on the structural and functional properties of the SAFs-Gly scaffolds was investigated. Scanning electron microscopy confirmed that the aligned pore size and distribution of the scaffolds, with diameters ranging of 10–50 μm, could be adjusted by varying the amount of glycerin. Increasing the glycerin content decreased the median pore size of the scaffolds and increased the strain at the yield point by up to 40%. Additionally, the water resistance of the scaffolds significantly increased without any obvious changes in morphology. Differential scanning calorimetry analysis revealed two glass transitions in the SAFs/glycerin systems, corresponding to the glycerin-rich and protein-rich domains, respectively. FTIR spectroscopy indicated the formation of hydroxyl interactions and intermolecular hydrogen bonding between SAFs and glycerin. X-ray diffraction and small-angle X-ray scattering suggested that glycerin can induce SAFs crystallization, structural separation, and exfoliation of β-sheets to some extent. These results demonstrate the prospective application of this approach in the preparation of food-based polymer scaffolds and its usefulness in the food industry.

Lateral compression of lipids drives transbilayer coupling of protein condensates.

Protein phase separation that is restricted to a two-dimensional lipid membrane has recently gained much attention due to its involvement in various cellular processes. Two popular in vitro model membrane systems, giant unilamellar vesicles (GUVs) and supported lipid bilayers (SLBs), have been used to study it. However, due to the accessibility to only one side of the membrane in these systems, protein phase separation on both sides of the membrane remains unexplored. Here, we introduce a freestanding planar lipid membrane array as an effective platform to study membrane-associated protein phase separation. Protein condensates on the membrane display liquid-like features, and environmental stimuli, such as salt concentration and temperature, effectively control the observed phase separation results in our system with the application of several intrinsically disordered proteins. Interestingly, we observe transbilayer coupling of protein condensates such that protein-rich domains on one side of the membrane are found to colocalize with those on the other side of the membrane. How do these protein phases communicate across the lipid bilayer? Based on data from lipid probe partitioning and photobleaching experiments, we hypothesize that protein condensation on the membrane exerts lateral compressive stress on the underlying lipids, driving the formation of lipid domains with reduced fluidity. Specifically, lipid domains with reduced fluidity and increased lipid packing, formed by protein condensation on each membrane leaflet, are attracted to one another, leading to transbilayer coupling of protein condensates. These findings suggest a previously unknown mechanism by which cellular signals from one membrane leaflet, triggered by membrane-associated protein phase separation, can be transferred to the opposing leaflet.

Thermodynamics of wetting, prewetting and surface phase transitions with surface binding

In living cells, protein-rich condensates can wet the cell membrane and surfaces of membrane-bound organelles. Interestingly, many phase-separating proteins also bind to membranes leading to a molecular layer of bound molecules. Here we investigate how binding to membranes affects wetting, prewetting and surface phase transitions. We derive a thermodynamic theory for a three-dimensional bulk in the presence of a two-dimensional, flat membrane. At phase coexistence, we find that membrane binding facilitates complete wetting and thus lowers the wetting angle. Moreover, below the saturation concentration, binding facilitates the formation of a thick layer at the membrane and thereby shifts the prewetting phase transition far below the saturation concentration. The distinction between bound and unbound molecules near the surface leads to a large variety of surface states and complex surface phase diagrams with a rich topology of phase transitions. Our work suggests that surface phase transitions combined with molecular binding represent a versatile mechanism to control the formation of protein-rich domains at intra-cellular surfaces.

Coacervation in pea protein solutions: The effect of pH, salt, and fractionation processing steps

Legumes such as pea and soy have the ability to form coacervates in solution, which depends on the pH and salt concentration. We studied the phenomenon of coacervation for pea protein solutions in which the proteins were exposed to different extents of fractionation (i.e., neutral extraction, neutral extraction followed by freeze drying, alkaline extraction and alkaline extraction followed by isoelectric precipitation). The pH of the dispersed fractions was varied between 5.5 and 6.5 and the salt concentration between 0 and 500 mM NaCl. Confocal microscopy and particle size measurements were used to confirm the presence of spherical shaped protein-rich domains; a signature of the concentrated coacervate phase. It was found that the mildest processed fraction – obtained after protein extraction from the flour and subsequent removal of solids by centrifugation – formed coacervates as well as aggregates (i.e., non-spherical domains), between pH 6.0 and 6.5. At pH 6.25 only coacervates were observed. When 50 mM NaCl was added at pH 6.25, the coacervate average diameter increased from approximately 1 to 5 μm. When the salt concentration was increased to ≥200 mM NaCl, no coacervates were observed anymore. The coacervates formed at pH 6.25 with 0 and 50 mM NaCl were examined further. It turned out that the coacervates contained pea globulins, with legumin being most abundant. Pea albumins were not found in the coacervates. The internal protein content of the coacervates formed at pH 6.25 was around 45 wt %. When the mildest processed fraction was freeze dried, coacervates could still be formed at pH 6.25 and low salt concentrations (≤50 mM NaCl). After alkaline extraction, dispersions with both aggregates and coacervates were observed at pH 6.25. Isoelectric precipitated pea protein isolate did not show coacervates at any of the conditions tested. Our research shows that mildly fractionated pea protein was most suitable to form coacervates.

The tumor suppressor gene TP53 and colorectal cancer: A review

TP53 is a gene and the p53 is its protein product. It is well known as a tumor suppressor gene, due to its inherent property of apoptosis and its role in the suppression of tumor formation. Its oncogenic property is due to p53 mutation. This oncogenic property is named “gain of oncogenic property”. The functions of p53 have been studied the world over, especially the oncogenic function. It is well known for its role to induce apoptosis, but now it is established that it is also a key player in cell cycle regulation. It controls and monitors the cell division, chromosomal segregation, and cellular senescence. Due to all these important cellular functions, this protein is labeled as the “guardian of the genome”. The p53 protein is a nuclear phosphoprotein. It is composed of 393 amino acids. The structural and functional domains are an N-terminal transactivation domain, a protein-rich regulatory domain, and a C-terminal domain involved in the regulation of DNA binding. The colorectal cancers (CRC) are a major cause of morbidity and mortality world over. This is the third most common cancer worldwide and the fourth most common cause of death. Although it is slow-growing cancer the five-year survival is low. It is a heterogeneous disease and is due to, a complex genetic and biochemical interplay. This review summarizes the functions of p53 and its role in CRC. Keywords: TP53, p53, colorectal cancers, CRC, Tumor suppressor gene, apoptosis, cellular senescence, guardian of the genome.

Amino acids, polyols and soluble fibres as sugar replacers in bakery applications: Egg white proteins denaturation controlled by hydrogen bond density of solutions

In this paper we demonstrate that the denaturation behavior, i.e. Tden, of egg white proteins in sugar and sugar replacer solutions is explained by the volumetric density of hydrogen bonds in the solutions, i.e. nOH,eff. The validity of the presented approach is demonstrated using 18 solutions comprising single compounds as well as 7 ternary/quaternary mixtures. Different classes of plasticizers are used at various concentrations and at various ratio with proteins. Sweet amino acids such as l-proline and glycine are included as novel alternatives to polyols. The experimental data are modelled with the Flory-Huggins (FH) theory for biopolymer melting. For such purpose, solutions are treated as a single solvent, which is described by the effective volume fraction of the solvent Φw,eff (⁓nOH,eff). Overall, the FH model can well describe the denaturation behavior of egg white proteins in sugar and sugar replacer solutions up to 30% concentration. Deviations from the model become particularly evident at high sugar concentrations (i.e. 50%), which relate to conditions of phase separation in a protein-rich and sugar-rich domain. In such conditions, Φw,eff does not reflect the composition of the solvent around the proteins. An elevation in Tden is observed due to a reduction in hydrogen bond density in the protein-rich domain. The results indicate that phase separation is driven by both the concentration and the molar volume density of effective hydroxyl groups NOH,s/vs of the plasticizers or plasticizer mixtures. Finally, the proposed approach can predict key phase-transitions which result in protein network formation in pound cake baking.

Towards understanding the mechanism of fibrous texture formation during high-moisture extrusion of meat substitutes

This paper investigates the physical mechanisms of structure formation during high-moisture extrusion of vegetable proteins. Our model starts from the observation that extrudates with fibrous, meat-like structures exhibit water-rich and protein-rich domains. The origin and structure of these domains is attributed to a spinodal phase-separation process which occurs upon cooling of the extrudate. We investigate the process using continuum-mechanics simulations, considering the combined effects of viscous flow, thermal diffusivity, and the mixing thermodynamics of water and protein. This multi-physics problem is numerically solved using an unconventional mesh-free approach, the material point method (MPM), combined with the Cahn- Hilliard model of phase separation. The method incorporates both Eulerian and Lagrangian aspects, and is well suited to model multicomponent flows of history-dependent materials. Our simulations show that fiber-like structures are obtained when the ratio of phase separation rate, heat conduction rate, and flow rate are matched within a narrow window. Our results predict that the shape of the temperature profile within the cooling channel determines the structure of the phase-separated state. These findings suggest that the physical mechanism which causes fibrous structure formation is given by spinodal phase separation under the influence of a temperature gradient.

Intermolecular interactions and microstructure of glycerol-plasticized soy protein materials at molecular and nanometer levels

Glycerol plasticized soy protein is an important kind of biodegradable materials among the possible soy protein systems. Fully realized performance relies on the investigation and understanding of the molecular structure of the plasticized soy protein. However, the relevant research is primarily limited in the microstructure and physical properties of the mixtures with a lack of knowledge on the intermolecular interactions at molecular and nano-scale levels. In this work, intermolecular interaction and molecular motion of soy protein in glycerol plasticized systems were investigated at molecular and nanometer levels. Several techniques including atomic force microscope (AFM), transmission electron microscope (TEM) and 13C solid-state nuclear magnetic resonance (13C NMR), have been effectively employed. Observations from AFM and TEM indicated that the glycerol plasticized soy protein materials were heterogeneous at nanometer scale and there were two important phases existing in the materials, i.e. protein-rich domain and glycerol-rich domain. The protein-rich domain was composed of compact aggregations of pan-like protein while the glycerol-rich domain possessed a loose chain-like structure. Further studies on the interactions between soy protein and glycerol at molecular level using NMR indicated that the strong hydrogen bonds between glycerol and protein molecules was greatly affected by amino acid residues on protein chains. The protein-rich micro-domains were mainly composed of amino acids with long alkane lateral chains or aromatic ring lateral groups, while amino acids with polar groups or short nonpolar lateral groups formed glycerol-rich micro-domains. These results would be beneficial for the development and realization of plasticized soy protein based nanocomposites and blend materials with high performance.

Deciphering Protein-Rich Domains Formed by Non-structured Proteins in the Nuclear Pore Complex

Deciphering Protein-Rich Domains Formed by Non-structured Proteins in the Nuclear Pore Complex

Pattern formation by curvature-inducing proteins on spherical membranes

Spatial organisation is a hallmark of all living cells, and recreating it in model systems is a necessary step in the creation of synthetic cells. It is therefore of both fundamental and practical interest to better understand the basic mechanisms underlying spatial organisation in cells. In this work, we use a continuum model of membrane and protein dynamics to study the behaviour of curvature-inducing proteins on membranes of spherical shape, such as living cells or lipid vesicles. We show that the interplay between curvature energy, entropic forces, and the geometric constraints on the membrane can result in the formation of patterns of highly-curved/protein-rich and weakly-curved/protein-poor domains on the membrane. The spontaneous formation of such patterns can be triggered either by an increase in the average density of curvature-inducing proteins, or by a relaxation of the geometric constraints on the membrane imposed by the membrane tension or by the tethering of the membrane to a rigid cell wall or cortex. These parameters can also be tuned to select the size and number of the protein-rich domains that arise upon pattern formation. The very general mechanism presented here could be related to protein self-organisation in many biological processes, ranging from (proto)cell division to the formation of membrane rafts.

In vivo analysis of protein crowding within the nuclear pore complex in interphase and mitosis

The central channel of the nuclear pore complex (NPC) is occupied by non-structured polypeptides with a high content of Phe-Gly (FG) motifs. This protein-rich environment functions as an entropic barrier that prevents the passage of molecules, as well as the binding sites for karyopherins, to regulate macromolecular traffic between the nucleoplasm and the cytoplasm. In this study, we expressed individual Nups fused with a crowding-sensitive probe (GimRET) to determine the spatial distribution of protein-rich domains within the central channel in vivo, and characterize the properties of the entropic barrier. Analyses of the probe signal revealed that the central channel contains two protein-rich domains at both the nucleoplasmic and cytoplasmic peripheries, and a less-crowded central cavity. Karyopherins and other soluble proteins are not the constituents of the protein-rich domains. The time-lapse observation of the post-mitotic reassembly process also revealed how individual protein-rich domains are constructed by a sequential assembly of nucleoporins.

In situ microscopic studies on the structures and phase behaviors of SF/PEG films using solid-state NMR and Raman imaging.

In order to overcome the drawbacks of silk fibroin (SF)-based materials, SF has been blended with some polymers. Before using the blend material, understanding of the structures and phase behaviors of the blend is thought to be essential. In this study, solid-state (13)C CP-MAS NMR and Raman imaging techniques were used to study the structures and phase behaviors of blends of SF with polyethylene glycol (PEG) at a molecular weight that varied from 2 to 20 kDa and a blend ratio of SF/PEG from 95/5 to 70/30 (w/w%) at the molecular and microscopic levels. It is found that the conformational transition of SF to the β-sheet increased as the PEG content increased, while the amount of the formed β-sheet conformers was decreased as the PEG molecular weight increased for a given content. It is also observed that SF was incompatible with PEG to some extent. The phase separation into "sea" and "island" domains took place in the SF/PEG blend films. SF was dominantly present in the "sea" domain, while PEG in the "island" domains. The conformation of SF in the interface between SF and PEG was changed to the β-sheet, while that in the protein-rich domain remained in the random coil and/or helix conformation. These observations suggest that the specifically expected materials, for example, the silk-based microspheres or scaffold materials can be manufactured by controlling the molecular weight and content of PEG in the blend system.

Role of Polarized G Protein Signaling in Tracking Pheromone Gradients

Yeast cells track gradients of pheromones to locate mating partners. Intuition suggests that uniform distribution of pheromone receptors over the cell surface would yield optimal gradient sensing. However, yeast cells display polarized receptors. The benefit of such polarization was unknown. During gradient tracking, cell growth is directed by a patch of polarity regulators that wanders around the cortex. Patch movement is sensitive to pheromone dose, with wandering reduced on the up-gradient side of the cell, resulting in net growth in that direction. Mathematical modeling suggests that active receptors and associated G proteins lag behind the polarity patch and act as an effective drag on patch movement. In vivo, the polarity patch is trailed by a G protein-rich domain, and this polarized distribution of G proteins is required to constrain patch wandering. Our findings explain why G protein polarization is beneficial and illuminate a novel mechanism for gradient tracking.

IRSp53 senses negative membrane curvature and phase separates along membrane tubules.

BAR domain proteins contribute to membrane deformation in diverse cellular processes. The inverted-BAR (I-BAR) protein IRSp53, for instance, is found on the inner leaflet of the tubular membrane of filopodia; however its role in the formation of these structures is incompletely understood. Here we develop an original assay in which proteins are encapsulated in giant unilamellar vesicles connected to membrane nanotubes. Our results demonstrate that I-BAR dimers sense negative membrane curvature. Experiment and theory reveal that the I-BAR displays a non-monotonic sorting with curvature, and expands the tube at high imposed tension while constricting it at low tension. Strikingly, at low protein density and tension, protein-rich domains appear along the tube. This peculiar behaviour is due to the shallow intrinsic curvature of I-BAR dimers. It allows constriction of weakly curved membranes coupled to local protein enrichment at biologically relevant conditions. This might explain how IRSp53 contributes in vivo to the initiation of filopodia.

Using Lipopolysaccharides to Create 3-D Multicomponent Biomimetic Membranes on Solid Supports

Cell membranes are complex, multi component systems that contain dynamic elements such as lipid rafts and protein rich domains. Lipid Bilayer Assemblies (LBAs) can be used as model membrane systems for investigating interactions and responses to different stimuli but the level of complexity that can be studied is limited. Previous studies have shown the ability to create these LBAs on glass microspheres as well as flat substrates. These microspheres introduce a new level of complexity by creating mobile samples that can be analyzed using new techniques and also provide a curved surface more similar to biological cells. In previous studies we have demonstrated that Lipopolysaccharides (LPS) from gram-negative bacteria can be used to pattern LBAs in a simple, iterative manner. In this study, we have applied this LPS-modification approach to introduce new elements into LBAs on glass microspheres. We have found that LPS can be used to form static holes throughout the membrane while keeping the membrane intact. These holes can be backfilled with proteins such as BSA, and lipids, allowing membrane repair and or introduction of new components into the system. This technique gives rise to a multicomponent system that can be manipulated while still maintaining lipid fluidity in three dimensions. These new findings are being used to better understand LPS-membrane interactions, lead to new approaches for potential biosensor design, and provide a new platform for investigating cell membrane interactions. Future directions include using these coated microspheres as an LPS biosensor that can be analyzed using flow cytometry, introducing additional components into the system, such as receptors, gel phase lipids, and complex proteins to investigate membrane interactions and binding events and exploring as a template for the generation of janus-type particles.

The effect of protein aggregate morphology on phase separation in mixtures with polysaccharides

The morphology of aggregates formed by heating the globular protein β-lactoglobulin ( β-lg) changes with the addition of a small amount of CaCl2, from small strands to larger spherical aggregates (microgels). We investigated the effect of this morphological transition on the structure of mixtures of β-lg aggregates with the polysaccharide κ-carrageenan (κ-car), using confocal laser scanning microscopy and dynamic light scattering. The change in the morphology of the β-lg aggregates strongly reduced the κ-car concentration at which the system phase separated. As a consequence a dramatic change in the structure of the mixtures occurred over a narrow range of the CaCl2 concentration. Phase separation leads to the formation of micron-sized protein rich domains that have a tendency to stick together in large flocs. There is a big difference between the protein concentrations in the two phases, but the κ-car concentration is only weakly lower in the protein rich phase. A comparison is made between mixtures prepared at room temperature, after separately heating β-lg, and heated mixtures of native β-lg and κ-car. The micro-phase separated structure of the two systems is similar, but the aggregates disperse upon dilution in the former case, while they are covalently bound within the domains in the latter case. Other, more subtle, differences were also observed. The results explain the very high sensitivity of the structure of β-lg/κ-car mixtures to calcium ions.

Dynamics of Phase Separation in Oat β-glucan/Milk Mixtures Studied with Ultrasonic and Diffusing Wave Spectroscopy

When high molecular weight β-glucan is added to milk, macroscopic phase separation occurs due to thermodynamic incompatibility between mixed-linkage β-glucan and milk proteins. In this research, the evolution of microphase separated domains was investigated using ultrasonic and diffusing wave spectroscopy. It was possible to follow the dynamics of phase separation, in situ, without dilution. Immediately after mixing, there was a significant decrease in the initial turbidity of the mixtures, measured using diffusing wave spectroscopy, even at concentrations below macroscopic phase separation. At low and intermediate concentrations of β-glucan (0.1–0.2 w/w) the turbidity parameter, 1/l*, changed after mixing, because of rearrangements within the protein rich domains and growth of the phase separated domains. At high concentrations of β-glucan (0.6 % w/w), after an initial change in 1/l*, no further changes in turbidity were observed, because of the high viscosity of the mobile phase and the low diffusivity of the micelles due to crowding within the phase separated domains. Similar conclusions were drawn by observing the changes in ultrasonic attenuation, when probing at larger length scales. By using these two spectroscopic techniques it was possible to obtain complementary information on the dynamics of phase separation in β-glucan milk mixtures, and determine which systems still show rearrangements within the domains after mixing.

Designing microstructure into xanthan gum-enriched acid milk gels

Abstract Designing various microstructures through the interplay between phase separation and acid-induced gelation processes was investigated. Acid skim milk/xanthan gum mixed gels were prepared at different xanthan gum and glucono-δ-lactone (GDL) concentrations in order to modulate the extent of phase separation and the gelation rate, respectively. A gradual change in acid milk gel microstructure was found by increasing xanthan gum concentration, from homogeneous protein network, to fibrous structures and finally to isolated protein-rich aggregates. Addition of xanthan gum in the milk led to an apparent depletion–flocculation with protein-rich domains dispersed in a continuous phase enriched in xanthan. The sedimenting denser protein-rich domains could be trapped by protein gelation, forming specific fibrous structures at 0.05 wt.% xanthan gum. Manipulating the microstructure through modification of the gelation rate resulted in a design of a broad range of fibrous morphologies varying from 20 to 400 μm long, which could be used to generate novel textures for food products. Industrial relevance As a response to industrial demand by the consumer for novel acid milk gels with superior structural properties, mouthfeel and potential health benefits, we present here a way to manipulate textures by designing specific fibrous microstructures. Protein–polysaccharide mixtures that are subject to phase separation and gelation are interesting not only for the creation of major differences in structure and rheological properties of mixed systems, but also because of resulting protein-enriched products (up to 4 times more concentrated than conventional yoghurt). This approach should make it possible to improve the nutritional benefits as well as formulate a wide range of acid milk gels with innovative structures and sensory properties.

Rheological and microstructural characteristics of lentil starch–lentil protein composite pastes and gels

The rheological and structural changes in heat induced pastes and gels of lentil starch and lentil protein composites were investigated at various starch to protein ratios. The starch fraction (φs) in the mixtures was varied from 0 to 1 and the total solid content was maintained at 25% (w/w). Results showed that the gel strength of the composite gels increased exponentially with the increase in lentil starch fraction. The pasting temperature increased and the paste viscosity decreased with the increase in the lentil protein fraction. The high starch composite gels (φs > 0.5) showed higher elastic (G′) and loss (G″) moduli at higher temperature (60 °C) than at lower temperature (10 °C). The high protein composite gels (φs ≤ 0.5) showed higher G′ and G″ values at lower temperature (10 °C) than at higher temperature (60 °C). Segregation of protein-rich domains was observed in the high starch gels (φs > 0.7) whereas low starch composite gels (φs ≤ 0.5) appeared more homogeneous. The microstructure of composite gels appeared to be more fragile with larger pore size and thinner wall compared to the microstructure of starch gel. Both non-covalent interactions (hydrophobic and hydrogen bonding) and covalent bonding were found to contribute to the gel structure and firmness of these composite gels. The NaCl concentration increased the paste viscosity and gel firmness of the composite gels up to 0.25 mM above which the magnitudes of these parameters were decreased. Both the paste viscosity and the gel firmness of the composite gels were found to be higher above the isoelectric point of lentil protein and vice versa. From this study, textural properties of the composite gel/paste were found to be strongly affected by the proportion of the starch and protein as well as the extrinsic factors (pH, ionic strength, presence of reducing agents). Therefore, understanding of gelling behaviour of lentil starch and protein in composite gel would be helpful for structure formation of these two biopolymers in mixtures and would help their application in new product development.

Structural Characterization of Nanoscale Meshworks within a Nucleoporin FG Hydrogel

The permeability barrier of nuclear pore complexes (NPCs) controls all exchange of macromolecules between the cytoplasm and the cell nucleus. It consists of phenylalanine-glycine (FG) repeat domains apparently organized as an FG hydrogel. It has previously been demonstrated that an FG hydrogel derived from the yeast nucleoporin Nsp1p reproduces the selectivity of authentic NPCs. Here we combined time-resolved optical spectroscopy and X-ray scattering techniques to characterize such a gel. The data suggest a hierarchy of structures that form during gelation at the expense of unstructured elements. On the largest scale, protein-rich domains with a correlation length of ~16.5 nm are evident. On a smaller length scale, aqueous channels with an average diameter of ~3 nm have been found, which possibly represent the physical structures accounting for the passive sieving effect of nuclear pores. The protein-rich domains contain characteristic β-structures with typical inter-β-strand and inter-β-sheet distances of 1.3 and 0.47 nm, respectively. During gelation, the formation of oligomeric associates is accompanied by the transfer of phenylalanines into a hydrophobic microenvironment, supporting the view that this process is driven by a hydrophobic collapse.