Soluble Protein Aggregates Research Articles

Exploration of the regulatory mechanism of pulsed electric field on the aggregation behavior of soybean protein isolates

As an emerging protein modification technology, pulsed electric field (PEF) technology in modifying soybean protein isolates (SPI) suffers from unclear mechanism and controversial changes in aggregation structure. To address these issues, the effects of PEF treatment with different electric field intensities (5–30 kV/cm) on the aggregation behavior and spatial structure of SPI were investigated. The results showed that the SPI aggregates exhibited a trend of depolymerization followed by reaggregation with the increase of PEF intensity. At 10 and 15 kV/cm, the polarization effect of PEF induced the unfolding of the tertiary structure of SPI, leading to the enhancement of the electronegativity of the side chains, and the electrostatic repulsive force between the molecules promoted the depolymerization of SPI aggregates. However, with the withdrawal of PEF, the structure of the SPI was partially reversible, resulting in a limited depolymerization effect. When the PEF intensity reached 20 kV/cm and above, SPI underwentstructure unfolding, subunit dissociation, continuous exposure of hydrophobic groups and sulfhydryl groups, and ultimately reaggregation mediated by hydrophobic interactions and disulfide bonding, resulting in the formation of high molecular weight soluble and insoluble protein aggregates. In addition, the formation of free radicals under strong electric fields (≥20 kV/cm) accelerated the oxidation of SPI and promoted the rapid formation of disulfide bonds. This study provides a theoretical basis for the targeted regulation of SPI aggregation structure by PEF.

An aptamer-guided fluorescence polarisation platform for extracellular vesicle liquid biopsy.

The translation of discoveries on extracellular vesicle (EV) based cancer biomarkers to personalised precision oncology requires the development of robust, sensitive and specific assays that are amenable to adoption in the clinical laboratory. Whilst a variety of elegant approaches for EV liquid biopsy have been developed, most of them remain as research prototypes due to the requirement of a high level of microfabrication and/or sophisticated instruments. Hence, this study is set to develop a simple DNA aptamer-enabled and fluorescence polarisation-based homogenous assay that eliminates the need to separate unbound detection ligands from the bound species for EV detection. High specificity is achieved by immobilising EVs with one set of antibodies and subsequently detecting them with a DNA aptamer targeting a distinct EV biomarker. This two-pronged strategy ensures the removal of most, if not all, non-EV substances in the input biofluids, including soluble proteins, protein aggregates or non-vesicular particles, prior to quantifying biomarker-positive EVs. A limit of detection of 5.0 × 106 EVs/mL was achieved with a linear quantification range of 5.0 × 108 to 2.0 × 1010 EVs/mL. Facilitated by a multiple parametric analysis strategy, this aptamer-guided fluorescence polarisation assay was capable of distinguishing EVs from three different types of solid cancer cells based on quantitative differences in the levels of the same sets of biomarkers on EVs. Given the simplicity of the method and its ease of implementation in automated clinical biochemistry analysers, this assay could be exploited for future EV-based continuous and real-time monitoring of the emergence of new macro- or micro-metastasis, cancer progression as well as the response to treatment throughout different stages of cancer management in the clinic.

Revealing the deterioration mechanism in gelling properties of pork myofibrillar protein gel induced by high-temperature treatments: Perspective on the protein aggregation and conformation

The purpose of the present study was to investigate the mechanism of gel deterioration of myofibrillar proteins (MP) gels induced by high-temperature treatments based on the protein aggregation and conformation. The results showed that the gel strength and water holding capacity of MP obviously increased and then decreased as the temperature increased, reaching the maximum value at 80 °C (P < 0.05). The microstructure analysis revealed that appropriate temperature (80 °C) contributed to the formation of a more homogeneous, denser, and smoother three-dimensional mesh structure when compared other treatment temperatures, whereas excessive temperature (95 °C) resulted in the formation of heterogeneous and large protein aggregates of MP, decreasing the continuity of gel networks. This was verified by the rheological properties of MP gels. The particle size (D4,3 and D3,2) of MP obviously increased with larger clusters at excessive temperature, and the surface hydrophobicity of MP decreased (P < 0.05), which has been linked to the formation of soluble or insoluble protein aggregates. Tertiary structure and secondary structure results revealed that the proteins had a tendency to be more stretched under higher temperature treatments, which resulted in a decrease in covalent interactions and non-covalent interactions, fostering the over-aggregation of MP. Therefore, our present study indicated that the degradation of MP gels treated at high temperatures was explained by protein aggregation and conformational changes in MP.

Use of turbidimetry for determination of heat treatment intensity applied at pasteurization of milk

Express methods for estimating the intensity of heat treatment of milk are necessary in industry and in research work. For this reason, there are many ways to measure this parameter, which are based on different physical principles, including turbidimetric methods. The Harland &amp; Ashworth turbidimetric method has been developed for a long time, however, due to its high reliability and ease of implementation, it is still used in practice. However, this method has a drawback: it takes a long time to perform the analysis. In this regard, the aim of the work is to develop an express method for evaluating the thermal class of milk based on the principle of measuring the concentration of soluble whey proteins. The result is achieved through the use of a turbidimetric measurement method with optimized sample preparation parameters and parameters for measuring the optical density of a suspension of protein aggregates. The method is implemented as follows. The milk sample is mixed with 0.1 N acetate buffer (pH 4.6) in a ratio that allows to obtain a concentration of soluble milk whey proteins from 0.05% to 0.1%. Recommended dilution coefficients: 1:3 for samples of ultra-pasteurized milk and pasteurized milk with high heat treatment intensity; 1:7 for samples of pasteurized milk with low heat treatment intensity and 1:14 for raw milk samples. The solution is filtered on a membrane filter with a pore size of 0.45 microns. The resulting filtrate is mixed with 24% trichloroacetic acid (TCA) in a 1:1 ratio to coagulate soluble whey proteins and form protein aggregates. After holding for 5–10 minutes, the optical density of the suspension of protein aggregates is measured at a wavelength of 650 nm. The content of water-soluble whey proteins in the sample can be calculated according to the calibration curve. The developed method allows to obtain the measurement result in less time than the Harland &amp; Ashworth turbidimetric method.

Correcting Ultraviolet-Visible Spectra for Baseline Artifacts

Rayleigh and Mie light scattering from particulates, soluble protein aggregates, or large proteins can lead to inaccuracy of concentration measurements using ultraviolet (UV) spectroscopy and Beer's Law. While a number of light scattering correction equations have been proposed in the literature, they can also lead to incorrect values if samples vary in particulate and/or soluble aggregate levels or depart in other ways from which the equations were developed. We propose a curve-fitting baseline subtraction approach based on fundamental Rayleigh and Mie scattering equations which also factors in instrument baseline artifacts. We validated this Rayleigh-Mie correction against a wide variety of positive and negative controls, including protein size standards, protein aggregates induced by forced degradation, lentivirus and polystyrene nanospheres.

Soluble-protein-aggregate-assisted improvements in heat-induced gel properties: Effect of genipin-mediated crosslinks on egg white protein

Genipin, a covalent crosslinker, has attracted significant attention from the food industry. Herein, the color, heat-induced gelling properties, and soluble aggregate character of the genipin-treated egg white were investigated to clarify the impact of soluble protein aggregates on the gel properties of egg white protein.After genipin pretreatment, the egg white protein turned dark blue and retained this color after heat-induced gelling. The genipin-pretreated egg white protein gel exhibited up to 2.9-fold and 13% higher breaking strength and strain, respectively, compared to non-treated egg white gel. The gel-breaking strength and strain correlated well with the color, before and after gelling. Gel electrophoresis, size exclusion, and anion exchange high-performance liquid chromatography revealed that a soluble protein aggregate with a negatively charged surface formed during pretreatment. The genipin-treated egg white protein formed a finely structured gel with high breaking strength and strain at all tested NaCl concentrations, indicating that the enhanced negative charge of the surface did not contribute to improving the gel properties.The soluble protein aggregates formed via covalent crosslinking are important for improving gel properties. The proposed method is a novel environmentally friendly method for enhancing the gel properties of egg white protein.

Protein aggregation impacts in vitro protein digestibility, peptide profiling and potential bioactive peptides of soymilk and dry-heated soybeans

Protein aggregates and in vitro digestibility of soymilk, 160 °C and 200 °C dry-heated soybeans (DHS) were investigated. The results showed that, different from the soluble protein aggregates in soymilk, thermal treatment led to formation of increased insoluble aggregates in 160 °C DHS via non-covalent bonds (hydrogen bonds and hydrophobic interactions). However, covalent bonds (protein oxidation-induced crosslinking) were predominant forces for insoluble aggregates in 200 °C DHS. The order of in vitro digestibility was 160 °C DHS > soymilk >200 °C DHS (p < 0.05). More types of peptides were identified from 160 °C DHS (2,230) than soymilk (2,104) and 200 °C DHS (2,130). Soymilk and 200 °C DHS had similar peptides distribution and sequence alignment; while 160 °C DHS generated unique peptides. Moreover, potential bioactive peptides and antimicrobial peptides were all found in the three samples. Thus, protein aggregates, caused by different cooking methods, affected protein digestibility and peptide composition.

Influence of Ionizing Radiation on Spontaneously Formed Aggregates in Proteins or Enzymes Solutions.

We have shown that many proteins and enzymes (ovalbumin, β-lactoglobulin, lysozyme, insulin, histone, papain) undergo concentration-dependent reversible aggregation as a result of the interaction of the studied biomolecules. Moreover, irradiation of those protein or enzyme solutions under oxidative stress conditions results in the formation of stable soluble protein aggregates. We assume that protein dimers are mainly formed. A pulse radiolysis study has been made to investigate the early stages of protein oxidation by N3• or •OH radicals. Reactions of the N3• radical with the studied proteins lead to the generation of aggregates stabilized by covalent bonds between tyrosine residues. The high reactivity of the •OH with amino acids contained within proteins is responsible for the formation of various covalent bonds (including C-C or C-O-C) between adjacent protein molecules. In the analysis of the formation of protein aggregates, intramolecular electron transfer from the tyrosine moiety to Trp• radical should be taken into account. Steady-state spectroscopic measurements with a detection of emission and absorbance, together with measurements of the dynamic scattering of laser light, made it possible to characterize the obtained aggregates. The identification of protein nanostructures generated by ionizing radiation using spectroscopic methods is difficult due to the spontaneous formation of protein aggregates before irradiation. The commonly used fluorescence detection of dityrosyl cross-linking (DT) as a marker of protein modification under the influence of ionizing radiation requires modification in the case of the tested objects. A precise photochemical lifetime measurement of the excited states of radiation-generated aggregates is useful in characterizing their structure. Resonance light scattering (RLS) has proven to be an extremely sensitive and useful technique to detect protein aggregates.

Soluble and insoluble protein aggregates, endoplasmic reticulum stress, and vascular dysfunction in Alzheimer's disease and cardiovascular diseases.

Dementia refers to a particular group of symptoms characterized by difficulties with memory, language, problem-solving, and other thinking skills that affect a person's ability to perform everyday activities. Alzheimer's disease (AD) is the most common form of dementia, affecting about 6.2 million Americans aged 65 years and older. Likewise, cardiovascular diseases (CVDs) are a major cause of disability and premature death, impacting 126.9 million adults in the USA, a number that increases with age. Consequently, CVDs and cardiovascular risk factors are associated with an increased risk of AD and cognitive impairment. They share important age-related cardiometabolic and lifestyle risk factors, that make them among the leading causes of death. Additionally, there are several premises and hypotheses about the mechanisms underlying the association between AD and CVD. Although AD and CVD may be considered deleterious to health, the study of their combination constitutes a clinical challenge, and investigations to understand the mechanistic pathways for the cause-effect and/or shared pathology between these two disease constellations remains an active area of research. AD pathology is propagated by the amyloid β(Aβ) peptides. These peptides give rise to small, toxic, and soluble Aβ oligomers (SPOs) that are nonfibrillar, and it is their levels that show a robust correlation with the extent of cognitive impairment. This review will elucidate the interplay between the effects of accumulating SPOs in AD and CVDs, the resulting ER stress response, and their role in vascular dysfunction. We will also address the potential underlying mechanisms, including the possibility that SPOs are among the causes of vascular injury in CVD associated with cognitive decline. By revealing common mechanistic underpinnings of AD and CVD, we hope that novel experimental therapeutics can be designed to reduce the burden of these devastating diseases. Graphical abstract Alzheimer's disease (AD) pathology leads to the release of Aβ peptides, and their accumulation in the peripheral organs has varying effects on various components of the cardiovascular system including endoplasmic reticulum (ER) stress and vascular damage. Image created with BioRender.com.

Impact of Cavitation Jet on the Structural, Emulsifying Features and Interfacial Features of Soluble Soybean Protein Oxidized Aggregates

A cavitation jet can enhance food proteins' functionalities by regulating solvable oxidized soybean protein accumulates (SOSPI). We investigated the impacts of cavitation jet treatment on the emulsifying, structural and interfacial features of soluble soybean protein oxidation accumulate. Findings have shown that radicals in an oxidative environment not only induce proteins to form insoluble oxidative aggregates with a large particle size and high molecular weight, but also attack the protein side chains to form soluble small molecular weight protein aggregates. Emulsion prepared by SOSPI shows worse interface properties than OSPI. A cavitation jet at a short treating time (<6 min) has been shown to break the core aggregation skeleton of soybean protein insoluble aggregates, and insoluble aggregates into soluble aggregates resulting in an increase of emulsion activity (EAI) and constancy (ESI), and a decrease of interfacial tension from 25.15 to 20.19 mN/m. However, a cavitation jet at a long treating time (>6 min) would cause soluble oxidized aggregates to reaggregate through an anti-parallel intermolecular β-sheet, which resulted in lower EAI and ESI, and a higher interfacial tension (22.44 mN/m). The results showed that suitable cavitation jet treatment could adjust the structural and functional features of SOSPI by targeted regulated transformation between the soluble and insoluble components.

Tardigrade small heat shock proteins can limit desiccation-induced protein aggregation

Small heat shock proteins (sHSPs) are chaperones with well-characterized roles in heat stress, but potential roles for sHSPs in desiccation tolerance have not been as thoroughly explored. We identified nine sHSPs from the tardigrade Hypsibius exemplaris, each containing a conserved alpha-crystallin domain flanked by disordered regions. Many of these sHSPs are highly expressed. Multiple tardigrade and human sHSPs could improve desiccation tolerance of E. coli, suggesting that the capacity to contribute to desicco-protection is a conserved property of some sHSPs. Purification and subsequent analysis of two tardigrade sHSPs, HSP21 and HSP24.6, revealed that these proteins can oligomerize in vitro. These proteins limited heat-induced aggregation of the model enzyme citrate synthase. Heterologous expression of HSP24.6 improved bacterial heat shock survival, and the protein significantly reduced heat-induced aggregation of soluble bacterial protein. Thus, HSP24.6 likely chaperones against protein aggregation to promote heat tolerance. Furthermore, HSP21 and HSP24.6 limited desiccation-induced aggregation and loss of function of citrate synthase. This suggests a mechanism by which tardigrade sHSPs promote desiccation tolerance, by limiting desiccation-induced protein aggregation, thereby maintaining proteostasis and supporting survival. These results suggest that sHSPs provide a mechanism of general stress resistance that can also be deployed to support survival during anhydrobiosis.

Phenolic compounds in walnut pellicle improve walnut (Juglans regia L.) protein solubility under pH-shifting condition

This study focused on the interaction of walnut protein with phenolic extracts of walnut pellicle (PEWP) under alkaline condition, leading to enhancement of protein solubility under neutral condition. First, the change of PEWP under alkaline condition was determined by RP-HPLC and mass spectrometry, and the results showed that most ellagitannins in PEWP could be retained under alkaline condition within 3 h. Interaction between PEWP and walnut protein under pH-shifting condition resulted in the remarkable increase of protein solubility (above 90%) at neutral pH. The results from SDS-PAGE and SEC showed that the improved solubility lied in the formation of large and soluble protein aggregates due to the covalent interaction among walnut protein and polyphenols. A significant change in tertiary structure of protein–phenolic complex was witnessed by fluorescence spectrum and near-UV circular dichroism. Meanwhile, walnut protein–polyphenol interaction led to a slight increase in β-turn while a slight decrease in β-sheet. Combined with amino acid composition, it could be illustrated that the covalent bonding for walnut protein with polyphenol mainly occurred at Lysine residues.

Structure-specific amyloid precipitation in biofluids.

The composition of soluble toxic protein aggregates formed in vivo is currently unknown in neurodegenerative diseases, due to their ultra-low concentration in human biofluids and their high degree of heterogeneity. Here we report a method to capture amyloid-containing aggregates in human biofluids in an unbiased way, a process we name amyloid precipitation. We use a structure-specific chemical dimer, a Y-shaped, bio-inspired small molecule with two capture groups, for amyloid precipitation to increase affinity. Our capture molecule for amyloid precipitation (CAP-1) consists of a derivative of Pittsburgh Compound B (dimer) to target the cross β-sheets of amyloids and a biotin moiety for surface immobilization. By coupling CAP-1 to magnetic beads, we demonstrate that we can target the amyloid structure of all protein aggregates present in human cerebrospinal fluid, isolate them for analysis and then characterize them using single-molecule fluorescence imaging and mass spectrometry. Amyloid precipitation enables unbiased determination of the molecular composition and structural features of the in vivo aggregates formed in neurodegenerative diseases.

Characterizing Soluble Protein Aggregates Using Native Mass Spectrometry Coupled with Temperature-Controlled Electrospray Ionization and Size-Excl usion Chromatography.

Characterization of soluble protein aggregates provides valuable information for revealing mechanisms of protein aggregation process and assessing the activity and safety of protein therapeutics. However, the noncovalent interaction, the transient nature and higher degree of structural heterogeneity of the soluble aggregation system hinders precise characterization at the molecular level. Here, we describe methods using native mass spectrometry coupled with temperature-control electrospray ionization and size-exclusion chromatography to monitor the aggregation process and profile the aggregates in detail.

The Female Impact in the World of Neurodegeneration.

Scientific research fuels advances in medicine, but the contributions of individual scientists, especially women, are often underappreciated. The field of neuroscience is vast, with many important gaps in knowledge remaining; thus, it is imperative for women to be able to contribute. A pivotal 2018 study revealed that women were still shockingly underrepresented in academic neurology, representing only 30.8% of academic neurologists (McDermott et al., 2018). Of additional concern, this gender gap grows with increasing academic rank, with only 13.8% of neurology professors being women (McDermott et al., 2018). Although women have faced much adversity in the field of neuroscience, there have been pioneers who have paved a path for other women. Moreover, these women made important contributions that enhanced scientific understanding of neurodegeneration and neurodegenerative diseases. The purpose of this paper is to highlight notable, but underappreciated, female neuroscientists and their important contributions to modern understanding of neurodegeneration. Overview of Neurodegeneration The term neurodegeneration is a mixture of two words—“neuro”, which refers to nerve cells and then “degeneration”, which refers to progressive damage. The term in its entirety, “neurodegeneration”, can be used to describe various conditions that result in the loss of nerve structure and function. This deterioration of neural structures results in a loss of cognitive abilities such as memory and decision making (Murman, 2015). Loss of neural function is a key hallmark in many neurodegenerative diseases, including Parkinson's disease (PD), Huntington's disease (HD), Multiple Sclerosis (MS), Alzheimer's disease (AD), and many rare disorders (Przedborski et al., 2003). An important area of research is exploring the mechanisms that underlie neurodegeneration and plasticity; this includes investigation of proteins implicated in AD (e.g., tau; amyloid-beta) and Parkinson's (e.g., alpha-synuclein). Tau is a protein found in the brain that serves to stabilize microtubules (Gao et al., 2018); tau hyperphosphorylation results in its aggregation into insoluble tangles, a characteristic biomarker of AD. Another characteristic feature of AD is the aggregation of amyloid beta protein into extracellular deposits known as amyloid plaques (Bloom, 2014). Alpha-synuclein is a neuronal protein that normally regulates trafficking of synaptic vesicles for neurotransmitter release. If mutated, the once soluble protein aggregates into insoluble fibrils forming Lewy bodies, a hallmark feature of PD (Stefanis, 2012). Notably, there are currently no known therapies that can prevent or reverse neurodegeneration. Rather, for each particular disease, medications may be available that reduce symptom burden and improve quality of life. Ongoing research efforts focus on the similarities between neurodegenerative these diseases in the hope of 1 day finding a cure. In this manuscript, notable contributions of women to neurodegeneration research are highlighted. These contributions are vast and include discoveries about neuronal pathways, proteins, vascular effects on neurodegeneration, nerve growth factor, and novel imaging techniques. In total, 12 female neuroscientists are celebrated below, namely: Rita Levi-Montalcini, Valina L. Dawson, Eva-Maria Mandelkow, Tara Spires-Jones, Maria Grazia Spillantini, Miia K. Kivipelto, Vivian Tabar, Marian Diamond, Elizabeth Roboz-Einstein, Cecile Vogt-Mugnier, Patricia Goldman-Rakic, and Anita Harding. A timeline of select key advancements in neurodegeneration research is provided below (see Figure 1). Open in a separate window Figure 1 The above timeline outlines a few key events that contributed to knowledge of neurodegeneration. Some of the events shown are the first descriptions of neurodegenerative diseases, others are key discoveries regarding the mechanisms of neurodegeneration, one is a clinical trial.

Formation of whey protein aggregates by partial hydrolysis and reduced thermal treatment

Thermal treatment to form functional, soluble whey protein aggregates often generates undesired off-flavours. The aim of the present study was to generate soluble aggregates of whey protein isolates (WPI) at lower temperature than traditionally used by introducing partial hydrolysis of WPI prior to thermal treatment. WPI was partially hydrolyzed either by Bacillus licheniformis protease (BLP) or by a trypsin type protease (TP) at pH 7.0 and 65 °C for 1–10 min prior to thermal treatment at 80 °C for 5–10 min to generate soluble protein aggregates. Partial hydrolysis by BLP and TP induced structural changes and exposure of free thiol groups, but no change of surface hydrophobicity of WPI. After incubation with BLP or TP and thermal treatment, the yield of protein aggregates was 36–48%, which was similar to the reference sample obtained by thermal treatment at 90 °C for 10 min. In addition to non-covalent interactions, disulfide bonds also contributed to the association of protein aggregates. The soluble protein aggregates obtained by BLP partial hydrolysis and thermal treatment, had particle size range of 10–100 nm in radius which was in the same range as observed for the reference sample; and samples obtained by TP treatment had a particle size range of 7–50 nm in radius. However, aggregates obtained by BLP or TP and thermal treatment were not UHT (140 °C/5 s) stable.

Do peripheral protein oligomers in neurodegenerative diseases shape T cell responses beyond the brain?

Neurodegenerative diseases place a devastating burden on affected individuals and their families, and new treatments are desperately needed for these common immune-mediated inflammatory conditions. While large aggregates of abnormal proteins in the brain cause significant damage, it is becoming clear that smaller soluble protein aggregates can also contribute to disease, by both direct and indirect mechanisms. These soluble protein oligomers can be found in patients' serum. Here, we describe recent research that identifies effects of model oligomer molecules on peripheral blood T cells, therefore providing an additional mechanism by which neurodegeneration may be worsened through amplification of peripheral adaptive immune responses.

Partial Removal of Phenolics Coupled with Alkaline pH Shift Improves Canola Protein Interfacial Properties and Emulsion in In Vitro Digestibility.

The effect of polyphenol removal (“dephenol”) combined with an alkaline pH shift treatment on the O/W interfacial and emulsifying properties of canola seed protein isolate (CPI) was investigated. Canola seed flour was subjected to solvent extraction to remove phenolic compounds, from which prepared CPI was exposed to a pH12 shift to modify the protein structure. Dephenoled CPI had a light color when compared with an intense dark color for the control CPI. Up to 53% of phenolics were removed from the CPI after the extraction with 70% ethanol. Dephenoled CPI showed a partially unfolded structure and increased surface hydrophobicity and solubility. The particle size increased slightly, indicating that soluble protein aggregates formed after the phenol removal. The pH12 shift induced further unfolding and decreased protein particle size. Dephenoled CPI had a reduced β subunit content but an enrichment of disulfide-linked oligopeptides. Dephenol improved the interfacial rheology and emulsifying properties of CPI. Although phenol removal did not promote peptic digestion and lipolysis, it facilitated tryptic disruption of the emulsion particles due to enhanced proteolysis. In summary, dephenol accentuated the effect of the pH shift to improve the overall emulsifying properties of CPI and emulsion in in vitro digestion.

Detection of Protein Aggregation using Fluorescence Correlation Spectroscopy.

Protein aggregation is a hallmark of neurodegenerative disorders such as amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), and so on. To detect and analyze soluble or diffuse protein oligomers or aggregates, fluorescence correlation spectroscopy (FCS), which can detect the diffusion speed and brightness of a single particle with a single molecule sensitivity, has been used. However, the proper procedure and know-how for protein aggregation detection have not been widely shared. Here, we show a standard procedure of FCS measurement for diffusion properties of aggregation-prone proteins in cell lysate and live cells: ALS-associated 25 kDa carboxyl-terminal fragment of TAR DNA/RNA-binding protein 43 kDa (TDP25) and superoxide dismutase 1 (SOD1). The representative results show that a part of aggregates of green fluorescent protein (GFP)-tagged TDP25 was slightly included in the soluble fraction of murine neuroblastoma Neuro2a cell lysate. Moreover, GFP-tagged SOD1 carrying ALS-associated mutation shows a slower diffusion in live cells. Accordingly, we here introduce the procedure to detect the protein aggregation via its diffusion property using FCS.

Improvement in the functional properties of quinoa (Chenopodium quinoa) protein isolates after the application of controlled heat-treatment: Effect on structural properties

Quinoa protein isolates (QPIs) were subjected to controlled heat-treatment (80−100 °C) at variable time (15−30 min). Results showed that heat-treatment induced structural changes in QPIs. Circular dichroism showed loss of secondary structure of QPIs, particularly α-helix and β-sheet. Total and exposed SH groups were significantly (p ≤ 0.05) lower in heat-treated QPIs. SDS-PAGE profile showed formation of soluble protein aggregates with smaller particle size. Particle size decreased when QPIs were heated at 80 °C for 30 min. Solubility and turbidity of heat-treated QPIs improved significantly. Decline in crystallinity was due to denaturation of protein molecules by heat-treatment. Water binding capacity, oil binding capacity, emulsifying activities and stabilities were found lower in QPIs heat-treated at higher thermal temperatures for longer time. This might be due to decline in total and exposed SH contents, accelerated conversion of SH groups into disulfide bonds and formation of large molecular weight polymers or protein aggregates due to polymerization reactions. Higher foaming capacity (89.99 %) and foaming stability values of 167.80 % (10 min) and 124.15 % (20 min) were found in QPIs heated at 80 °C for 30 min. The results thus suggests that heat-treatment carried out at lower heating temperatures had improved the functional properties of QPIs.