Search Results (140)

Ganoderma sichuanense is a widely studied medicinal mushroom, but the production of its antioxidant proteins has been scarcely evaluated. We assess the influence of different concentrations of culture media components under submerged fermentation, with and without agitation, on production of proteins with antioxidant activity from the mycelial biomass of G. sichuanense. Protein extracts were characterized by scanning electron microscopy, X-ray diffraction, and attenuated total reflectance Fourier-transform infrared spectroscopy. They were also analyzed for total protein and phenolic contents, antioxidant activities (ABTS^•+, DPPH^•, chelating ability, and reducing power), and electrophoretic profiles by SDS-PAGE. The most active extract was tested for cytoprotective potential under H₂O₂-induced oxidative stress in Saccharomyces cerevisiae. Growth kinetics of the best fermentation condition were also analyzed. Microstructural differences ranged from fibrillar to aggregated forms, depending on cultivation. Crystallinity was unaffected, but chemical differences and secondary structure organization were confirmed by infrared spectroscopy. The extract from the static culture with 10 g·L⁻¹ glucose, 5 g·L⁻¹ yeast extract, and 2.5 g·L⁻¹ soy peptone (referred as CM1S) showed the highest protein and phenolic contents and the strongest antioxidant activity (IC₅₀ = 4.8 and 24.0 µg of protein·mL⁻¹ for ABTS^•+ and DPPH^•, respectively). SDS-PAGE revealed higher protein band intensities in static cultures. CM1S showed potential to protect yeast cells from oxidative stress. The Gompertz model estimated a specific growth rate of 0.0068 h⁻¹ in CM1S. The findings highlight a cultivation strategy that modulates fungal metabolism and improves the recovery of antioxidant proteins from G. sichuanense biomass. Full article

Antibiotic persistence is a transient phenotype in which a subset of genetically susceptible bacteria survives lethal antibiotic exposure without acquiring resistance. However, survival alone does not define a persister cell—only cells that successfully recover, resume growth, and produce viable progeny complete the persister cycle. Recent studies in Escherichia coli show that persister awakening is a multistage process shaped by dormancy depth, metabolic state, and antibiotic-induced damage. Upstream induction mechanisms, including stringent-response signaling and toxin–antitoxin–mediated growth arrest, primarily determine dormancy depth but do not directly control awakening kinetics. During the lag phase, persister cells undergo coordinated recovery involving detoxification of residual antibiotics, ATP restoration, dissolution of protein aggregates, and ribosome reactivation. After exposure to fluoroquinolones, awakening additionally requires SOS-driven DNA repair via homologous recombination or transcription-coupled repair. In contrast, β-lactam–exposed persister cells rely mainly on efflux-mediated detoxification and asymmetric damage partitioning. Failure to restore proteostasis or resolve damage results in abortive recovery or cell death. Only after damage processing and metabolic reactivation can persister cells resume division and generate viable progeny. This review integrates current molecular insights into persister cell recovery in E. coli, highlighting the lag phase as the critical barrier between survival and true persistence. Full article

The formation of mixed adsorption layers of amyloid fibrils of a plant protein, oat globulin (OG), and a strong polyelectrolyte, sodium polystyrene sulfonate (PSS), at the liquid–gas interface was studied by measurements of the kinetic dependencies of surface tension, dynamic surface elasticity, and ellipsometric angle. The micromorphology of the layers was determined by atomic force microscopy. A strong increase in the surface elasticity was discovered when both components had similar concentrations and formed a network of threadlike aggregates at the interface, thereby explaining the high foam stability in this concentration range. The sequential adsorption of PSS and OG resulted in the formation of thick mixed multilayers and the surface elasticity increased with the number of duplex layers. Full article

Neurodegenerative diseases such as Alzheimer’s disease, Parkinson’s disease, and Huntington’s disease are characterized by progressive neuronal loss, driven mainly by the misfolding, aggregation, and accumulation of each disease’s specific proteins. These pathogenic aggregates, including tau, α-synuclein, TDP-43, and huntingtin, disrupt cellular proteostasis and initiate cascades of neuroinflammation, oxidative stress, mitochondrial dysfunction, and synaptic failure. While protein aggregation has been a long-recognized hallmark of these disorders, growing evidence points towards a more complex interplay of initial molecular pathways with defects in RNA processing, stress granule pathology, and cell-type-specific vulnerability. Notably, such events may manifest differentially with respect to sex and are further modulated by age-related loss of the protein quality control processes like the ubiquitin–proteasome pathway, autophagy–lysosome pathway, and molecular chaperones. This review synthesizes current insights into the structural and functional dynamics of protein aggregation and its significance for neuronal well-being. It highlights the role of post-translational modifications, prion-like transmission, and aggregation kinetics in the regulation of toxicity. The review further discusses promising therapeutic strategies centered on restoring proteostasis, including small molecules that inhibit aggregation, protein clearance pathway enhancers, immunotherapy, antioxidant therapy, and diagnostic prospects such as the identification of reliable molecular signatures in bodily fluids that can reflect pathological changes even before clinical symptoms emerge. Advancements in single-cell transcriptomics and multi-omics platforms, which are changing our understanding of disease onset and progression and opening avenues for precision medicine and personalized treatments, were also discussed. Ultimately, deciphering the molecular logic that distinguishes physiological from pathological protein assemblies and understanding how cellular systems fail to adapt under stress will be key to the development of effective, disease-modifying therapies for these debilitating disorders. Full article

The kinetic dependencies of the surface pressure, the dilational dynamic surface elasticity and ellipsometric angles of cupin-1.2 and vicilin solutions in 8M urea were measured at different concentrations. The analysis of these kinetics dependencies and the obtained master curves allowed us to determine a few adsorption steps in the investigated systems and showed that the master curves are individual characteristics of the protein for a given solvent. At the same time, the shape of these curves can be different for adsorbed and spread layers of plant proteins indicating different structures of these layers. The dependencies of the dynamic surface elasticity on surface pressure are non-monotonic, unlike the corresponding results for most of the solutions of the investigated plant proteins. The extremums of these dependencies can be connected to the formation of the distal region of the surface layer in agreement with the theory for the surface viscoelasticity of polymer solutions. Full article

This study explores the structural transitions and aggregation behaviour of recombinant β- and γ-synucleins from five vertebrate species—Cyprinus carpio, Danio rerio, Xenopus laevis, Anolis carolinensis, and Homo sapiens—using thioflavin T fluorescence and circular dichroism spectroscopy, with and without copper ions. Although synucleins are well-conserved proteins among vertebrates, species-specific differences in amino acid composition and predicted secondary structures were observed, particularly within β-strand-forming regions. During a six-day incubation, human β-synuclein exhibited a time-dependent increase in β-sheet-rich structures, while non-mammalian β-synucleins showed limited variation. In contrast, γ-synucleins from all species displayed greater aggregation propensity, with variations in kinetics and magnitude. The presence of copper reduced the rate of aggregation in human β-synuclein, likely due to high-affinity metal-binding sites, whereas γ-synuclein aggregation was only mildly affected. Notably, copper enhanced late-phase aggregation in A. carolinensis β-synuclein. These findings suggest that sequence divergence among synuclein isoforms may underlie species-specific aggregation mechanisms and metal sensitivity. The differential aggregation behaviour observed across taxa may reflect evolutionary adaptations in synuclein function and folding propensity, with implications for understanding the molecular basis of synucleinopathies and their potential modulation by metal ions. Full article

The kinetics of insulin aggregation and fibril formation were studied in vitro using Scanning Electron Microscopy (SEM) and Fourier Transform Infrared (FTIR) spectroscopy. Our investigation centered on the protein’s morphological and structural changes to better understand the transient molecular configurations that occur during the lag phase. SEM images showed that, already at early incubation stages, a network of disordered pseudo-filaments, ranging in length between 200 and 500 nanometers, develops on the surface of large aggregates. At later stages, fibrils catalyzed by protein aggregates were observed. Principal Component Analysis (PCA) of the FTIR data identified signatures of intramolecular β-sheet secondary structures forming during the lag phase and at the onset of the exponential growth phase. These absorption bands are linked to secondary nucleation mechanisms due to their transient nature. This interpretation is further supported by a chemical equilibrium model, which yielded a reliable secondary nucleation rate constant, K₂, on the order of 10⁴ M⁻² s⁻¹. Full article

The underlying structural features of the mismatch between catalytic and cytostatic properties in L-asparaginase from Rhodospirillum rubrum (RrA) and three of its mutants were investigated. The rationale for selecting the specific mutations (RrA_{A64V, E67K}; RrA_{R118H, G120R}; RrA_{E149R, V150P, F151T}) is to elucidate the role of inter-subunit interaction in RrA and its impact on catalytic efficiency and stability. Bioinformatic modeling revealed a predominantly negative surface charge on RrA with limited positive charge clusters in the vicinity of the interface region. Thus, some negatively charged groups were replaced with positively charged ones to enhance the electrostatic interactions and stabilize the enzyme quaternary structure. RrA_{A64V, E67K} and RrA_{R118H, G120R} additionally contained an N-terminal 17-amino acid capsid peptide derived from the bacteriophage T7 (MASMTGGQQMGRGSSRQ), which could potentially affect the conformational stability of theenzymes. Circular dichroism (CD) spectroscopy was applied to the kinetic parameters analysis of Asn hydrolysis and showed that native RrA displayed a V_max of 30 U/mg and a K_M of 4.5 ± 0.5 mM. RrA_{E149R, V150P, and F151T} exhibited a substantially increased V_max of 57 U/mg. The catalytic efficiency of V_max/K_M also improved compared to the native enzyme: the V_max/K_M increased from approximately 7 U/mg × mM⁻¹ (for the native enzyme) to 9 U/mg × mM⁻¹ for Mut3. Other mutants exhibited less pronounced changes. Thermo-denaturation studies allowed us to determine the phase transition parameters of the RrA variants in comparison with commercial reference sample EcA. RrA_{A64V, E67K} and RrA_{R118H, G120R} exhibited the most favorable phase transition parameters, with melting temperatures (T_m) of 60.3 °C and 59.4 °C, respectively, exceeding that of the wild-type RrA (54.6 °C) and RrA_{E149R, V150P, F151T} (52 °C). The EcA demonstrated a slightly superior thermal stability, with a T_m of 62 °C. The mutations showed a significant effect on protein stability during trypsinolysis. Therefore, RrA_{E149R, V150P, F151T} showed higher resistance (45% activity remaining after 30 min of trypsin exposure) compared to the native RrA retained 20% activity. EcA preparations exhibited lower stability to trypsinolysis (losing over 90% activity in 15 min). The cytostatic effects were evaluated using MTT assays against K562 (leukemic) and A549 (lung carcinoma) cell lines. The MTT assays with K562 cells revealed that RrA_{E149R, V150P, F151T} (IC₅₀ of 10 U/mL) and RrA_{R118H, G120R} (IC₅₀ of 11.5 U/mL) exhibited superior antiproliferative activity compared to native enzymes RrA (IC₅₀ of 15 U/mL) and EcA (24 U/mL). RrA_{E149R, V150P, F151T} showed the most significant improvement in cytostatic activity. The results obtained indicate that the substitutions in RrA_{E149R, V150P, F151T} resulted in the improvement of the enzyme biocatalytic properties and an increase in the resistance to aggregation and trypsinolysis. This highlights the role of electrostatic interactions in stabilizing the oligomeric structure of the enzyme, which eventually translates into an improvement in cytostatic efficiency and antiproliferative forces. Full article

The tetrameric protein transthyretin (TTR) transports the hormone thyroxine in plasma and cerebrospinal fluid. Certain point mutations of TTR, including the Val122Ile mutation investigated here, destabilize the tetramer leading to its dissociation, misfolding, aggregation, and the eventual buildup of amyloid fibrils in the myocardium. Cioffi et al. reported the design and synthesis of a novel TTR kinetic stabilizing ligand, referred to here as TKS14, that inhibited TTR dissociation and amyloid fibril formation. In this study, molecular dynamics simulations were used to investigate the binding of TKS14 and eight TSK14 derivatives to the Val122Ile TTR mutant. For each complex, the ligand’s solvent accessible surface area (SASA), ligand–receptor hydrogen-bonding interactions, and the free energy of ligand-binding to TTR were investigated. The goal of this study was to identify the TSK14 functional groups that contributed to TTR stabilization. TKS14 was found to form a stable, two-point interaction with TTR by hydrogen bonding to Ser-117 residues in the inner receptor binding pocket and interacting through hydrogen bonds and electrostatically with Lys-15 residues near the receptor’s surface. The free energy of TKS14-TTR binding was −18.0 kcal mol⁻¹ and the ligand’s average SASA value decreased by over 80% upon binding to the receptor. The thermodynamic favorability of TTR binding decreased when TKS14 derivatives contained either methyl ester, amide, tetrazole, or N-methyl functional groups that disrupted the above two-point interaction. One derivative in which a tetrazole ring was added to TKS14 was found to form hydrogen bonds with Thr-106, Thr-119, Ser-117, and Lys-15 residues. This derivative had a free energy of TTR binding of −21.4 kcal mol⁻¹. Overall, the molecular dynamics simulations showed that the functional groups within the TKS14 structural template can be tuned to optimize the thermodynamic favorability of ligand binding. Full article

The self-assembly mechanisms of various complex biological structures, including viral capsids and carboxysomes, have been theoretically studied through numerous kinetic models. However, most of these models focus on the equilibrium aspects of a simplified kinetic description in terms of a single reaction coordinate, typically the number of proteins in a growing aggregate, which is often insufficient to describe the size and shape of the resulting structure. In this article, we use mesoscopic non-equilibrium thermodynamics (MNET) to derive the equations governing the non-equilibrium kinetics of viral capsid formation. The resulting kinetic equation is a Fokker–Planck equation, which considers viral capsid self-assembly as a diffusive process in the space of the relevant reaction coordinates. We discuss in detail the case of the self-assembly of a spherical (icosahedral) capsid with a fixed radius, which corresponds to a single degree of freedom, and indicate how to extend this approach to the self-assembly of spherical capsids that exhibit radial fluctuations, as well as to tubular structures and systems with higher degrees of freedom. Finally, we indicate how these equations can be solved in terms of the equivalent Langevin equations and be used to determine the rate of formation and size distribution of closed capsids, opening the door to the better understanding and control of the self- assembly process. Full article

Investigating amyloid–β (Aβ) peptides in solution is essential during the initial stages of developing lead compounds that can influence Aβ fibrillation while the peptide is still in a soluble state. The tendency of the Aβ(1–42) peptide to misfold in solution, correlated to the aetiology of Alzheimer’s disease (AD), is one of the main hindrances to characterising its aggregation kinetics in a cell-mimetic environment. Moreover, the Aβ(1–42) aggregation triggers the unfolded protein response (UPR) in the endoplasmic reticulum (ER), leading to cellular dysfunction and multiple cell death modalities, exacerbated by reactive oxygen species (ROS), which damage cellular components and trigger inflammation. Antioxidants like curcumin, a derivative of Curcuma longa, help mitigate ER stress by scavenging ROS and enhancing antioxidant enzymes. Furthermore, evidence in the literature highlights the effect of curcumin on the secondary structure of Aβ(1–42). This explorative study investigates the Aβ(1–42) peptide conformational behaviour in the presence of curcumin and six derivatives using circular dichroism (CD) to explore their interactions with lipid bilayers, potentially preventing aggregate formation. The results suggest that the synthetic tetrahydrocurcumin (THC) derivative interacts with the amyloid peptide in all the systems presented, while cyclocurcumin (CYC) and bisdemethoxycurcumin (BMDC) only interact when the peptide is in a less stable conformation. Molecular dynamics simulations helped visualise the curcuminoids’ effect in an aqueous system and hypothesise the importance of the peptide surface exposition to the solvent, differently modulated by the curcumin derivatives. Full article

The misfolding and amyloid aggregation of proteins have been attracting scientific interest for a few decades, due to their link with several diseases, particularly neurodegenerative diseases. Proteins can assemble and result in insoluble aggregates that, together with intermediate oligomeric species, modify the extracellular environment. Many efforts have been and are devoted to the search for cosolvents and cosolutes able to interfere with amyloid aggregation. In this work, we intensively study the effect of saponins, bioactive compounds, on human insulin aggregation. To monitor the kinetic of amyloid aggregation following secondary structure changes, we perform fluorescence and UV-Visible absorption spectroscopies, using Thioflavin T and Congo Red as amyloid specific probes, and Circular Dichroism. To study the overall structural features and size of aggregates, we perform Synchrotron Small-Angle X-ray Scattering and Dynamic Light Scattering experiments. The morphology of the aggregates was assessed by Atomic Force Microscopy. To deepen the understanding of the saponins interaction with insulin, a Molecular Dynamics investigation is performed, too. The reported data demonstrate that saponins interfere with the amyloid aggregation by inducing a strong inhibition on the formation of insulin fibrils, likely through specific interactions with insulin monomers. A dose-dependent effect is evident, and amyloid inhibition is already clear when saponins are just 0.01% w/w in solution. We suggest that saponins, which are natural metabolites present in a wide range of foods ranging from grains, pulses, and green leaves to sea stars and cucumbers, can be promising metabolites to inhibit human insulin aggregation. This basic research work can pave the way to further investigations concerning insulin amyloidosis, suggesting the use of saponins as amyloid inhibitors and/or stabilizing agents in solution. Full article

Controlling protein crystallization processes is essential for improving downstream processing in biotechnology. This study investigates the combination of machine learning-based image analysis and in situ microscopy for real-time monitoring of protein crystallization kinetics. The experimental research is focused on the batch crystallization of an alcohol dehydrogenase from Lactobacillus brevis (LbADH) and two selected rational crystal contact mutants. Technical protein crystallization experiments were performed in a 1 L stirred crystallizer by adding polyethyleneglycol 550 monomethyl ether (PEG 550 MME). The estimated crystal volumes from online microscopy correlated well with the offline measured protein concentrations in solution. In addition, in situ microscopy was superior to offline data if amorphous protein precipitation occurred. Real-time image analysis provides the data basis for online estimation of important batch crystallization performance indicators like yield, crystallization kinetics, crystal size distributions, and number of protein crystals. Surprisingly, one of the LbADH mutants, which should theoretically crystallize more slowly than the wild type based on molecular dynamics (MD) simulations, showed better crystallization performance except for the yield. Thus, online monitoring of scalable protein crystallization processes with in situ microscopy and real-time image analysis improves the precision of crystallization studies for industrial settings by providing comprehensive data, reducing the limitations of traditional analytical techniques, and enabling new insights into protein crystallization process dynamics. Full article

Polyampholytes (PAs) exhibit complex behaviors in various environments influenced by their charge distribution. This study focuses on the kinetics of dimerization of PAs, aiming to elucidate the underlying mechanisms and clarify relevant characteristics of the charge sequence. We focus on PAs with non-zero net charges, employing molecular dynamics simulations and theoretical analyses to examine how charge sequences influence the rates of dimer formation and dissociation. Our findings reveal that the charge sequence of tails and the blockiness of the minority charge group markedly influence the kinetics of dimerization: large blockiness and tails with a high number of majority-type charges slow down the dissociation of dimers. Additionally, the presence of an extended (central) block of the majority charge promotes structural diversity. Within dimer states, blocks alternate between intra- and inter-chain contacts. The duration times in the dimer states are significantly longer than the typical dwell times of block inter-contacts, with a notable extension when multiple blocks are engaged. Intrinsically disordered proteins (IDPs) play crucial roles in cellular functions, primarily due to their ability to undergo rapid conformational changes and form transient complexes. These properties largely depend on the sequence of charged residues. We provide insights into the fundamental principles governing the structural and dynamical properties of polyampholytic IDP, emphasizing the importance of sequence-specific effects on both aggregation and dissociation. Full article

Tau is an intrinsically disordered protein involved in several neurodegenerative diseases where a common hallmark is the appearance of tau aggregates in the brain. One common approach to elucidate the mechanisms behind the aggregation of tau has been to recapitulate in vitro the self-assembly process in a fast and reproducible manner. While the seeding of tau aggregation is prompted by negatively charged cofactors, the obtained fibrils are morphologically distinct from those found in vivo. The Tau AD core fragment (TADC, tau 306–378) has emerged as a new model and potential solution for the cofactor-free in vitro aggregation of tau. Here, we use TADC to further study this process combining multiple amyloid-detecting fluorophores and fibril bioimaging. We confirmed by transmission electron microscopy that this fragment forms fibrils after quiescent incubation at 37 °C. We then employed a panel of eight amyloid-binding fluorophores to query the formed species by acquiring their emission spectra. The results obtained showed that nearly all dyes detect TADC self-assembled species. However, the successful monitoring of TADC aggregation kinetics was limited to three fluorophores (X-34, Bis-ANS, and pFTAA) which yielded sigmoidal curves but different aggregation half-times, hinting to different species being detected. Altogether, this study highlights the potential of using multiple extrinsic fluorescent probes, alone or in combination, as tools to further clarify mechanisms behind the aggregation of amyloidogenic proteins. Full article