Protein-rich Domains Research Articles

Microstructure and properties of glycerol plasticized soy protein plastics containing castor oil

A series of glycerol-plasticized soy protein plastics containing castor oil were prepared by intensive mixing and hot pressing. The microstructure and properties of the resulting plastics were characterized with scanning electron microscopy (SEM), dynamic mechanical analysis (DMA), thermogravimetric analysis (TGA), and mechanical tests. With small amount of castor oil incorporated (glycerol/oil ratio above 8:2), castor oil dispersed in the protein matrix homogeneously. At high concentrations, phase separation occurred. The adding of castor oil led to significant increase of storage modulus as well as glass transition temperatures attributed to glycerol-rich and protein-rich domains. Plastics containing castor oil exhibited improved tensile strength and Young’s modulus under high humidity (75% RH), compared with neat glycerol plasticized protein plastics. Especially, incorporation of low content of castor oil (glycerol/oil ratio=9:1) would result in the simultaneous enhancement of tensile strength, elongation at break and Young’s modulus. Increasing castor oil content also enhanced the thermal stability of the protein plastics.

Processing and Characterization of Glycerol-Plasticized Soy Protein Plastics Reinforced with Citric Acid-Modified Starch Nanoparticles

Citric acid-modified starch nanoparticles with an average size of 82 nm were prepared through precipitation from gelatinized starch solution by ethanol and further modification with citric acid. When being incorporated in glycerol-plasticized soy protein plastics, citric acid-modified starch nanoparticles displayed dramatic reinforcing effect. The resulted nanocomposite plastics exhibited improvement in mechanical performance. Also, the water uptake decreased, indicating an increase of water resistance. The modified starch nanoparticles had a good compatibility with soy protein matrix. Possessing a relative hydrophobic surface, the filler would prefer to interact with protein-rich domains in glycerol-plasticized soy protein. The work provided a green approach of biodegradable materials based on naturally occurring biopolymers.

Evidence for a Second, High Affinity Gβγ Binding Site on Gαi1(GDP) Subunits

It is well known that Galpha(i1)(GDP) binds strongly to Gbetagamma subunits to form the Galpha(i1)(GDP)-Gbetagamma heterotrimer, and that activation to Galpha(i1)(GTP) results in conformational changes that reduces its affinity for Gbetagamma subunits. Previous studies of G protein subunit interactions have used stoichiometric amounts of the proteins. Here, we have found that Galpha(i1)(GDP) can bind a second Gbetagamma subunit with an affinity only 10-fold weaker than the primary site and close to the affinity between activated Galpha(i1) and Gbetagamma subunits. Also, we find that phospholipase Cbeta2, an effector of Gbetagamma, does not compete with the second binding site implying that effectors can be bound to the Galpha(i1)(GDP)-(Gbetagamma)(2) complex. Biophysical measurements and molecular docking studies suggest that this second site is distant from the primary one. A synthetic peptide having a sequence identical to the putative second binding site on Galpha(i1) competes with binding of the second Gbetagamma subunit. Injection of this peptide into cultured cells expressing eYFP-Galpha(i1)(GDP) and eCFP-Gbetagamma reduces the overall association of the subunits suggesting this site is operative in cells. We propose that this second binding site serves to promote and stabilize G protein subunit interactions in the presence of competing cellular proteins.

Micro-phase separation explains the abrupt structural change of denatured globular protein gels on varying the ionic strength or the pH

Aqueous solutions of globular proteins gel after heat-induced denaturation. The structure of β-lactoglobulin gels was studied as a function of the pH (2–8) and the NaCl concentration (0–1 M) over a wide range of length scales (1 nm-100 µm) using a combination of scattering techniques and microscopy. The gel structure depended on the strength of the electrostatic interaction that was varied by changing the charge density of the proteins (pH) or the screening length (ionic strength). Homogeneous so-called finely stranded gels were formed at strong interaction, while more heterogeneous so-called particulate gels were formed at weak interaction. It is shown that phase separation of growing irreversibly bound protein aggregates explains the abrupt transition between the two structures for small changes of the ionic strength or the pH. Phase separation was limited to the formation of protein rich domains with a radius of several microns that associated into the particulate gels. The structure of homogeneous gels is shown to be determined by the amplitude of the (critical) concentration fluctuations of protein aggregates that were frozen-in by gelation. The characteristic length scales of the homogeneous gels varied between about 20 nm and 1 µm.

Microstructural features of composite whey protein/polysaccharide gels characterized at different length scales

Mixed biopolymer gels are often used to model semi-solid food products. Understanding of their functional properties requires knowledge about structural elements composing these systems at various length scales. This study has been focused on investigating the structural features of mixed cold-set gels consisting of whey protein isolate and different polysaccharides at different length scales by using confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM). Whey protein cold-set gels were prepared at different concentrations to emulate stiffness of various semi-solid foods. Mixed gels contained different concentrations of gellan gum, high methyl pectin or locust bean gum. Results obtained with CLSM, at the micrometer length scale, indicated the homogeneous nature of the investigated gels. Results obtained with SEM, at the sub-micron length scale, indicated the presence of spherical protein aggregates. During the gel preparation (acidification), the presence of polysaccharides in the whey protein gels led to on initially segragative phase separation into a gelled protein phase and a polysaccharide/serum phase at a micrometer length scale. At the final pH of the gels (pH 4.8, i.e. below the pI of whey proteins), the negatively charged polysaccharides interacted with the protein phase and their spatial distribution was effected by charge density. Polysaccharides with a higher charge density were more homogeneously distributed within the protein phase. Neutral polysaccharide, locust bean gum, did not interact with the protein aggregates but was present in the serum phase. Using SEM, a new type of microstructure formed in the whey protein/polysaccharide gels was characterized. It composed of a protein continuous, porous network at the length scale of 100 μm, coexisting next to the pools of serum which contained spherical protein-rich domains. Heterogeneity of the structure strongly related to the macroscopic behavior of the gels under large deformation. Upon uniaxial compression these heterogeneous gels releases a large amount of serum. Combination of the results of two microscopic techniques, CLSM and SEM, appeared to offer unique possibilities to characterize the structural elements of whey protein/polysaccharide cold-set gels over a wide range of length scales.

Suppression of Polyglutamine Toxicity by the Yeast Sup35 Prion Domain in Drosophila

The propensity of proteins to form beta-sheet-rich amyloid fibrils is related to a variety of biological phenomena, including a number of human neurodegenerative diseases and prions. A subset of amyloidogenic proteins forms amyloid fibrils through glutamine/asparagine (Q/N)-rich domains, such as pathogenic polyglutamine (poly(Q)) proteins involved in neurodegenerative disease, as well as yeast prions. In the former, the propensity of an expanded poly(Q) tract to abnormally fold confers toxicity on the respective protein, leading to neuronal dysfunction. In the latter, Q/N-rich prion domains mediate protein aggregation important for epigenetic regulation. Here, we investigated the relationship between the pathogenic ataxin-3 protein of the human disease spinocerebellar ataxia type 3 (SCA3) and the yeast prion Sup35, using Drosophila as a model system. We found that the capacity of the Sup35 prion domain to mediate protein aggregation is conserved in Drosophila. Although select yeast prions enhance poly(Q) toxicity in yeast, the Sup35N prion domain suppressed poly(Q) toxicity in the fly. Suppression required the oligopeptide repeat of the Sup35N prion domain, which is critical for prion properties in yeast. These results suggest a trans effect of prion domains on pathogenic poly(Q) disease proteins in a multicellular environment and raise the possibility that Drosophila may allow studies of prion mechanisms.

Effects of Moisture on Glass Transition and Microstructure of Glycerol‐Plasticized Soy Protein

The glass transition behavior of the glycerol-plasticized soy protein sheets (SL series) at various relative humidity (RH) was investigated by using differential scanning calorimetry with the aluminum pan and O-ring-sealed stainless steel capsule, and the microstructure of these sheets was detected on small-angle X-ray scattering. The results revealed that there were three glass transitions (Tg1, Tg2 and Tg3), corresponding to glycerol-rich, protein-rich and protein-water domains, in the protein-glycerol-water ternary system. The Tg1 values of the SL-series sheets at 75% RH decreased from -49.3 to -83.8 degrees C with an increase of glycerol content from 10 to 50 wt.-%, whereas Tg2 and Tg3 were almost invariable at about 60 degrees C and 3 degrees C, respectively. In addition, the Tg1, Tg2 and Tg3 values of the SL-25 containing 25 wt.-% glycerol at 0%, 35%, 58%, 75% and 98% RH were in the range of -12.7 - -83.2 degrees C, 65.8 - 53.1 degrees C and 3.5 - 1.9 degrees C, respectively. The result from small-angle X-ray scattering indicated that the radii of gyration (Rg) of protein-rich domain were in the range of 60-63 nm; this suggested the existence of protein macromolecules as aggregates in the stable protein-rich and protein-water domains. With an increase of RH, the tensile strength and Tg values of the SL-series sheets decreased, but the elongation at break increased. In view of the results above, the moisture in ambient environment significantly influenced the Tg values and microstructures of the glycerol-plasticized soy protein sheets, leading to the changes of the mechanical and thermal properties.

New Evidences of Glass Transitions and Microstructures of Soy Protein Plasticized with Glycerol

Soy protein isolate (SPI) and glycerol were mixed under mild (L series) and severe (H series) mixing conditions, respectively, and then were compression-molded at 140 degrees C and 20 MPa to prepare the sheets (SL and SH series). The glass transition behaviors and microstructures of the soy protein plasticized with glycerol were investigated carefully by using differential scanning calorimetry and small-angle X-ray scattering. The results revealed that there were two glass transitions in the SPI/glycerol systems. When the glycerol contents ranged from 25 to 40 wt.-%, all of the SL- and SH-series sheets showed two glass transition temperatures (T(g1) and T(g2)) corresponding to glycerol-rich and protein-rich domains, respectively. The T(g1) values of the sheets decreased from -28.5 to -65.2 degrees C with an increase of glycerol content from 25 to 50 wt.-%, whereas the T(g2) values were almost invariable at about 44 degrees C. The results from wide-angle X-ray diffraction and small-angle X-ray scattering indicated that both protein-rich and glycerol-rich domains existed as amorphous morphologies, and the radii of gyration (R(g)) of the protein-rich domains were around 60 nm, a result suggesting the existence of stable protein domains. The results above suggest that protein-rich domains were composed of the compact chains of protein with relatively low compatibility to glycerol and glycerol-rich domains consisted of relative loose chains that possessed good compatibility with glycerol. The significant microphase separation occurred in the SPI sheets containing more than 25 wt.-% glycerol, with a rapid decrease of the tensile strength and Young's modulus. [illustration in text].

Fourier transform infrared microspectroscopic study of the chemical microstructure of corn and oat flour-based extrudates

The spatial distribution of starch, protein, and lipid domains (chemical microstructure) in corn and oat flour-based extrudates was investigated in 8μm thick cross-sections of the products by FTIR-microspectroscopy mapping experiments. The results reveal an even starch distribution in all extrudates investigated confirming that starch forms a continuous phase in cereal-based extrudates. Proteins were the least evenly distributed among the three components. Also, small, protein rich domains of 70μm diameter were detected in the microstructure of the extrudates. Starch and protein distributions were always inversely related to each other. The lipids in oat flour-based extrudates were less evenly distributed than the starch, but more evenly than the proteins. Lipid distribution was neither correlated with starch nor with protein distribution.

Layer formation of a lipid-tagged single-chain antibody and the interaction with antigen

A phospholipid monolayer was prepared at the air-water interface and different amounts of a lipid-tagged genetically fragmented antibody were incorporated into the lipid matrix. The molecular surface area of the film increased with antibody concentration and reached a maximum at an antibody mole fraction of 0.03. Atomic force microscopy showed that the antibody fragment was segregated into protein-rich domains with a mean diameter of about 12 nm. In-situ measurements with surface plasmon resonance displayed a specific binding of antigen—a binding that was not observed to the pure lipid film.

Synthetic lipid-anchored receptors based on the binding site of a monoclonal antibody

Highly specific ligand receptor interactions generally characterize molecular recognition at cell surfaces and other biological systems. In this study we simulate a membrane receptor by fusing a monoclonal antibody fragment to a phospholipid. A sulfhydryl group in the hinge region of a monoclonal antibody fragment, was covalently linked to derivatives of phosphatidylethanolamines and phosphatidylserine via three different hydrophilic spacer arms. We investigated and characterized these lipid-anchored Fab-fragments which we have named ‘Fab-lipids’ in liposomal and monolayer systems. Methods for the monomolecular assembling of such films at the air/water interface and techniques used for their manipulation are outlined. We describe two possibilities for building a monomolecular receptor layer, consisting of two-dimensioal pattern of oriented Fab-fragments with their artificial hydrophobic anchor embedded in a lipid matrix. In the first method a monomolecular film at the air/water interface was allowed to form from a vesicular suspension and driven into a phase separation, resulting in protein rich domains embedded in a protein depleted phase. This film was transferred onto a solid support in such a way that the established pattern was preserved. Alternatively, a recognition pattern was formed by directly cross-linking the Fab-fragments to preformed planar membranes composed of the reactive spacer-lipids and an inert matrix lipid. Specificity as well as contrast of the binding activity of the receptor layers were quantified using micro-fluorimetry.

Two-dimensional recognition pattern of lipid-anchored Fab' fragments

A two-dimensional pattern of oriented antibody fragments was formed at the air-water interface and transferred onto a solid support. The Fab'-fragments of a monoclonal antibody against the hapten dinitrophenyl (DNP) were covalently linked via a hydrophilic spacer to phospholipid vesicles. A monomolecular lipid-protein layer at equilibrium with these vesicles was allowed to form at the air-water interface. The monolayer was separated from the vesicle phase and transferred to a Langmuir-Blodgett trough. By cooling and compressing, the previously homogeneous lipid-protein film was driven into a two-dimensional phase separation resulting in protein-rich domains and a second phase consisting mainly of lipid. This film was transferred onto a solid support in a way that preserved the protein-lipid pattern. The specificity as well as the contrast in the binding activity of the two different separated phases were then quantified using microfluorometry. DNP conjugated to fluorescein-labeled bovine serum albumin (BSA) showed virtually no binding to the lipid regions, but gave a ratio of bound DNP-BSA to Fab'-lipid of greater than 50% in the protein-rich domains proving that the Fab'-moiety retained its biological activity. This demonstrates that the technique presented here is well suited to modify different solid surfaces with a pattern of a given biological function. The optional control of lateral packing and orientation of the components in the monolayer makes it a general tool for the reconstitution of supported lipid-protein membranes and might also open new ways for the two-dimensional crystallization of proteins at membranes.

Micrometer-scale domains in fibroblast plasma membranes.

We have used the technique of fluorescence photobleaching recovery to measure the lateral diffusion coefficients and the mobile fractions of a fluorescent lipid probe, 1-acyl-2-(12-[(7-nitro-2-1, 3-benzoxadiazol-4-yl)aminododecanoyl]) phosphatidylcholine (NBD-PC), and of labeled membrane proteins of human fibroblasts. Values for mobile fractions decrease monotonically with increasing size of the laser spot used for the measurements, over a range of 0.35-5.0 microns. Values for NBD-PC diffusion coefficients increase in part of this range to reach a plateau at larger laser spots. This variation is not an artifact of the measuring system, since the effects are not seen if diffusion of the probe is measured in liposomes. We also find that the distribution of diffusion coefficients measured with small laser spots is heterogeneous indicating that these small spots can sample different regions of the membrane. These regions appear to differ in protein concentration. Our data strongly indicate that fibroblast surface membranes consist of protein-rich domains approximately 1 micron in diameter, embedded in a relatively protein-poor lipid continuum. These features appear in photographs of labeled cell surfaces illuminated by the expanded laser beam.

Experimental and theoretical considerations of mechanisms controlling cation effects on thylakoid membrane stacking and chlorophyll fluorescence

The roles of specific cation binding, charge neutralization and electrostatic screening mechanisms in controlling salt-induced stacking and chlorophyll fluorescence changes in thylakoid membranes are examined in the light of new experimental evidence and theoretical calculations of the forces between membrane surfaces. A comparison of the biphasic stacking and fluorescence phenomena generated by organic mono- and divalent cations known sterically to inhibit specific binding with the effects generated by inorganic mono- and divalent cations suggests that the observed salt-induced changes at pH ⩾ 7.5 are predominantly governed by the electrostatic screening mechanism in agreement with previous work (e.g. Barber, J., Mills, J.D. and Love, A. (1977) FEBS Lett. 74, 174–181). Detailed calculations of the coulombic double layer repulsive force between negatively charged membrane surfaces immersed in a mixed electrolyte of valence type Z 1+/Z 1−, Z 2+/Z 1− were performed both under the constraints of fixed surface charge density and fixed surface potential. From a close comparison of the theoretical results with new experimental data on salt-induced stacking and fluorescence changes and a consideration of the contributions of the ‘hydration’ repulsive force and the van der Waals attractive force, it is argued that a reduction in surface charge density alone by lateral diffusion is probably insufficient to realize membrane stacking and that an increase in the van der Waals attractive force is necessary to account for the experimental observations perhaps through the formation of protein rich domains. In view of the complexity of the thylakoid membranes, the conclusions are to be considered qualitative. Nevertheless, these calculations give support to a model in which the cation induced chlorophyll fluorescence and stacking changes can be explained by lateral diffusion of two types of pigment protein complexes in the lipid matrix of the membrane. Such diffusion gives rise to changes in energy transfer between Photosystem II and Photosystem I and also to the creation of domains having low and high electrical surface charge density.