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CapG, an enzyme expressed by Staphylococcus aureus , catalyzes an epimerization reaction to synthesize N -acetyl-L-fucosamine, a constituent of capsule involved in pathogenesis. This protein has two domains, exists as the homohexamers in the solution, and usually produces products at hundred-nanomolar concentrations. To determine the folding-unfolding mechanism and the oligomeric form of CapG, particularly at low concentrations, we have investigated a recombinant CapG (rCapG) using different probes. The results show that rCapG in the aqueous solution is well-structured and exists as a mixture of different homo-oligomers such as dimer, trimer, tetramer, and hexamer. A considerable amount of rCapG also remained as the monomers at 0.5-5 µM concentrations. However, its trimeric forms are predominant at 5-100 µM concentrations. The formation of trimers is induced at higher concentrations of rCapG. Besides, rCapG at 0-7 M urea was reversibly unfolded by forming three structurally dissimilar intermediates. The first intermediate was possibly formed by the partial disruption of native rCapG trimers to dimers and monomers, whereas the second intermediate was likely produced due to the swelling and additional dissociation of the first intermediate. Further dissociation/swelling may have generated a third intermediate from the second intermediate. Additionally, both domains of rCapG started unfolding at the same urea concentrations. However, its C-terminal domain mostly completed unfolding at 7 M urea. Collectively, the study has provided new clues about the oligomeric state and the folding mechanism of CapG and also set up a foundation for discovering new anti-CapG molecules in the future.

The neutron spectrum was measured at two locations in the spent fuel storage facility of the Temelín nuclear power plant. The measurement had two primary objectives: to map the neutron -γ field by quantifying the ambient dose equivalent H∗(10) and to identify methods that could improve the quality of the adjusted neutron spectrum using a Bonner Sphere Spectrometer (BSS). Three spectrometers were used: a BSS and two proton recoil spectrometers. Hydrogen-filled proportional counters and an EJ309 scintillator were used to construct the a priori spectrum for BSS adjustment. The details of this process and its results are discussed. The a posteriori spectrum was used to calculate the ambient dose equivalent H∗(10). The resulting spectrum is highly thermalised, but the predominant contribution to H∗(10) was in the 100 keV-1.3 MeV range. The use of hydrogen-proportional counters in combination with the BSS is recommended., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

The inner membrane associated protein of 30 kDa (IM30), a member of the endosomal sorting complex required for transport (ESCRT-III) superfamily, is crucially involved in the biogenesis and maintenance of thylakoid membranes in cyanobacteria and chloroplasts. In solution, IM30 assembles into various large oligomeric barrel- or tube-like structures, whereas upon membrane binding it forms large, flat carpet structures. Dynamic localization of the protein in solution, to membranes and changes of the oligomeric states are crucial for its in vivo function. ESCRT-III proteins are known to form oligomeric structures that are dynamically assembled from monomeric/smaller oligomeric proteins, and thus these smaller building blocks must be assembled sequentially in a highly orchestrated manner, a still poorly understood process. The impact of IM30 oligomerization on function remains difficult to study due to its high intrinsic tendency to homo-oligomerize. Here, we used molecular dynamics simulations to investigate the stability of individual helices in IM30 and identified unstable regions that may provide structural flexibility. Urea-mediated disassembly of the IM30 barrel structures was spectroscopically monitored, as well as changes in the protein's tertiary and secondary structure. The experimental data were finally compared to a three-state model that describes oligomer disassembly and monomer unfolding. In this study, we identified a highly stable conserved structural core of ESCRT-III proteins and discuss the advantages of having flexible intermediate structures and their putative relevance for ESCRT-III proteins., (© 2024 The Author(s). Protein Science published by Wiley Periodicals LLC on behalf of The Protein Society.)

A key challenge in the analysis of microbiome data is the integration of multi-omic datasets and the discovery of interactions between microbial taxa, their expressed genes, and the metabolites they consume and/or produce. In an effort to improve the state of the art in inferring biologically meaningful multi-omic interactions, we sought to address some of the most fundamental issues in causal inference from longitudinal multi-omics microbiome data sets. We developed METALICA, a suite of tools and techniques that can infer interactions between microbiome entities. METALICA introduces novel unrolling and de-confounding techniques used to uncover multi-omic entities that are believed to act as confounders for some of the relationships that may be inferred using standard causal inferencing tools. The results lend support to predictions about biological models and processes by which microbial taxa interact with each other in a microbiome. The unrolling process helps identify putative intermediaries (genes and/or metabolites) to explain the interactions between microbes; the de-confounding process identifies putative common causes that may lead to spurious relationships to be inferred. METALICA was applied to the networks inferred by existing causal discovery, and network inference algorithms were applied to a multi-omics data set resulting from a longitudinal study of IBD microbiomes. The most significant unrollings and de-confoundings were manually validated using the existing literature and databases., Importance: We have developed a suite of tools and techniques capable of inferring interactions between microbiome entities. METALICA introduces novel techniques called unrolling and de-confounding that are employed to uncover multi-omic entities considered to be confounders for some of the relationships that may be inferred using standard causal inferencing tools. To evaluate our method, we conducted tests on the inflammatory bowel disease (IBD) dataset from the iHMP longitudinal study, which we pre-processed in accordance with our previous work. From this dataset, we generated various subsets, encompassing different combinations of metagenomics, metabolomics, and metatranscriptomics datasets. Using these multi-omics datasets, we demonstrate how the unrolling process aids in the identification of putative intermediaries (genes and/or metabolites) to explain the interactions between microbes. Additionally, the de-confounding process identifies potential common causes that may give rise to spurious relationships to be inferred. The most significant unrollings and de-confoundings were manually validated using the existing literature and databases., Competing Interests: The authors declare no conflict of interest.

In this study, the interaction of the human hemoglobin with cost effective and chemically fabricated CdS quantum dots (QDs) (average sizes ≈3nm) has been investigated. The semiconductor QDs showed maximum visible absorption at 445 nm with excitonic formation and band gap of ≈ 2.88 eV along with hexagonal crystalline phase. The binding of QDs-Hb occurs through corona formation to the ground sate complex formation. The life time of the heme pocket binding and reorganization were found to be t 1  = 43 min and t 2  = 642 min, respectively. The emission quenching of the Hb has been indicated large energy transfer between CdS QDs and Hb with tertiary deformation of Hb. The binding thermodynamics showed highly exothermic nature. The ultrafast decay during corona formation was studied from TCSPC. The results showed that the energy transfer efficiency increases with the increase of the QDs concentration and maximum ≈71.5 % energy transfer occurs and average ultrafast lifetime varies from 5.45 ns to1.51 ns. The deformation and unfolding of the secondary structure of Hb with changes of the α-helix (≈74 % to ≈51.07 %) and β-sheets (≈8.63 % to ≈10.25 %) have been observed from circular dichroism spectrum. The SAXS spectrum showed that the radius of gyration of CdS QDs-Hb bioconjugate increased (up to 23 ± 0.45 nm) with the increase of the concentration of QDs compare with pure Hb (11 ± 0.23 nm) and Hb becoming more unfolded., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Alzheimer's disease (AD) is a prevalent neurodegenerative disorder, impacting millions of individuals worldwide. Among its defining characteristics is the accumulation of senile plaques within the brain's gray matter, formed through the self-assembly of misfolded proteins contributing to the progressive symptoms of AD. This study investigates a polymorphic Aβ fibril under static and oscillating electric fields using molecular dynamics simulation. Specifically, we utilized a polymorphic fibrillar complex composed of two intertwined pentamer-strands of the Aβ1-40 peptide with the Osaka mutation (E22Δ), known for its toxicity and stable structure. Our findings demonstrate that a 0.3 and 0.4 V/nm electric field combined with a 0.20 GHz frequency effectively disrupts the polymorphic conformation of Aβ fibrils. Furthermore, we elucidate the molecular mechanisms underlying this disruption, providing insights into the potential therapeutic use of oscillating electric fields for AD. This research offers valuable insights into novel therapeutic approaches for combating AD pathology., (© 2024. The Author(s).)

The effect of resveratrol (RESV) on α-lactalbumin (α-LA) thermal stability was evaluated using differential scanning calorimetry (DSC), circular dichroism (CD) and dynamic light scattering (DLS) measurements. Complementary information offered by molecular docking served to identify the binding site of the ligand on the native structure of protein and the type of interacting forces. DSC thermograms revealed a double-endotherm pattern with partial overlapping of the two components. The most relevant effect of RESV is manifested in the narrowing of the protein thermal fingerprint: the first process (peak temperature T 1 ) is shifted to higher temperatures while the second one (peak temperature T 2 ). On further heating, unfolding of the α-helix domain takes place at T 1 , resulting in a β-sheet richer intermediate (BSRI) with an unaffected, native-like α-helix backbone. The RESV influence on this process may be defined as slightly demoting, at least within DSC conditions (linear heating rate of 1 K min -1 ). On further heating, unfolding of the α-helix domain takes place at T 2 , with RESV acting as a promoter of the process. Long time incubation at 333 K produced the same type of BSRI: no significant effect of RESV on the secondary structure content was detected by CD spectroscopy. Nevertheless, the size distribution of the protein population obtained from DLS measurements revealed the free (non-bound) RESV action manifested in the developing of larger size aggregates.

The red macroalgae Porphyra, commonly known as Nori, is widely used as food around the world due to its high nutrient content, including the significant abundance of colored phycobiliproteins (PBPs). Among these, R-phycocyanin (R-PC) stands out for its vibrant purple color and numerous bioactive properties, making it a valuable protein for the food industry. However, R-PC's limited thermal stability necessitates alternative processing methods to preserve its color and bioactive properties. Our study aimed to investigate the in-situ stability of oligomeric R-PC under high pressure (HP) conditions (up to 4000 bar) using a combination of absorption, fluorescence, and small-angle X-ray scattering (SAXS) techniques. The unfolding of R-PC is a multiphase process. Initially, low pressure induces conformational changes in the R-PC oligomeric form (trimers). As pressure increases above 1600 bar, these trimers dissociate into monomers, and at pressures above 3000 bar, the subunits begin to unfold. When returned to atmospheric pressure, R-PC partially refolds, retaining 50% of its original color absorbance. In contrast, heat treatment causes irreversible and detrimental effects on R-PC color, highlighting the advantages of HP treatment in preserving both the color and bioactive properties of R-PC compared to heat treatment., (© 2024 The Protein Society.)

In this work, neutron spectra are unfolded using artificial neural networks (ANNs). The neutron response of the NE213 scintillator detector, characterized by the pulse height distribution, is calculated to obtain the necessary data for unfolding the energy spectrum. This is achieved using both analytical response functions and response functions generated by the MCNPX-PHOTRACK code. In this query, the Levenberg-Marquardt method (LMM), which has a high computational speed in the learning method, is used to train the network. The performance of the ANN for unfolding the neutron energy spectrum of the NE213 scintillation detector was evaluated by comparing its results to the established Gravel method. The ANN method consistently produced spectra with a single peak closely matching the incident energy, while the Gravel method showed additional peaks and distortions. Quantitative analysis revealed a lower relative energy peak difference (indicating higher accuracy) for the ANN method compared to Gravel, particularly when noise was introduced into the data. These results suggest that ANNs offer a more robust and accurate approach for neutron spectrum unfolding., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Understanding how shear affects whey protein stability is crucial to deal with typical industrial issues occurring at the bulk solution/surface interface, such as fouling during heat treatments. However, at the state of the art, this effect remains unclear, contrary to that of temperature. This article presents a novel strategy to study the impact of shear rate and concentration on the accumulation of whey protein surficial deposits. It consists in applying a range of shear rates (0-200 s -1 ) at controlled temperature (65 °C) on whey protein solutions (5-10 wt%) by a parallel plate rheometer equipped with a glass disc, thus allowing the off-line characterization of the deposits by microscopy. Our results highlight an unequivocal effect of increasing shear stress. At 5 wt%, it fosters the formation of primary deposits (≈ 10 μm), whereas at 10 wt% it results in the development of complex branched structures (≈ 50 μm) especially for shear rates ranging from 140 s -1 to 200 s -1 . Based on the classification by size of the observed populations, we discuss possible hypotheses for the deposit growth kinetics, involving the interplay of different physico-chemical protein-surface interactions and paving the way to future further investigations., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Glycation is an effective strategy for the application of myofibrillar protein (MP) in beverage formulas by improving water solubility. In conventional glycation, the efficiency was limited as MP-saccharides conjugates mostly produced at low temperature due to thermosensitivity. This study was aimed to explore unfolding/aggregation kinetics of MP, including aggregate behavior, structural characteristics, and micromorphology, which guided the selection of temperature for glycation. It was shown that 40 °C/47.5 °C were critical temperature for MP unfolding/aggregation, respectively. Accordingly, an innovative technology of glycation (cyclic continuous glycation, CCG) was established by combining such temperatures. The results confirmed that cyclic continuous heating (CCH) inhibited excessive exposure of sulfhydryl and hydrophobic groups impeding protein aggregation. Importantly, it was revealed that rational designed CCG promoted covalent binding of MP to glucose by regulating unfolding-aggregation balance, exhibiting higher glycation degree. Overall, CCG-modified MP is expected to motivate the application of meat proteins in food formulations., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

The ATP-dependent molecular chaperone Cdc48 (in yeast) and its human counterpart p97 (also known as VCP), are essential for a variety of cellular processes, including the removal of DNA-protein crosslinks (DPCs) from the DNA. Growing evidence demonstrates in the last years that Cdc48/p97 is pivotal in targeting ubiquitinated and SUMOylated substrates on chromatin, thereby supporting the DNA damage response. Along with its cofactors, notably Ufd1-Npl4, Cdc48/p97 has emerged as a central player in the unfolding and processing of DPCs. This review introduces the detailed structure, mechanism and cellular functions of Cdc48/p97 with an emphasis on the current knowledge of DNA-protein crosslink repair pathways across several organisms. The review concludes by discussing the potential therapeutic relevance of targeting p97 in DPC repair., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Fluorinated graphene, a two-dimensional nanomaterial composed of three atomic layers, a central carbon layer sandwiched between two layers of fluorine atoms, has attracted considerable attention across various fields, particularly for its potential use in biomedical applications. Nonetheless, scant effort has been devoted to assessing the potential toxicological implications of this nanomaterial. In this study, we scrutinize the potential impact of fluorinated graphene on a protein model, HP35 by utilizing extensive molecular dynamics (MD) simulation methods. Our MD results elucidate that upon adsorption to the nanomaterial, HP35 undergoes a denaturation process initiated by the unraveling of the second helix of the protein and the loss of the proteins hydrophobic core. In detail, substantial alterations in various structural features of HP35 ensue, including alterations in hydrogen bonding, Q value, and RMSD. Subsequent analyses underscore that hydrophobic and van der Waals interactions (predominant), alongside electrostatic energy (subordinate), exert influence over the adsorption of HP35 on the fluorinated graphene surface. Mechanistic scrutiny attests that the unrestrained lateral mobility of HP35 on the fluorinated graphene nanomaterial primarily causes the exposure of HP35's hydrophobic core, resulting in the eventual structural denaturation of HP35. A trend in the features of 2D nanostructures is proposed that may facilitate the denaturation process. Our findings not only substantiate the potential toxicity of fluorinated graphene but also unveil the underlying molecular mechanism, which thereby holds significance for the prospective utilization of such nanomaterials in the field of biomedicine., (© 2024. The Author(s).)

High hydrostatic pressure (HHP) has gained prominence in the food processing industry over the last decade. In addition to the effectiveness of microbial and enzymatic inactivation, HHP directly impacts protein structures and properties. Accordingly, this review article aims to consolidate relevant research findings elucidating the effects of HHP on protein structure, allergenicity, bioactivities, and functional properties across diverse protein sources. They encompass cereals, legumes, nuts, meat, poultry products, milk, eggs, seafood, algae, insects, seeds, and vegetables. This review provides insights into the consistent trends of HHP effects on each protein source. In conclusion, HHP induces alterations in non-covalent bonds within protein structures, leading to the unfolding of their interior regions and consequential changes in their properties. Remarkably, the allergenicity of cereals, legumes, and nuts decreases while their bioactivities and digestibility escalate. The disruption of non-covalent bonds during HHP results in the exposure of the interior hydrophobic regions to the surface microenvironment, thereby enhancing the surface hydrophobicity of proteins, particularly those derived from seeds and vegetables. HHP weakens the allergenicity and elevates the foaming properties of proteins from dairy products, including improving the gelling properties and antioxidant activities of egg proteins. Texture profiles of meat and poultry, particularly hardness, are enhanced. Furthermore, HHP demonstrates the potential to diminish the allergenicity of seafood proteins and augment insect protein bioactivities. Lastly, HHP enhances the extraction of algal bioactive components, improving their nutritional quality.

Protein degradation is a major concern for protein-based therapeutics. It may alter the biological activity of the product and raise the potential for undesirable effects on the patients. Among the numerous drivers of protein degradation, shear stress has been the focus around which much work has revolved since the 1970s. In the pharmaceutical realm, the product is often processed through several unit operations, which include mixing, pumping, filtration, filling, and atomization. Nonetheless, the drug might be exposed to significant shear stresses, which might cooperatively contribute to product degradation, together with interfacial stress. This review presents fundamentals of shear stress about protein structure, followed by an overview of the drivers of product degradation. The impact of shear stress on protein stability in different unit operations is then presented, and recommendations for limiting the adverse effects on the biopharmaceutical formulations are outlined. Finally, several devices used to explore the effects of shear stress are discussed., Competing Interests: Declaration of competing interest Camilla Moino holds a Doctorate studentship and collaborates with GSK as part of her PhD training., (Copyright © 2023 The Author(s). Published by Elsevier B.V. All rights reserved.)

Biometrical sciences and disease diagnosis in particular, are often concerned with the analysis of associations for cross-classified data, for which distance association models give us a graphical interpretation for non-sparse matrices with a low number of categories. In this framework, usually binary exploratory and response variables are present, with analysis based on individual profiles being of great interest. For saturated models, we show the usual linear relationship for log-linear models is preserved in full dimension for the distance association parameterization. This enables a two-step procedure to facilitate the analysis and the interpretation of associations in terms of unfolding after the overall and main effects are removed. The proposed procedure can deal with cross-classified data for profiles by binary variables, and it is easy to implement using traditional statistical software. For disease diagnosis, the problems of a degenerate solution in the unfolding representation, and that of determining significant differences between the profile locations are addressed. A hypothesis test of independence based on odds ratio is considered. Furthermore, a procedure is proposed to determine the causes of the significance of the test, avoiding the problem of error propagation. The equivalence between a test for equality of odds ratio pairs and the test for equality of location for two profiles in the unfolding representation in the disease diagnosis is shown. The results have been applied to a real example on the diagnosis of coronary disease, relating the odds ratios with performance parameters of the diagnostic test., (© 2023 The Authors. Statistics in Medicine published by John Wiley & Sons Ltd.)

Ferguson plot was used to characterize the multiple intermediate species of bovine serum albumin (BSA) upon thermal unfolding. Differential scanning calorimetry showed an irreversible melting of BSA in Tris-HCl and phosphate buffers with a mid-transition temperature, Tm, of ∼68 °C. Thermally unfolded BSA was analyzed by agarose native gel electrophoresis stained by Coomassie blue and SYPRO Orange staining as a function of pH or protein concentration. SYPRO Orange was used to stain unfolded proteins. BSA heated at 70 and 80 °C, i.e., above the Tm, formed multiple intermediate species, which depended on the pH between 7.0 and 8.0, protein concentration and which buffer was used. These intermediate species were analyzed by Ferguson plot, which showed that BSA heated at 60 °C had a similar size to the native BSA, indicating that they are either native or native-like state consistent with no SYPRO Orange staining. The intermediate species observed at higher temperatures with the mobility less than that of the native BSA showed a steeper Ferguson plot and were stained by SYPRO Orange, indicating that these species had a larger hydrodynamic size than the native BSA and were unfolded., Competing Interests: Declaration of Competing Interest This work was supported by Kyokuto Pharmaceutical Industrial Co., Ltd. Y.T., M.N., and T.A. (Teruo Akuta) are employees of the for-profit company Kyokuto Pharmaceuticals. S·N and K.T. are staffs of University of Tokyo and have no conflict of interest. Tsutomu Arakawa used to belong to the for-profit company Alliance Protein Laboratories but currently has no conflict of interest., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Boron neutron capture therapy (BNCT) is a promising cancer treatment that uses energetic ions released from 10 B(n, α) 7 Li reactions. Accurate assessment of neutron energy spectra is important for simulation-based evaluation of neutron doses during BNCT. In this study, a proof-of-concept study was conducted for a neutron spectrometry technique that involves the use of a water phantom, which is commonly used for quality assurance in BNCT, as a moderator. The technique involves applying unfolding to the count rate distribution of the thermal neutron counter measured within the phantom to derive the energy spectrum. We performed experiments using a spherical 3 He proportional counter in neutron fields generated by 252 Cf and 241 Am-Be sources. The results demonstrated that the spectrometer reasonably reproduced neutron spectra and showed the potential of using a water phantom as a moderator for such a technique., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Mycobacterium Aspartate beta semialdehyde dehydrogenase (ASADH) was studied using various spectroscopic techniques and size exclusion chromatography to examine the unfolding of free (apo) and NADP/H-bound (holo) forms of ASADH. Non-cooperative guanidinium chloride (GdnHCl)-induced unfolding of the apo ASADH was discovered, and no partially folded intermediate structures were stabilized. On the other hand, it was observed that GdnHCl's unfolding of holoenzyme was a cooperative process without any stable intermediate structure. The native form of holoenzyme is found to be stable against the lower concentration of GdnHCl only (namely up to 1.25 M GdnHCl). The tryptophan environment appears to unfold cooperatively in case of the holoenzyme and is in well coordination with the overall unfolding of the holoenzyme. The presence of NADP/H shows a stabilizing effect on the tryptophan environment as well as on the native NADP/H-bound enzyme. Δ G Solvent o values reveal nearly two-fold (∼1.9) conformationally more stable folded holoenzyme compared to its native apo state. The C m for the apo and holo forms of ASADH are 1.3 and 1.9 M, respectively. Novel drug leads targeting the NADP/H binding domain of ASADH could offer promising drugs against extremely infective Mycobacterium tuberculosis. Communicated by Ramaswamy H. Sarma.

Integrin, as a mechanotransducer, establishes the mechanical reciprocity between the extracellular matrix (ECM) and cells at integrin-mediated adhesion sites. This study used steered molecular dynamics (SMD) simulations to investigate the mechanical responses of integrin α v β ion and ligand based on the application of force in the folding and unfolding directions of integrin. Furthermore, these SMD simulation results were used to predict the mechanical properties of integrin underlying the mechanism of integrin-based adhesion. The evaluation of integrin mechanics provides new insights into understanding the mechanotransmission (force transmission) between cells and ECM and contributes to developing an accurate model for integrin-mediated adhesion. This article is part of a discussion meeting issue 'Supercomputing simulations of advanced materials'.3 with and without 10th type III fibronectin (FnIII 10 ) binding for tensile, bending and torsional loading conditions. The ligand-binding integrin confirmed the integrin activation during equilibration and altered the integrin dynamics by changing the interface interaction between β-tail, hybrid and epidermal growth factor domains during initial tensile loading. The tensile deformation in integrin molecules indicated that fibronectin ligand binding modulates its mechanical responses in the folded and unfolded conformation states. The bending deformation responses of extended integrin models reveal the change in behaviour of integrin molecules in the presence of Mn 2+ ion and ligand based on the application of force in the folding and unfolding directions of integrin. Furthermore, these SMD simulation results were used to predict the mechanical properties of integrin underlying the mechanism of integrin-based adhesion. The evaluation of integrin mechanics provides new insights into understanding the mechanotransmission (force transmission) between cells and ECM and contributes to developing an accurate model for integrin-mediated adhesion. This article is part of a discussion meeting issue 'Supercomputing simulations of advanced materials'.

Protein unfolding is a ubiquitous process responsible for the loss of protein functionality (denaturation), which, in turn, can be accompanied by the death of cells and organisms. The nature of enthalpy-entropy compensation (EEC) in the kinetics of protein unfolding is a subject of debate. In order to investigate the nature of EEC, the "completely loose" transition state (TS) model has been applied to calculate the Arrhenius parameters for the unfolding of polyglycine dimers as a model process. The calculated Arrhenius parameters increase with increasing dimer length and demonstrate enthalpy-entropy compensation. It is shown that EEC results from the linear correlations of enthalpy and entropy of activation with dimer length, which are derived directly from the properties of the transition state. It is shown that EEC in solvated (hydrated, etc.) proteins is a direct consequence of EEC in proteins themselves. The suggested model allows us also to reproduce and explain "exotic" very high values of the pre-exponential factor measured for the proteins unfolding, which are drastically higher than those known for unimolecular reactions of organic molecules. A similar approach can be applied to analyzing the nature of EEC phenomena observed in other areas of chemistry.

The extraneuronally deposited senile plaques, composed of neurotoxic aggregates of Aβ fibril, define Alzheimer's disease (AD). Natural compounds have been tested for their destabilization potential on Aβ fibril, thereby curing AD. However, the resultant destabilized Aβ fibril, needs to be checked for its irreversibility to the native organized state after removal of the ligand. Herein, we assessed the stability of a destabilized fibril after the ligand (ellagic acid represented as REF) is removed from the complex. The study has been conducted via Molecular Dynamics (MD) simulation of 1 μs for both Aβ-Water (control) and Aβ-REF″ (test or REF removed) system. The increased value of RMSD, R g , SASA, lower β-sheet content and reduced number of H-bonds explains enhanced destabilization observed in Aβ-REF″ system. The increased inter-chain distance demonstrates breaking of the residual contacts, testifying the drift of terminal chains from the pentamer. The increased SASA along with the ∆G ps (polar solvation energy) accounts for the reduced interaction amongst residues, and more with solvent molecules, governing irreversibility to native state. The higher Gibb's free energy of the misaligned structure of Aβ-REF″ ensures irreversibility to the organized structure due to its inability to cross such high energy barrier. The observed stability of the disaggregated structure, despite ligand elimination, establishes the effectiveness of the destabilization technique as a promising therapeutic approach towards treating AD., Competing Interests: Declaration of competing interest The authors declare no competing financial interest., (Copyright © 2023 Elsevier B.V. All rights reserved.)

We present an improved application of a recently proposed computational method designed to evaluate the change of free energy as a function of the average value of a suitably chosen collective variable in proteins. The method is based on a full atomistic description of the protein and its environment. The goal is to understand how the protein melting temperature changes upon single-point mutations, because the sign of the temperature variation will allow us to discriminate stabilizing vs. destabilizing mutations in protein sequences. In this refined application the method is based on altruistic well-tempered metadynamics, a variant of multiple-walkers metadynamics. The resulting metastatistics is then modulated by the maximal constrained entropy principle. The latter turns out to be especially helpful in free-energy calculations as it is able to alleviate the severe limitations of metadynamics in properly sampling folded and unfolded configurations. In this work we apply the computational strategy outlined above in the case of the bovine pancreatic trypsin inhibitor, a well-studied small protein, which is a reference for computer simulations since decades. We compute the variation of the melting temperature characterizing the folding-unfolding process between the wild-type protein and two of its single-point mutations that are seen to have opposite effect on the free energy changes. The same approach is used for free energy difference calculations between a truncated form of frataxin and a set of five of its variants. Simulation data are compared to in vitro experiments. In all cases the sign of the change of melting temperature is reproduced, under the further approximation of using an empirical effective mean-field to average out protein-solvent interactions., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Nobili, Botticelli, La Penna, Morante, Rossi and Salina.)

Heat shock proteins 90 (Hsp90) are chaperones that promote the proper folding of other proteins under high temperature stress situations. Hsp90s are highly conserved and ubiquitous proteins, and in mammalian cells, they are localized in the cytoplasm, endoplasmic reticulum, and mitochondria. Cytoplasmic Hsp90 are named Hsp90α and Hsp90β and differ mainly in their expression pattern: Hsp90α is expressed under stress conditions, while Hsp90β is a constitutive protein. Structurally, both share the same characteristics by presenting three well-conserved domains, one of which, the N-terminal domain, has a binding site for ATP to which various drugs targeting this protein, including radicicol, can bind. The protein is mainly found in dimeric form and adopts different conformations depending on the presence of ligands, co-chaperones and client proteins. In this study, some aspects of structure and thermal unfolding of cytoplasmic human Hsp90 were analysed by infrared spectroscopy. The effect on Hsp90β of binding with a non-hydrolysable ATP analogue and radicicol was also examined. The results obtained showed that despite the high similarity in secondary structure the two isoforms exhibit substantial differences in their behaviour during thermal unfolding, as Hsp90α exhibits higher thermal stability, slower denaturation process and different event sequence during unfolding. Ligand binding strongly stabilizes Hsp90β and slightly modifies the secondary structure of the protein as well. Most likely, these structural and thermostability characteristics are closely related to the conformational cycling of the chaperone and its propensity to exist in monomer or dimer form., (Copyright © 2023 The Author. Published by Elsevier Inc. All rights reserved.)

We treat protein folding as molecular self-assembly, while unfolding is viewed as disassembly. Fracture is typically a much faster process than self-assembly. Self-assembly is often an exponentially decaying process, since energy relaxes due to dissipation, while fracture is a constant-rate process as the driving force is opposed by damping. Protein folding takes two orders of magnitude longer than unfolding. We suggest a mathematical transformation of variables, which makes it possible to view self-assembly as time-reversed disassembly, thus folding can be studied as reversed unfolding. We investigate the molecular dynamics modelling of folding and unfolding of the short Trp-cage protein. Folding time constitutes about 800 ns, while unfolding (denaturation) takes only about 5.0 ns and, therefore, fewer computational resources are needed for its simulation. This RetroFold approach can be used for the design of a novel computation algorithm, which, while approximate, is less time-consuming than traditional folding algorithms., Competing Interests: The authors declare no competing interest., (© 2023 The Authors.)

Interfacial adsorption is a molecular process occurring during the production, purification, transport, and storage of antibodies, with a direct impact on their structural stability and subsequent implications on their bioactivities. While the average conformational orientation of an adsorbed protein can be readily determined, its associated structures are more complex to characterize. Neutron reflection has been used in this work to investigate the conformational orientations of the monoclonal antibody COE-3 and its Fab and Fc fragments at the oil/water and air/water interfaces. Rigid body rotation modeling was found to be suitable for globular and relatively rigid proteins such as the Fab and Fc fragments but less so for relatively flexible proteins such as full COE-3. Fab and Fc fragments adopted a 'flat-on' orientation at the air/water interface, minimizing the thickness of the protein layer, but they adopted a substantially tilted orientation at the oil/water interface with increased layer thickness. In contrast, COE-3 was found to adsorb in tilted orientations at both interfaces, with one fragment protruding into the solution. This work demonstrates that rigid-body modeling can provide additional insights into protein layers at various interfaces relevant to bioprocess engineering.

Aggregation of proteins is a critical quality attribute and a major concern during the purification of therapeutic proteins, like monoclonal antibodies. In-solution experiments applying different stress scenarios, e.g., mechanical, or physical stresses, can determine the overall conformational stability of the protein to enhance drug product shelf-life. Several groups have reported surface-induced unfolding and aggregation of monoclonal antibodies and their derivatives during cation exchange chromatography, which results in a two-peak elution behavior of the protein and its species. We have investigated universal influencing factors, like temperature and hold time, on this phenomenon. The formation of the second peak is a kinetic process, which is strongly influenced by temperature during the hold time. However, our main focus was the application of excipients and their influence on the two-peak elution behavior. We compared the on-column screening results with results obtained through a "traditional" in-solution screening using nanoDSF. Mostly, stabilizing excipients, like Sucrose, show their stabilizing abilities in both systems, but some discrepancies, e.g., using Arginine, between the two orthogonal techniques show the potential of the on-column screening system to lead to unexpected results, which would not necessarily be visible in in-solution experiments., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Introduction: Sensitive methods are necessary to identify the residual structure in an unfolded protein, which may be similar to the functionally native structure. Signal intensity in NMR experiments is useful for analyzing the line width for a dynamic structure; however, another contribution is contained., Methods: Here, the signal-intensity difference along the sequence was used for probability to calculate the standard deviation., Results: The relative values of the standard deviations were 0.57, 0.57, and 0.66 for alpha-synuclein wild-type, A53T, and A30P, respectively. This revealed that the flexible region was mainly in the Cterminal region of alpha-synuclein at higher temperatures as observed by the amide-proton exchange studies., Conclusion: In particular, the flexible structure was induced by the A30P mutation., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Logistic regression models are a powerful research tool for the analysis of cross-classified data in which a categorical response variable is involved. In a logistic model, the effect of a covariate refers to odds, and the simple relationship between the coefficients and the odds ratio often makes these the parameters of interest due to their easy interpretation. In this article we present a distance-based logistic model that allows a simple graphical interpretation of the association coefficients using the odds ratio in a contingency table. Two configurations are estimated, one for the rows and one for the columns, as the categories of a polytomous predictor and a nominal response variable respectively, such that the local odds ratio and the distances between the predictor and response categories are inversely related. The associations in terms of the odds ratios, or the ratios of the odds to their geometric means, are interpreted through distances for the most common coding schemes of the predictor variable, and the relationship between the distances related to different codings is investigated in its full dimension. The performance of the estimation procedure is analysed with a Monte Carlo experiment. The interpretation of the model and its performance, as well as its comparison with a two-step procedure involving first a logistic regression and then unfolding, is illustrated using real data sets., (© 2022 The Authors. British Journal of Mathematical and Statistical Psychology published by John Wiley & Sons Ltd on behalf of British Psychological Society.)

The recombinant human keratinocyte growth factor (rhKGF) is a highly aggregation-prone therapeutic protein. The high aggregation liability of rhKGF is manifested by loss of the monomeric state, and accumulation of the aggregated species even at moderate temperatures. Here, we analyzed the rhKGF for its vulnerability toward aggregation by detection of aggregation-prone regions (APRs) using several sequence-based computational tools including TANGO, ZipperDB, AGGRESCAN, Zyggregator, Camsol, PASTA, SALSA, WALTZ, SODA, Amylpred, AMYPDB, and structure-based tools including SolubiS, CamSol structurally corrected, Aggrescan3D and spatial aggregation propensity (SAP) algorithm. The sequence-based prediction of APRs in rhKGF indicated that they are mainly located at positions 10-30, 40-60, 61-66, 88-120, and 130-140. Mapping on the rhKGF structure revealed that most of these residues including F16-R25, I43, E45, R47-I56, F61, Y62, N66, L88-E91, E108-F110, A112, N114, T131, and H133-T140 are surface-exposed in the native state which can promote aggregation without major unfolding event, or the conformational change may occur in the oligomers. The other regions are buried in the native state and their contribution to non-native aggregation is mediated by a preceding unfolding event. The structure-based prediction of APRs using the SAP tool limited the number of identified APRs to the dynamically-exposed hydrophobic residues including V12, A50, V51, L88, I89, L90, I118, L135, and I139 mediating the native-state aggregation. Our analysis of APRs in rhKGF identified the regions determining the intrinsic aggregation propensity of the rhKGF which are the candidate positions for engineering the rhKGF to reduce its aggregation tendency.Communicated by Ramaswamy H. Sarma.

The functional structure of proteins results from marginally stable folded conformations. Reversible unfolding, irreversible denaturation, and deterioration can be caused by chemical and physical agents due to changes in the physicochemical conditions of pH, ionic strength, temperature, pressure, and electric field or due to the presence of a cosolvent that perturbs the delicate balance between stabilizing and destabilizing interactions and eventually induces chemical modifications. For most proteins, denaturation is a complex process involving transient intermediates in several reversible and eventually irreversible steps. Knowledge of protein stability and denaturation processes is mandatory for the development of enzymes as industrial catalysts, biopharmaceuticals, analytical and medical bioreagents, and safe industrial food. Electrophoresis techniques operating under extreme conditions are convenient tools for analyzing unfolding transitions, trapping transient intermediates, and gaining insight into the mechanisms of denaturation processes. Moreover, quantitative analysis of electrophoretic mobility transition curves allows the estimation of the conformational stability of proteins. These approaches include polyacrylamide gel electrophoresis and capillary zone electrophoresis under cold, heat, and hydrostatic pressure and in the presence of non-ionic denaturing agents or stabilizers such as polyols and heavy water. Lastly, after exposure to extremes of physical conditions, electrophoresis under standard conditions provides information on irreversible processes, slow conformational drifts, and slow renaturation processes. The impressive developments of enzyme technology with multiple applications in fine chemistry, biopharmaceutics, and nanomedicine prompted us to revisit the potentialities of these electrophoretic approaches. This feature review is illustrated with published and unpublished results obtained by the authors on cholinesterases and paraoxonase, two physiologically and toxicologically important enzymes.

An attempt was made to specifically stain unfolded proteins on agarose native gels. SYPRO Orange is routinely used to detect unfolded protein in differential scanning fluorimetry, which is based on the enhanced fluorescence intensity upon binding to the unfolded protein. We demonstrated that this dye barely bound to the native proteins, resulting in no or faint staining of the native bands, but bound to and stained the unfolded proteins, on agarose native gels. Using bovine serum albumin (BSA), it was shown that staining did not depend on whether BSA was thermally unfolded in the presence of SYPRO Orange or stained after electrophoresis. On the contrary, SYPRO Orange dye stained protein bands in the presence of sodium dodecylsulfate (SDS) due to incorporation of the dye into SDS micelles that bound to the unfolded proteins. This staining resulted in detection of new, intermediately unfolded structure of BSA during thermal unfolding. Such intermediate structure occurred at higher temperature in the presence of ATP., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Γ-Crystallins play a major role in age-related lens transparency. Their destabilization by mutations and physical chemical insults are associated with cataract formation. Therefore, drugs that increase their stability should have anticataract properties. To this end, we screened 2560 Federal Drug Agency-approved drugs and natural compounds for their ability to suppress or worsen H 2 O 2  and/or heat-mediated aggregation of bovine γ-crystallins. The top two drugs, closantel (C), an antihelminthic drug, and gambogic acid (G), a xanthonoid, attenuated thermal-induced protein unfolding and aggregation as shown by turbidimetry fluorescence spectroscopy dynamic light scattering and electron microscopy of human or mouse recombinant crystallins. Furthermore, binding studies using fluorescence inhibition and hydrophobic pocket-binding molecule bis-8-anilino-1-naphthalene sulfonic acid revealed static binding of C and G to hydrophobic sites with medium-to-low affinity. Molecular docking to HγD and other γ-crystallins revealed two binding sites, one in the "NC pocket" (residues 50-150) of HγD and one spanning the "NC tail" (residues 56-61 to 168-174 in the C-terminal domain). Multiple binding sites overlap with those of the protective mini αA-crystallin chaperone MAC peptide. Mechanistic studies using bis-8-anilino-1-naphthalene sulfonic acid as a proxy drug showed that it bound to MAC sites, improved T m  of both H 2 O 2  oxidized and native human gamma D, and suppressed turbidity of oxidized HγD, most likely by trapping exposed hydrophobic sites. The extent to which these drugs act as α-crystallin mimetics and reduce cataract progression remains to be demonstrated. This study provides initial insights into binding properties of C and G to γ-crystallins., Competing Interests: Conflict of interest V. M. M. is on the Scientific Advisory Board of Revel Pharmaceuticals. Inc. All other authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Cation exchange chromatography, as part of the monoclonal antibody purification train, is known as a mild polishing technique. However, in the last couple of years, more and more publications have shown unusual elution behavior, resulting from e.g. on-column (reversible) unfolding and aggregation of the predominantly mAb molecules. The stability of the investigated protein seems to play a significant role in this phenomenon. We have used a glycosylated IgG1 antibody as a model protein and investigated several influencing factors, including pH value and ligand density variations of three prototype Fractogel® cation exchange resins. Ligand density, pH and salt concentration are the main contributing factors in the Donnan effect, i.e. distribution of ions, between resin pore volume and bulk volume. This leads to a significantly lower pH value the protein is subjected to during the on-column hold time and therefore influences the conformational stability of our protein. Nano-DSF and kinetic SEC measurements show that the protein is destabilized at low pH values, but also, that the binding to the CEX resin and the elution with increasing salt concentration is responsible for the resulting two-peak elution behavior and partially reversible unfolding and aggregation., Competing Interests: Declaration of Competing Interest None., (Copyright © 2022. Published by Elsevier B.V.)

In this paper a simple but effective procedure to avoid degeneracies in ordinal Unfolding for preference rank data based on the Kemeny distance is proposed. Considering Unfolding as a particular MDS procedure with missing within-set proximities, unknown proximities are first estimated using correlations related to the Kemeny distance, and then the complete proximity matrix is analyzed in a standard MDS framework. A simulation study shows that our proposal is able to both recover the order of the preferences and reproduce the position of both rankings and objects in a geometrical space. Several applications on real data sets show that our procedure returns non-degenerate Unfolding solutions.

Misfolding and aggregation of proteins occur in many pathological states. Because of the inherent disorder involved, these processes are difficult to study. We attempted to capture aggregation intermediates of γS-crystallin, a highly stable, internally symmetrical monomeric protein, by crystallization under mildly acidic and oxidizing conditions. Here we describe novel oligomerization through strained domain-swapping and partial intermolecular disulfide formation. This forms an octamer built from asymmetric tetramers, each of which comprises an asymmetric pair of twisted, domain-swapped dimers. Each tetramer shows patterns of acquired disorder among subunits, ranging from local loss of secondary structure to regions of intrinsic disorder. The octamer ring is tied together by partial intermolecular disulfide bonds, which may contribute to strain and disorder in the octamer. Oligomerization in this structure is self-limited by the distorted octamer ring. In a more heterogeneous environment, the disordered regions could serve as seeds for cascading interactions with other proteins. Indeed, solubilized protein from crystals retain many features observed in the crystal and are prone to further oligomerization and precipitation. This structure illustrates modes of loss of organized structure and aggregation that are relevant for cataract and for other disorders involving deposition of formerly well-folded proteins., (Published by Elsevier Ltd.)

The successful design of a hydrogel for tissue engineering requires a profound understanding of its constituents' structural and molecular properties, as well as the proper selection of components. If the engineered processes are in line with the procedures that natural materials undergo to achieve the best network structure necessary for the formation of the hydrogel with desired properties, the failure rate of tissue engineering projects will be significantly reduced. In this review, we examine the behavior of proteins as an essential and effective component of hydrogels, and describe the factors that can enhance the protein-based hydrogels' structure. Furthermore, we outline the fabrication route of protein-based hydrogels from protein microstructure and the selection of appropriate materials according to recent research to growth factors, crucial members of the protein family, and their delivery approaches. Finally, the unmet needs and current challenges in developing the ideal biomaterials for protein-based hydrogels are discussed, and emerging strategies in this area are highlighted.

The hexameric Cdc48 ATPase (p97 or VCP in mammals) cooperates with its cofactor Ufd1/Npl4 to extract polyubiquitinated proteins from membranes or macromolecular complexes for degradation by the proteasome. Here, we clarify how the Cdc48 complex unfolds its substrates and translocates polypeptides with branchpoints. The Cdc48 complex recognizes primarily polyubiquitin chains rather than the attached substrate. Cdc48 and Ufd1/Npl4 cooperatively bind the polyubiquitin chain, resulting in the unfolding of one ubiquitin molecule (initiator). Next, the ATPase pulls on the initiator ubiquitin and moves all ubiquitin molecules linked to its C terminus through the central pore of the hexameric double ring, causing transient ubiquitin unfolding. When the ATPase reaches the isopeptide bond of the substrate, it can translocate and unfold both N- and C-terminal segments. Ubiquitins linked to the branchpoint of the initiator dissociate from Ufd1/Npl4 and move outside the central pore, resulting in the release of unfolded, polyubiquitinated substrate from Cdc48., Competing Interests: Declaration of interests J.A.M. serves on the scientific advisory board of 908 Devices and receives sponsored research support from AstraZeneca and Vertex. All other authors declare no competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

α-Amylase plays an important role in food processing and in-vivo digestion. Many biological functions of α-amylase are affected by unfolding. The pre-steady-state rapid unfolding kinetics of α-amylase remains unknown. In this study, the rapid unfolding kinetics of porcine pancreatic α-amylase (PPA) with guanidine hydrochloride (GdmHCl) were investigated by stopped-flow spectroscopy. Structural characterization of PPA by fluorescence spectroscopy, and molecular dynamics simulation showed that the unfolding process of PPA might start from the internal active center, where the β-sheet structure was destroyed, followed by the exposure of hydrophobic amino acid residues. Further research revealed that GdmHCl denaturized PPA not by complexing with PPA. The surrounding H-bond network of water was changed by GdmHCl. This research improves our understanding of the unfolding kinetics of the PPA on the microsecond scale. It also provides the evidence experimentally of the surrounding water contribution to protein denaturization., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Human γD-crystallin (HGDC) is an abundant lens protein residing in the nucleus of the human lens. Aggregation of this and other structural proteins within the lens leads to the development of cataract. Much has been explored on the stability and aggregation of HGDC and where detailed investigation at the atomic resolution was needed, the X-ray structure was used as an initial starting conformer for molecular modeling. In this study, we implemented NMR-solution HGDC structures as starting conformers for molecular dynamics simulations to provide the missing pieces of the puzzle on the very early stages of HGDC unfolding leading up to the domain swap theories proposed by past studies. The high-resolution details of the conformational dynamics also revealed additional insights to possible early intervention for cataractogenesis.

Enhanced 'Antenna effect' of a suitably designed ternary complex of Eu(III), Tetracycline hydrochloride (TC) and globular proteins viz bovine serum albumin (BSA), human serum albumin (HSA) and β-lactoglobulin A (BLGA) in aqueous medium is employed to characterize the different partially unfolded states along with investigation of the micro- heterogeneous environment of the proteins during their stepwise unfolding. The zone-wise perturbation for the proteins upon denaturation by Urea and Guanidine hydrochloride (Gdn. HCl) is followed by the emission of Eu(III) through 'Antenna Effect' and that of the tryptophan (Trp) residues of the proteins as a function of denaturants both by steady state and time resolved emission study. With Gdn. HCl as denaturant, both BSA and BLGA show quenching of Eu(III) emission compared to pure protein while HSA exhibits an enhancement of antenna effect during unfolding as compared to that in its absence. In the presence of Urea, HSA and BSA show enhancement of antenna effect accompanied by Stark splitting of the 5 D 0 → 7 F 2 transition of Eu(III) although BLGA follows the similar pattern of quenching of Eu(III) emission as observed with Gdn. HCl without any Stark splitting. The proteins exhibit a two state transition with ΔG D values of ~ 2-3 kcal mol -1 . Thus the use of Eu(III) emission as an efficient probe is advocating here to rationalize the microenvironment of the proteins during their stepwise unfolding., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021. Published by Elsevier B.V.)

Amyloid fibrillar aggregates play a critical role in many neurodegenerative disorders. Conversion of globular proteins into fibrils is associated with global conformational rearrangement and involves the transformation of α-helices to β-sheets. In the present work, the accelerated molecular dynamics technique was applied to study the unfolding of hen egg-white lysozyme at elevated temperatures, and the transformation of the native structure to a disordered one was analyzed. The influence of the disulfide bonds on the conformational dynamics and the energy landscape of denaturation process was considered. Our results show that formation of the metastable β-enriched conformers of individual protein molecules may precede the aggregation process. These β-rich intermediates can play a role of bricks making up fibrils.Communicated by Ramaswamy H. Sarma.

This study aimed to better characterize a recently purified stable extracellular alkaline peptidase produced by Penicillium aurantiogriseum (URM 4622) through fluorescence spectroscopy, far-UV circular dichroism, kinetic and thermodynamic models to understand its' structure-activity and denaturation. Fluorescence data showed that changing pH leads to tryptophan residues exposure to more hydrophilic environments at optimum activity pH 9.0 and 10.0. When thermally treated, it displayed less unfolding at these pH values, along with 4-fold less photoproducts formation than at neutral pH. Different pH CD spectra showed more β-sheet (21.5-43.0%) than α-helix (1-6.2%). At pH9.0, more than 2-fold higher α-helix content than any other pH. The melting temperature ( T m ) was observed between 50 and 60 °C at all pH studied, with lower T m at pH 9.0-11.0 (54.9-50.3 °C). The protease displayed two phase transition, with two energies of denaturation, and a 4-fold higher thermal stability (ΔH° m ) than reports for other microorganism's proteases. An irreversible folding transition occurs between 50 and 60 °C. It displayed energies of denaturation suggesting higher thermal stability than reported for other microorganism's proteases. These results help elucidating the applicability of this new stable protease.

CapF, a capsule-producing enzyme expressed by Staphylococcus aureus , binds NADPH and exists as a dimer in the aqueous solution. Many other capsule-producing virulent bacteria also express CapF orthologs. To understand the folding-unfolding mechanism of S. aureus CapF, herein a recombinant CapF (rCapF) was individually investigated using urea and guanidine hydrochloride (GdnCl). Unfolding of rCapF by both the denaturants was reversible but proceeded via the synthesis of a different number of intermediates. While two dimeric intermediates (rCapF4 and rCapF5) were formed at 0.5 M and 1.5 M GdnCl, three dimeric intermediates (rCapF1, rCapF2, and rCapF3) were produced at 1 M, 2 M, and 3 M urea, respectively. rCapF5 showed 3.6 fold less NADPH binding activity, whereas other intermediates retained full NADPH binding activity. Compared to rCapF, all of the intermediates (except rCapF3) had a compressed shape. Conversely, rCapF3 possessed a native protein-like shape. The maximum shape loss was in rCapF4 though its secondary structure remained unperturbed. Additionally, the tertiary structure and hydrophobic surface area of the intermediates neither matched with each other nor with those of the native rCapF. Of the four Trp residues in rCapF, one or more Trp residues in the intermediates may have higher solvent accessibility. Using sequence alignment and a tertiary structural model of CapF, we have demonstrated that the region around Trp 137 of CapF may be most sensitive to unfolding, whereas the NADPH binding motif carrying region at the N-terminal end of this protein may be resistant to unfolding, particularly at the low denaturant concentrations.Communicated by Ramaswamy H. Sarma.

In bacteria, Lon is a large hexameric ATP-dependent protease that targets misfolded and also folded substrates, some of which are involved in cell division and survival of cellular stress. The N-terminal domain of Lon facilitates substrate recognition, but how the domains confer such activity has remained unclear. Here, we report the full-length structure of Lon protease from Thermus thermophilus at 3.9 Å resolution in a substrate-engaged state. The six N-terminal domains are arranged in three pairs, stabilized by coiled-coil segments and forming an additional channel for substrate sensing and entry into the AAA+ ring. Sequence conservation analysis and proteolysis assays confirm that this architecture is required for the degradation of both folded and unfolded substrates in bacteria., (© 2021 MRC Laboratory of Molecular Biology. FEBS Letters published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

The SARS-CoV-2 spike is the primary target of virus-neutralizing antibodies and critical to the development of effective vaccines against COVID-19. Here, we demonstrate that the prefusion-stabilized two-proline "S2P" spike-widely employed for laboratory work and clinical studies-unfolds when stored at 4 °C, physiological pH, as observed by electron microscopy (EM) and differential scanning calorimetry, but that its trimeric, native-like conformation can be reacquired by low pH treatment. When stored for approximately 1 week, this unfolding does not significantly alter antigenic characteristics; however, longer storage diminishes antibody binding, and month-old spike elicits virtually no neutralization in mice despite inducing high ELISA-binding titers. Cryo-EM structures reveal the folded fraction of spike to decrease with aging; however, its structure remains largely similar, although with varying mobility of the receptor-binding domain. Thus, the SARS-CoV-2 spike is susceptible to unfolding, which affects immunogenicity, highlighting the need to monitor its integrity., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Published by Elsevier Inc.)

The widespread use of nanoparticles raises many serious concerns about the safety and environmental impact of nanoparticles. Therefore, risk assessments of specific nanoparticles in occupational and environmental exposure are essential before their large-scale production and applications, especially in medicine and for usage in household items. In this study, the effects of five different metal nanoparticles on the structure, stability, and function of four metabolic enzymes were evaluated using various biophysical techniques. Our results show that Cu nanoparticles exhibited the most significant adverse effects on the structures, stability, and activities of all the metabolic enzymes. Zn nanoparticles caused moderate adverse effects on these enzymes. The rest of the metal (Al, Fe, and Ni) nanoparticles had a relatively lower impact on the metabolic enzymes. Our data indicated that Cu nanoparticles promote metal-catalyzed disulfide bond formation in these proteins. In summary, some metal nanoparticles can cause adverse effects on the structure, function, and stability of metabolic enzymes. In addition, metal nanoparticles may affect protein homeostasis in the cytosol or extracellular fluids., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Contrary to the radionuclide neutron sources of 252 Cf and 241 Am-Be type, which are most widely used for the dosimetric instrumentation calibrations, the spectral source strength and spectrum averaged fluence to dose equivalent conversion coefficients of Pu-Be source are not recommended by an international standard. This work describes the determination of those parameters for the Pu-Be source used at Czech Metrology Institute (CMI) by means of a Bonner spectrometer in combination with neutron emission rate measurement in a manganese bath., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

The scientific study of consciousness is currently undergoing a critical transition in the form of a rapidly evolving scientific debate regarding whether or not currently proposed theories can be assessed for their scientific validity. At the forefront of this debate is Integrated Information Theory (IIT), widely regarded as the preeminent theory of consciousness because it quantified subjective experience in a scalar mathematical measure called Φ that is in principle measurable. Epistemological issues in the form of the "unfolding argument" have provided a concrete refutation of IIT by demonstrating how it permits functionally identical systems to have differences in their predicted consciousness. The implication is that IIT and any other proposed theory based on a physical system's causal structure may already be falsified even in the absence of experimental refutation. However, so far many of these arguments surrounding the epistemological foundations of falsification arguments, such as the unfolding argument, are too abstract to determine the full scope of their implications. Here, we make these abstract arguments concrete, by providing a simple example of functionally equivalent machines realizable with table-top electronics that take the form of isomorphic digital circuits with and without feedback. This allows us to explicitly demonstrate the different levels of abstraction at which a theory of consciousness can be assessed. Within this computational hierarchy, we show how IIT is simultaneously falsified at the finite-state automaton level and unfalsifiable at the combinatorial-state automaton level. We use this example to illustrate a more general set of falsification criteria for theories of consciousness: to avoid being already falsified, or conversely unfalsifiable, scientific theories of consciousness must be invariant with respect to changes that leave the inference procedure fixed at a particular level in a computational hierarchy., (© The Author(s) 2021. Published by Oxford University Press.)