Accelerates Protein Aggregation Research Articles

Starch hydration properties in relation to kinetic modelling of mass transfer and properties of deep-frying batter

The hydration properties of starch were examined in relation to mass transfer and the characteristics of deep-frying batter. With extended deep-frying, the oil content in all samples increased, while the moisture content exhibited an inverse trend. Analyses of zeta potential, disulfide bonds, secondary structure, particle size of the proteins, thermal properties, and water status indicated that prolonged deep-frying time facilitated protein aggregation and led to a decrease in both gelatinization enthalpy (ΔH) and the percentage of bound water. Additionally, deep-frying batter containing starch with medium hydration properties (carboxymethyl freeze-thawed tapioca starch), accelerated protein aggregation, delayed the dissipation of bound water, and significantly reduced both gelatinization and ΔH. This ultimately weakened the intensity of mass transfer, thereby diminishing oil distribution. The Pearson’s correlation test confirmed a negative correlation between mass transfer intensity and starch hydration properties, indicating that both parameters significantly influenced the characteristics of the deep-frying batter.

Polyamines putrescine and spermidine as modulators of protein aggregation rate: The effect on DTT-induced aggregation of α-lactalbumin

Protein aggregation is undesirable for cells due to its possible toxicity, and is also undesirable in biotechnology and pharmaceuticals. Polyamines are known to be capable of both suppressing and stimulating protein aggregation. In the present work polyamines (spermidine, putrescine) have been shown to alter the pathway of α-lactalbumin aggregation induced by dithiothreitol, leading to the formation of larger protein particles during the initial stages of aggregation and promoting the later stage of sticking of aggregates. According to the aggregation kinetics data, polyamines accelerate protein aggregation in a concentration-dependent manner, with a maximum at 50 mM spermidine and 100 mM putrescine. With a further increase in polyamines concentration the effect of aggregation acceleration decreased, thus, the modulation of the aggregation rate by polyamines was shown. A comparison of the aggregation kinetics and hydrodynamic radii growth data registered by dynamic light scattering with the data obtained by asymmetric flow field-flow fractionation and analytical ultracentrifugation allowed us to describe the early stages of aggregation and formation of initial α-lactalbumin clusters. Our results provide a deeper insight into the mechanism of amorphous aggregation of α-lactalbumin and polyamines action on protein aggregation and protein-protein interaction in general.

Copper toxicity and deficiency: the vicious cycle at the core of protein aggregation in ALS.

The pathophysiology of ALS involves many signs of a disruption in copper homeostasis, with both excess free levels and functional deficiency likely occurring simultaneously. This is crucial, as many important physiological functions are performed by cuproenzymes. While it is unsurprising that many ALS symptoms are related to signs of copper deficiency, resulting in vascular, antioxidant system and mitochondrial oxidative respiration deficiencies, there are also signs of copper toxicity such as ROS generation and enhanced protein aggregation. We discuss how copper also plays a key role in proteostasis and interacts either directly or indirectly with many of the key aggregate-prone proteins implicated in ALS, such as TDP-43, C9ORF72, SOD1 and FUS as well as the effect of their aggregation on copper homeostasis. We suggest that loss of cuproprotein function is at the core of ALS pathology, a condition that is driven by a combination of unbound copper and ROS that can either initiate and/or accelerate protein aggregation. This could trigger a positive feedback cycle whereby protein aggregates trigger the aggregation of other proteins in a chain reaction that eventually captures elements of the proteostatic mechanisms in place to counteract them. The end result is an abundance of aggregated non-functional cuproproteins and chaperones alongside depleted intracellular copper stores, resulting in a general lack of cuproenzyme function. We then discuss the possible aetiology of ALS and illustrate how strong risk factors including environmental toxins such as BMAA and heavy metals can functionally behave to promote protein aggregation and disturb copper metabolism that likely drives this vicious cycle in sporadic ALS. From this synthesis, we propose restoration of copper balance using copper delivery agents in combination with chaperones/chaperone mimetics, perhaps in conjunction with the neuroprotective amino acid serine, as a promising strategy in the treatment of this incurable disease.

Impact of Confinement within a Hydrogel Mesh on Protein Thermodynamic Stability and Aggregation Kinetics.

Though protein stability and aggregation have been well characterized in dilute solutions, the influence of a confining environment that exists (e.g., in intercellular and tissue spaces and therapeutic formulations) on the protein structure is largely unknown. Herein, the effects of confinement on stability and aggregation were explored for proteins of different sizes, stability, and hydrophobicity when encapsulated in hydrophilic poly(ethylene glycol) hydrogels. Denaturation curves show linear correlations between confinement size (mesh size) and thermodynamic stability, i.e., unfolding free energy and surface area accessible for solvation (represented by m-value). Two clusters of protein types are identifiable from these correlations; the clusters are defined by differences in protein stability, surface area, and aggregation propensity. Proteins with higher stability, larger surface area, and lower aggregation propensity (e.g., lysozyme and myoglobin) are less affected by the confinement imposed by mesh size than proteins with lower stability, smaller surface area, and higher aggregation propensity (e.g., growth hormone and aldehyde dehydrogenase). According to aggregation kinetics measured by thioflavin T fluorescence, confinement in smaller mesh sizes resulted in slower aggregation rates than that in larger mesh sizes. Compared to that in buffer solution, the confinement of a hydrophobic protein (e.g., human insulin) in the hydrogels accelerates protein aggregation. Conversely, the confinement of a hydrophilic protein (e.g., human amylin) in the hydrogels decelerates or prevents aggregation, with the rates of aggregation inversely proportional to mesh size. These findings provide new insights into protein conformational stability in confined microenvironments relevant to various cellular, tissue, and therapeutics scenarios.

The metabolic slowdown caused by the deletion of pspA accelerates protein aggregation during stationary phase facilitating antibiotic persistence.

Entering a dormant state is a prevailing mechanism used by bacterial cells to transiently evade antibiotic attacks and become persisters. The dynamic progression of bacterial dormancy depths driven by protein aggregation has been found to be critical for antibiotic persistence in recent years. However, our current understanding of the endogenous genes that affects dormancy depth remains limited. Here, we discovered a novel role of phage shock protein A (pspA) gene in modulating bacterial dormancy depth. Deletion of pspA of Escherichia coli resulted in increased bacterial dormancy depths and prolonged lag times for resuscitation during the stationary phase. ∆pspA exhibited a higher persister ratio compared to the wild type when challenged with various antibiotics. Microscopic images revealed that ∆pspA showed accelerated formation of protein aggresomes, which were collections of endogenous protein aggregates. Time-lapse imaging established the positive correlation between protein aggregation and antibiotic persistence of ∆pspA at the single-cell level. To investigate the molecular mechanism underlying accelerated protein aggregation, we performed transcriptome profiling and found the increased abundance of chaperons and a general metabolic slowdown in the absence of pspA. Consistent with the transcriptomic results, the ∆pspA strain showed a decreased cellular ATP level, which could be rescued by glucose supplementation. Then, we verified that replenishment of cellular ATP levels by adding glucose could inhibit protein aggregation and reduce persister formation in ∆pspA. This study highlights the novel role of pspA in maintaining proteostasis, regulating dormancy depth, and affecting antibiotic persistence during stationary phase.

Protein Compartments Modulate Fibrillar Self-Assembly.

A notable feature of complex cellular environments is protein-rich compartments that are formed via liquid-liquid phase separation. Recent studies have shown that these biomolecular condensates can play both promoting and inhibitory roles in fibrillar protein self-assembly, a process that is linked to Alzheimer's, Parkinson's, Huntington's, and various prion diseases. Yet, the exact regulatory role of these condensates in protein aggregation remains unknown. By employing microfluidics to create artificial protein compartments, the self-assembly behavior of the fibrillar protein lysozyme within them can be characterized. It is observed that the volumetric parameters of protein-rich compartments can change the kinetics of protein self-assembly. Depending on the change in compartment parameters, the lysozyme fibrillation process either accelerated or decelerated. Furthermore, the results confirm that the volumetric parameters govern not only the nucleation and growth phases of the fibrillar aggregates but also affect the crosstalk between the protein-rich and protein-poor phases. The appearance of phase-separated compartments in the vicinity of natively folded protein complexes triggers their abrupt percolation into the compartments' core and further accelerates protein aggregation. Overall, the results of the study shed more light on the complex behavior and functions of protein-rich phases and, importantly, on their interaction with the surrounding environment.

Condensate interfaces can accelerate protein aggregation

Protein aggregates, formed from the assembly of aberrant, misfolded proteins, are a hallmark of neurodegenerative diseases. Disease-associated aggregates such as mutant Huntingtin polyQ inclusions, are typically enriched in p62/SQSTM1, an oligomeric protein that binds to and sequesters aberrant proteins. p62 has been suggested to sequester proteins through formation of liquid-like biomolecular condensates, but the physical mechanisms by which p62 condensates may regulate pathological protein aggregation remain unclear. Here, we use a light-inducible biomimetic condensate system to show that p62 condensates enhance coarsening of mutant polyQ aggregates through interface-mediated sequestration, which accelerates polyQ accumulation into larger aggregates. However, the resulting large aggregates accumulate polyubiquitinated proteins, which depletes free p62, ultimately suppressing further p62 condensation. This dynamic interplay between interface-mediated coarsening of solid aggregates and downstream consequences on the phase behavior of associated regulatory proteins could contribute to the onset and progression of protein aggregation diseases.

Elucidation of the Role of Lipids in Late Endosomes on the Aggregation of Insulin.

Abrupt aggregation of misfolded proteins is the underlying molecular cause of numerous pathologies including diabetes type 2 and injection amyloidosis. Although the exact cause of this process is unclear, a growing body of evidence suggests that protein aggregation is linked to a high protein concentration and the presence of lipid membranes. Endosomes are cell organelles that often possess high concentrations of proteins due to their uptake from the extracellular space. However, the role of endosomes in amyloid pathologies remains unclear. In this study, we used a set of biophysical methods to determine the role of bis(monoacylglycero)phosphate (BMP), the major lipid constituent of late endosomes on the aggregation properties of insulin. We found that both saturated and unsaturated BMP accelerated protein aggregation. However, very little if any changes in the secondary structure of insulin fibrils grown in the presence of BMP were observed. Therefore, no changes in the toxicity of these aggregates compared to the fibrils formed in the lipid-free environment were observed. We also found that the toxicity of insulin oligomers formed in the presence of a 77:23 mol/mol ratio of BMP/PC, which represents the lipid composition of late endosomes, was slightly higher than the toxicity of insulin oligomers formed in the lipid-free environment. However, the toxicity of mature insulin fibrils formed in the presence of BMP/PC mixture was found to be lower or similar to the toxicity of insulin fibrils formed in the lipid-free environment. These results suggest that late endosomes are unlikely to be the source of highly toxic protein aggregates if amyloid proteins aggregate in them.

The application of Melissa officinalis L. essential oil nanoemulsions protects sea bass (Lateolabrax japonicus) against myofibrillar protein and lipid oxidation during refrigeration

Abstract Objectives The nutrient rich sea bass is prone to oxidation of lipid and protein during refrigeration. Materials and Methods The research was to investigate the effect of different concentrations of Melissa officinalis L. essential oil (MOEO) nanoemulsions on myofibrillar protein (MP) and lipid oxidation in sea bass (Lateolabrax japonicus) during refrigeration at 4 °C. Results The results of thiobarbituric acid reactive substances and mitochondrial membrane potential showed that carboxymethyl chitosan/locust bean gum active coating solutions incorporating 2% MOEO nanoemulsions (C/L-2M) was the most effective in inhibiting lipid oxidation that occurred in sea bass under attack by reactive oxygen species. Low-field nuclear magnetic resonance results showed that C/L-2M maximally slowed the conversion of bound water to free water during storage. The oxidation of lipids and MP disrupted the secondary and tertiary conformations of MP and accelerated protein aggregation and degradation. Conclusions C/L-2M slowed the oxidation of lipids and proteins by inhibiting the oxidation of reactive oxygen species. C/L-2M is a very promising preservative emulsion for the preservation of sea bass.

Effects of grain storage time and storage temperature on flour quality parameters of “5 + 10” wheat cultivar and “2 + 12” wheat cultivar

AbstractBackgrounds and ObjectivesPhysicochemical changes that take place inside the wheat kernel during storage make the quality stable. Storage temperature will affect the different mechanisms of changes. The newly harvested “2 + 12” wheat cultivar (H4399) and “5 + 10” wheat cultivar (XM26) grains were stored under ambient temperature and 4°C for 120 days, respectively, and sampled for grinding every 30 days.FindingsWith the extension of storage time, the content of crude protein and glutenin macropolymer (GMP), Gʹ and G" of GMP gel, maximum resistance, and extensibility of both varieties increased to higher value and then decreased slightly. The higher value appeared at 90 or 120 days under ambient temperature, and at 30 or 60 days under 4°C, approximately 30–60 days earlier than under ambient temperature. During storage, the relative increasing of protein and GMP content, sedimentation index, G", tan δ, stability, maximum resistance, and extendibility of dough of “2 + 12” wheat cultivar (H4399) were bigger than “5 + 10” wheat cultivar (XM26). The relative increasing of Gʹ, water absorption, development time of dough were smaller than “5 + 10” wheat cultivar (XM26).ConclusionsIt is concluded that low‐temperature storage can accelerate protein aggregation in wheat grains and shorten the time to steady state. Increasing in GMP stiffness and dough development time is more sensitive to postharvest storage in a “5 + 10” wheat cultivar (XM26) versus a “2 + 12” wheat cultivar (H4399).Significance and NoveltyThe changes of protein physicochemical properties of different varieties under different storage conditions explained the main mechanism of protein aggregation during wheat storage.

Imaging Cataract-Specific Peptides in Human Lenses.

Age-related protein truncation is a common process in long-lived proteins such as proteins found in the ocular lens. Major truncation products have been reported for soluble and membrane proteins of the lens, including small peptides that can accelerate protein aggregation. However, the spatial localization of age-related protein fragments in the lens has received only limited study. Imaging mass spectrometry (IMS) is an ideal tool for examining the spatial localization of protein products in tissues. In this study we used IMS to determine the spatial localization of small crystallin fragments in aged and cataractous lenses. Consistent with previous reports, the pro-aggregatory αA-crystallin 66-80 peptide as well as αA-crystallin 67-80 and γS-crystallin 167-178 were detected in normal lenses, but found to be increased in nuclear cataract regions. In addition, a series of γS-crystallin C-terminal peptides were observed to be mainly localized to cataractous regions and barely detected in transparent lenses. Other peptides, including abundant αA3-crystallin peptides were present in both normal and cataract lenses. The functional properties of these crystallin peptides remain unstudied; however, their cataract-specific localization suggests further studies are warranted.

Identifying C. elegans lifespan mutants by screening for early-onset protein aggregation

Genetic screens are widely used to identify genes that control specific biological functions. In Caenorhabditis elegans, forward genetic screens rely on the isolation of reproductively active mutants that can self-propagate clonal populations. Screens that target post-reproductive phenotypes, such as lifespan, are thus challenging. We combine microfluidic technologies and image processing to perform high-throughput automated screening for short-lived mutants using protein aggregation as a marker for aging. We take advantage of microfluidics for maintaining a reproductively active adult mutagenized population and for performing serial high-throughput analysis and sorting of animals with increased protein aggregation, using fluorescently-labeled PAB-1 as a readout. We demonstrate that lifespan mutants can be identified by screening for accelerated protein aggregation through quantitative analysis of fluorescently labeled aggregates while avoiding conditional sterilization or manual separation of parental and progeny populations. We also show that aged wildtypes and premature aggregation mutants differ in aggregate morphology, suggesting aggregate growth is time-dependent.

The effect of sodium tartrate and sodium citrate on quality changes of squid (Dosidicus gigas) surimi gel.

The yield of squid has grown gradually; however, the lack of intensive processing has led to the slow development of the squid industry. In a previous study, some organic salts were found to improve the quality of squid surimi gel. Therefore, this research focused on the effects of sodium citrate (SC) and sodium tartrate (ST) on the quality of squid surimi gel. Physical measurements, such as gel texture, water-holding capacity, and color of squid surimi gel, were conducted. The addition of 2.5% SC and ST significantly improved the gel strength by 40.7, 57.0, 22.9, and 58.1%, respectively, of gel strength compared with the addition of: 1.5 SC, 3.5 SC, 1.5 ST, and 3.5% ST alone. Rheological measurements revealed that the addition of 2.5% SC or ST shortened the gel degradation temperature range (i.e., 2.5% SC or ST: 40-53°C; other treatments: 40-60°C) of the squid paste during heating. Results of chemical force analysis showed that the addition of a high quantity of salt accelerated protein aggregation and reduced hydrophobic interactions and disulfide bond formation. Finally, an increase in the number of β-sheets and a decrease in the bulk water content demonstrated that the addition of 2.5% organic salt could form squid gel with a better network structure. The findings provide a scientific basis for the development of high-quality squid surimi gel.

Peripheral neuropathy, protein aggregation and serotonergic neurotransmission: Distinctive bio-interactions of thiacloprid and thiamethoxam in the nematode Caenorhabditis elegans

Due to worldwide production, sales and application, neonicotinoids dominate the global use of insecticides. While, neonicotinoids are considered as pinpoint neurotoxicants that impair cholinergic neurotransmission in pest insects, the sublethal effects on nontarget organisms and other neurotransmitters remain poorly understood. Thus, we investigated long-term neurological outcomes in the decomposer nematode Caenorhabditis elegans. In the adult roundworm the neonicotinoid thiacloprid impaired serotonergic and dopaminergic neuromuscular behaviors, while respective exposures to thiamethoxam showed no effects. Thiacloprid caused a concentration-dependent delay of the transition between swimming and crawling locomotion that is controlled by dopaminergic and serotonergic neurotransmission. Age-resolved analyses revealed that impairment of locomotion occurred in young as well as middle-aged worms. Treatment with exogenous serotonin rescued thiacloprid-induced swimming deficits in young worms, whereas additional exposure with silica nanoparticles enhanced the reduction of swimming behavior. Delay of forward locomotion was partly caused by a new paralysis pattern that identified thiacloprid as an agent promoting a specific rigidity of posterior body wall muscle cells and peripheral neuropathy in the nematode (lowest-observed-effect-level 10 ng/ml). On the molecular level exposure with thiacloprid accelerated protein aggregation in body wall muscle cells of polyglutamine disease reporter worms indicating proteotoxic stress. The results from the soil nematode Caenorhabditis elegans show that assessment of neurotoxicity by neonicotinoids requires acknowledgment and deeper research into dopaminergic and serotonergic neurochemistry of nontarget organisms. Likewise, it has to be considered more that different neonicotinoids may promote diverse neural end points.

Enhanced protein aggregation suppressor activity of N-acetyl-l-arginine for agitation-induced aggregation with silicone oil and its impact on innate immune responses

Previously, N-acetyl-l-arginine (NALA) suppressed the aggregation of intravenous immunoglobulins (IVIG) more effectively and with a minimum decrease in transition temperature (Tm) than arginine monohydrochloride. In this study, we performed a comparative study with etanercept (commercial product: Enbrel®), where 25 mM arginine monohydrochloride (arginine) was added to the prefilled syringe. The biophysical properties were investigated using differential scanning calorimetry (DSC), dynamic light scattering (DLS), size-exclusion chromatography (SEC), and flow-imaging microscopy (FI). NALA retained the transition temperature of etanercept better than arginine, where arginine significantly reduced the Tm by increasing its concentration. End-over-end rotation was applied to each formulation for 5 days to accelerate protein aggregation and subvisible particle formation. Higher monomeric content was retained with NALA with a decrease in particle level. Higher aggregation onset temperature (Tagg) was detected for etanercept with NALA than arginine. The results of this comparative study were consistent with previous study, suggesting that NALA could be a better excipient for liquid protein formulations. Agitated IVIG and etanercept were injected into C57BL/6J female mice to observe immunogenic response after 24 h. In the presence of silicone oil, NALA dramatically reduced IL-1 expression, implying that decreased aggregation was related to reduced immunogenicity of both etanercept and IVIG.

Failure Mode Identification of Insulin Drug Products – Impact of Relevant Stress Conditions on the Quality of the Drug

Fast-acting insulin drug products (DPs) are carried and administered by diabetic patients to maintain their blood glucose level throughout the day, exposing the DPs to stress conditions. Apidra, Novolog, and Humalog insulin DPs were tested under various stress conditions. Dynamic light scattering (DLS), and size exclusion chromatography (SEC) were used to monitor the stability and aggregation. Thermal stress alone did not influence the stability. However, 24 hr exposure to vigorous mechanical stress shifted the DLS size peaks of Novolog and Humalog from 5 ± 1 nm to > 50.9 ± 25.6 nm, and the SEC native protein peak areas decreased 52% for Novolog and 18.4% for Humalog. Combined stress accelerated protein aggregation more drastically. Novolog and Humalog size shifted (>75 nm) after 3 hr and the peak area decreased > 97.9% after 6 hr exposure, indicating that high temperature accelerated the aggregation triggered by agitation. Soluble aggregates were captured by DLS early on compared to SEC. Apidra was comparably stable indicating DP formulation plays a critical role in stability. Our study provides a greater understanding of potential failure modes patients and care givers may encounter while handling insulin DPs.

Probing the Role of Cu(II) Ions on Protein Aggregation Using Two Model Proteins.

Copper is an essential trace element for human biology where its metal dyshomeostasis accounts for an increased level of serum copper, which accelerates protein aggregation. Protein aggregation is a notable feature for many neurodegenerative disorders. Herein, we report an experimental study using two model proteins, bovine serum albumin (BSA) and human serum albumin (HSA), to elucidate the mechanistic pathway by which serum albumins get converted from a fully folded globular protein to a fibril and an amorphous aggregate upon interaction with copper. Steady-state fluorescence, time-resolved fluorescence studies, and Raman spectroscopy were used to monitor the unfolding of serum albumin with increasing copper concentrations. Steady-state fluorescence studies have revealed that the fluorescence quenching of BSA/HSA by Cu(II) has occurred through a static quenching mechanism, and we have evaluated both the quenching constants individually. The binding constants of BSA–Cu(II) and HSA–Cu(II) were found to be 2.42 × 104 and 0.05 × 104 M–1, respectively. Further nanoscale morphological changes of BSA mediated by oligomers to fibril and HSA to amorphous aggregate formation were studied using atomic force microscopy. This aggregation process correlates with the Stern–Volmer plots in the absence of discernible lag phase. Raman spectroscopy results obtained are in good agreement with the increase in antiparallel β-sheet structures formed during the aggregation of BSA in the presence of Cu(II) ions. However, an increase in α-helical fractions is observed for the amorphous aggregate formed from HSA.

Improvement of ultrasound-assisted thermal treatment on organoleptic quality, rheological behavior and flavor of defective dry-cured ham

Intense adhesiveness, softness and sourness are usually organoleptic and textural defects in dry-cured ham. To improve sensory and flavor qualities of defective dry-cured ham, biceps femoris of Jinhua ham with sensory defects were subjected to different levels of ultrasound-assisted heating (control group, 300 W & 40 °C, 300 W & 50 °C, 1000 W & 40 °C, and 1000 W & 50 °C). Sensory scores, texture, dynamic rheology, thermostability, water distribution and volatile compounds of ham samples were investigated among these treatments. The treatment (1000 W & 50 °C) improved sensory scores than that of other treatments (P < 0.001) and a decrease of 0.65-folds in adhesiveness values was observed at the group of 1000 W & 50 °C compared with the control; the enthalpy of actin decreased and the population of free water from 2.71% of control group increased to 11.35% of 1000 W & 50 °C, indicating that intense denaturation of proteins could accelerate protein aggregation and decrease the interaction between water and protein molecules, which further contributed to the improvement of texture of defective dry-cured ham after ultrasound. Furthermore, the treatment of 1000 W & 50 °C decreased the content of rancid and sour compounds, and accelerated the formation of esters. Partial least square regression confirmed that the decrease of rancid and sour compounds improved flavor quality of defective dry-cured ham.

Theoretical Studies on the Effect of Isomerized Aspartic Acid Residues on the Three-Dimensional Structures of Bovine Pancreatic Ribonucleases A

Isomerized aspartic acid (Asp) residues have previously been identified in various aging tissues, and are suspected to contribute to age-related diseases. Asp-residue isomerization occurs nonenzymatically under physiological conditions, resulting in the formation of three types of isomerized Asp (i.e., L-isoAsp, D-Asp, and D-isoAsp) residues. Asp-residue isomerization often accelerates protein aggregation and insolubilization, making structural biology analyses difficult. Recently, Sakaue et al. reported the synthesis of a ribonuclease A (RNase A) in which Asp121 was artificially replaced with different isomerized Asp residues, and experimentally demonstrated that the enzymatic activities of these artificial mutants were completely lost. However, their structural features have not yet been elucidated. In the present study, the three-dimensional (3D) structures of these artificial-mutant RNases A were predicted using molecular dynamics (MD) simulations. The 3D structures of wild-type and artificial-mutant RNases A were converged by 3000-ns MD simulations. Our computational data show that the structures of the active site and the formation frequencies of the appropriate catalytic dyad structures in the artificial-mutant RNases A were quite different from wild-type RNase A. These computational findings may provide an explanation for the experimental data which show that artificial-mutant RNases A lack enzymatic activity. Herein, MD simulations have been used to evaluate the influences of isomerized Asp residues on the 3D structures of proteins.

Effect of temperature and pH on the gelation, rheology, texture, and structural properties of whey protein and sugar gels based on Maillard reaction.

This study aimed to determine the effect of initial pH and temperature on whey protein gel formation via the Maillard reaction, including changes in gel structure, rheological and texture properties. The color changes in the whey protein and glucose gels were not significant with increasing heat temperature. High temperature and alkaline conditions promoted exposure to hydrophobic groups such as -SH, which accelerated protein aggregation and gel formation. Moreover, the increased particle size and additional hydrophobic groups contributed to higher elastic modulus (G') in the whey protein gel. Fluorescence measurements revealed that more tryptophan on the protein surface decreased with increasing temperature, which indicated that exposure to tryptophan could increase the hydrophobicity of the protein gels. Whey proteins formed stronger, gummier, more elastic, and more cohesive gels at 70 ℃ under initial pH 9 conditions, which also increased with the addition of fructose.