Precooked state based on protein denaturation kinetics impacts moisture status, protein oxidation and texture of prepared chicken breast.

The quality of meat in prepared dishes deteriorates due to excessive protein denaturation resulting from precooking, freezing, and recooking. This study aimed to link the precooked state with chicken breast's recooked quality. Cooked Value (CV), based on protein denaturation kinetics, was established to indicate the doneness of meat during pre-heating. The effects of CVs after pre-heating on recooked qualities were investigated compared to fully pre-heated samples (control). Mild pre-heating reduced water migration and loss. While full pre-heating inhibited protein oxidation during freezing, intense oxidation during pre-heating led to higher oxidation levels. Surface hydrophobicity analysis revealed that mild pre-heating suppressed aggregation during recooking. These factors contributed to a better texture and microstructure of prepared meat with mild pre-heating. Finally, a potential mechanism of how pre-heating affects final qualities was depicted. This study underlines the need for finely controlling the industrial precooking process to regulate the quality of prepared meat.

Collagen denaturation in post-run Achilles tendons and Achilles tendinopathy: In vivo mechanophysiology and magnetic resonance imaging.

Achilles tendinopathy is often attributed to overuse, but its pathophysiology remains poorly understood. Disruption to the molecular structure of collagen is fundamental for the onset and progression of tendinopathy but has mostly been investigated in vitro. Here, we interrogated the in vivo molecular structure changes of collagen in rat Achilles tendons following treadmill running. Unexpectedly, the tendons' collagen molecules were not mechanically unfolded by running but denatured through proteolysis during physiological post-run remodeling. We further revealed that running induces inflammatory gene expressions in Achilles tendons and that long-term running causes prolonged, elevated collagen degradation, leading to the accumulation of denatured collagen and tendinopathy development. For applications, we demonstrated magnetic resonance imaging of collagenase-induced Achilles tendon injury in vivo using a denatured collagen targeting contrast agent. Our findings may help close the knowledge gaps in the mechanobiology and pathogenesis of Achilles tendinopathy and initiate new strategies for its imaging-based diagnosis.

A Highly Specific and Versatile Biochip for Ultra-Sensitive Quantification of Denatured Collagen in Assessing Collagen Quality.

Collagen, a widely used biomaterial, is susceptible to denaturation during production from native tissues, posing serious challenges for its applications in tissue engineering. Accurate quantification of denatured collagen (DC) is essential for evaluating the quality of collagen-based biomaterials, yet quantitative methods for assessing collagen denaturation are lacking. Here, we for the first time present a highly specific biochip for sensitive quantification of denatured collagen levels (Ldc), addressing this critical need in collagen quality analysis. The denatured collagen-specific chip (DCSC) features an intrinsically nontrimerizing peptide probe, F-GOP-14, targeting denatured collagen and a fully denatured collagen-coated capture surface. The DCSC demonstrates exceptional sensitivity and accuracy in quantifying DC concentration (Cdc) and total collagen concentration (Ctc), enabling precise calculation of Ldc. Importantly, DCSC is versatile, detecting Ldc across various denaturing scenarios, including UV radiation, thermal environments, and decellularization. This denatured collagen-specific biochip offers a robust method for accurately analyzing Ldc, with significant potential for enhancing collagen quality assessment in biomaterial development and production.

In vitro biological activities of selected medicinal plants and their synergistic effects.

The present work aimed to use the methanol extracts of Croton bonplandianus (Cb) and Tithonia diversifolia (Td) and the synergistic activity of Croton bonplandianus and Tithonia diversifolia (CbTd) for the phytochemical screening, anti-microbial, anti-oxidant and anti-inflammatory activities. Phytochemical screening was done by the standard protocols. In vitro antimicrobial, antioxidant and anti-inflammation, were assayed by using disc diffusion, total antioxidant activity, DPPH method, HRBC (human red blood cells) membrane stabilization and anti-protein denaturation tests. The synergistic approach of the two plants showed potent antimicrobial, antioxidant and anti-inflammation activity. In vitro antibacterial activity was done against Pseudomonas aeroginosa, Escherichia coli, Bacillus subtilis and Staphylococcus aureus at different concentrations. The maximum zone of inhibition (21 mm) was observed in the combinatorial approach. The maximum inhibition of free radicals is observed in CbTd with a low IC50, i.e., 7.64 mg/ml, followed by Cb with an IC50 value of 11.8mg/ml and Td with an IC50 of 28.3 mg/ml. The percentage inhibition of hemolysis and protein denaturation is high in CbTd (93% and 69%). The experimental analysis reveals the effectiveness of the synergistic effect of these plants with anti-microbial, anti-oxidant and anti-inflammatory activity, further it can be used in the formulations against infectious human pathogens.

On the Stabilizing Effect of Aspartate and Glutamate and Its Counteraction by Common Denaturants.

By performing differential scanning calorimetry(DSC) measurements on RNase A, we studied the stabilization provided by the addition of potassium aspartate(KAsp) or potassium glutamate (KGlu) and found that it leads to a significant increase in the denaturation temperature of the protein. The stabilization proves to be mainly entropic in origin. A counteraction of the stabilization provided by KAsp or KGlu is obtained by adding common denaturants such as urea, guanidinium chloride, or guanidinium thiocyanate. A rationalization of the experimental data is devised on the basis of a theoretical approach developed by one of the authors. The main contribution to the conformational stability of globular proteins comes from the gain in translational entropy of water and co-solute ions and/or molecules for the decrease in solvent-excluded volume associated with polypeptide folding (i.e., there is a large decrease in solvent-accessible surface area). The magnitude of this entropic contribution increases with the number density and volume packing density of the solution. The two destabilizing contributions come from the conformational entropy of the chain, which should not depend significantly on the presence of co-solutes, and from the direct energetic interactions between co-solutes and the protein surface in both the native and denatured states. It is the magnitude of the latter that discriminates between stabilizing and destabilizing agents.

Conservation of kinetic stability, but not the unfolding mechanism, between human transthyretin and a transthyretin-related enzyme.

Kinetic stability is thought to be an attribute of proteins that require a long lifetime, such as the transporter of thyroxine and holo retinol-binding protein or transthyretin (TTR) functioning in the bloodstream, cerebrospinal fluid, and vitreous humor. TTR evolved from ancestral enzymes known as TTR-related proteins (TRPs). Here, we develop a rate-expansion approach that allows unfolding rates to be measured directly at low denaturant concentration, revealing that kinetic stability exists in the Escherichia coli TRP (EcTRP), even though the enzyme structure is more energetically frustrated and has a more mutation-sensitive folding mechanism than human TTR. Thus, the ancient tetrameric enzyme may already have been poised to mutate into a kinetically stable human transporter. An extensive mutational study that exchanges residues at key sites within the TTR and EcTRP dimer-dimer interface shows that tyrosine 111, replaced by a threonine in TTR, is the gatekeeper of frustration in EcTRP because it is critical for function. Frustration, virtually absent in TTR, occurs at multiple sites in EcTRP and even cooperatively for certain pairs of mutations. We present evidence that evolution at the C terminus of TTR was a compensatory event to maintain the preexisting kinetic stability while reducing frustration and sensitivity to mutation. We propose an "overcompensation" pathway from EcTRPs to functional hybrids to modern TTRs that is consistent with the biophysics discussed here. An alternative plausible pathway is also presented.

Characterization of a Monoclonal Antibody by Native and Denaturing Top-Down Mass Spectrometry.

Established in recent years as an important approach to unraveling the heterogeneity of intact monoclonal antibodies, native mass spectrometry has been rarely utilized for sequencing these complex biomolecules via tandem mass spectrometry. Typically, top-down mass spectrometry has been performed starting from highly charged precursor ions obtained via electrospray ionization under denaturing conditions (i.e., in the presence of organic solvents and acidic pH). Here we systematically benchmark four distinct ion dissociation methods─namely, higher-energy collisional dissociation, electron transfer dissociation, electron transfer dissociation/higher-energy collisional dissociation, and 213 nm ultraviolet photodissociation─in their capability to characterize a therapeutic monoclonal antibody, trastuzumab, starting from denatured and native-like precursor ions. Interestingly, native top-down mass spectrometry results in higher sequence coverage than the experiments carried out under denaturing conditions, with the exception of ultraviolet photodissociation. Globally, electron transfer dissociation followed by collision-based activation of product ions generates the largest number of backbone cleavages in disulfide protected regions, including the complementarity determining regions, regardless of electrospray ionization conditions. Overall, these findings suggest that native mass spectrometry can certainly be used for the gas-phase sequencing of whole monoclonal antibodies, although the dissociation of denatured precursor ions still returns a few backbone cleavages not identified in native experiments. Finally, a comparison of the fragmentation maps obtained under denaturing and native conditions strongly points toward disulfide bonds as the primary reason behind the largely overlapping dissociation patterns.

Relationship between thermal stability of collagens and the fraction of hydrophobic residues in their molecules.

In this study, a database of the thermal stability of collagens and their synthetic analogues has been compiled taking into account literature sources. In total, our database includes 1200 records. As a result of a comparative theoretical analysis of the collected experimental data, the relationship between the melting temperature (Tm) or denaturation temperature (Td) of collagens and the fraction of hydrophobic residues (f) in their molecules has been established. It is shown that this relationship is linear: the larger the f value, the higher the denaturation or melting temperature of a given collagen.

Enhanced stability of Pickering emulsions through co-stabilization with nanoliposomes and thermally denatured ovalbumin.

Pickering emulsions were co-stabilized by nanoliposome (NL) and thermally denatured ovalbumin (DOVA) based on the induction of OVA with strong particle characteristics through thermal denaturation. DOVA-NL particles were spherical and their sizes were mainly distributed between 50 and 100 nm. The surface tension and interfacial tension of DOVA-NL were significantly reduced, and the surface hydrophobicity, amphiphilicity and free -SH content of DOVA were enhanced after complexation with NL. The content of α-helix and ß-sheet in DOVA decreased, whereas the content of ß-turn and random coil increased after complexation with NL. Hydrophobic interactions, hydrogen bonding and electrostatic forces played a vital role in the interactions between NL and DOVA, leading to conformational changes in DOVA. The number of binding sites between NL and DOVA was more than one, and the interaction between NL and DOVA was exothermic and spontaneous. The emulsification index showed that DOVA-NL-stabilized Pickering emulsions (DNPE) were significantly more stable than DOVA-stabilized emulsions. DOVA-NL particles adsorbed at the oil-water interface and the droplet size of DNPE was smaller than that of DOVA-stabilized emulsions. This study suggests that it may be an effective strategy to improve the stability of Pickering emulsions through co-stabilization with NL and DOVA.

Using transglutaminase to cross-link complexes of lactoferrin and α-lactalbumin to increase thermal stability.

The poor thermal stability of lactoferrin (LF) hinders its bioavailability and use in commercial food products. To preserve LF from thermal denaturation, complexation with other biopolymers has been studied. Here we present the complex formation conditions, structural stability, and functional protection of LF by α-lactalbumin (α-LA). The formation of the LF-α-LA complexes was dependent on pH, mass ratio, and ionic strength. Changing the formation conditions and cross-linking by transglutaminase impacted the turbidity, particle size, and zeta-potential of the resulting complexes. Electrophoresis, Fourier-transform infrared spectroscopy, and circular dichroism measurements suggest that the secondary structure of LF in the LF-α-LA complex was maintained after complexation and subsequent thermal treatments. At pH 7, the LF-α-LA complex protected LF from thermal aggregation and denaturation, and the LF retained its functional and structural properties, including antibacterial capacity of LF after thermal treatments. The improved thermal stability and functional properties of LF in the LF-α-LA complex are of interest to the food industry.

Destabilization of the D2 domain of Thermotoga maritima arginine binding protein induced by guanidinium thiocyanate and its counteraction by stabilizing agents.

D2 is a structural and cooperative domain of Thermotoga maritima Arginine Binding Protein, that possesses a remarkable conformational stability, with a denaturation temperature of 102.6°C, at pH 7.4. The addition of potassium thiocyanate causes a significant decrease in the D2 denaturation temperature. The interactions of thiocyanate ions with D2 have been studied by means of isothermal titration calorimetry measurements and molecular dynamics simulations. It emerged that: (a) 20-30 thiocyanate ions interact with the D2 surface and are present in its first solvation shell; (b) each of them makes several contacts with protein groups, both polar and nonpolar ones. The addition of guanidinium thiocyanate causes a marked destabilization of the D2 native state, because both the ions are denaturing agents. However, on adding to the solution containing D2 and guanidinium thiocyanate a stabilizing agent, such as TMAO, sucrose or sodium sulfate, a significant increase in denaturation temperature occurs. The present results confirm that counteraction is a general phenomenon for globular proteins.

A simplified non-reduced peptide mapping method with faster and efficient enzymatic digestion for characterization of native disulfide bonds in monoclonal and bispecific antibodies.

Development of monoclonal and bispecific antibody-based protein therapeutics requires detailed characterization of native disulfide linkages, which is commonly achieved through peptide mapping under non-reducing conditions followed by liquid chromatography-mass spectrometry (LC-MS) analysis. One major challenge of this method is incomplete protein digestion due to insufficient denaturation of antibodies under non-reducing conditions. For a long time, researchers have explored various strategies with the aim of efficiently digesting antibody drugs when the disulfide bonds remain intact, but few could achieve this by using a simple and generic approach with well controlled disulfide scrambling artifacts. Here, we report a simple method for fast and efficient mapping of native disulfides of monoclonal and bispecific antibody-based protein therapeutics. The method was optimized to achieve optimal digestion efficiency by denaturing proteins with 8 M urea plus 0-1.25 M guanidine-HCl at elevated temperature (50 °C), followed by two-step digestion with trypsin/Lys-C mix using a one-pot reaction. The only parameter that needs to be optimized for different proteins is the concentration of guanidine-HCl present. This simplified sample preparation eliminated buffer exchange and can be completed within three hours. By using this new method, all native disulfide bonds were confirmed for these monoclonal and bispecific antibodies with high confidence. When compared with a commercial kit utilizing low-pH digestion condition, the new method demonstrated higher digestion efficiency and shorter sample preparation time. These results suggest this new one-pot-two-step digestion method is suitable for the characterization of antibody disulfide bonds, particularly for those antibodies with digestion-resistant domains under typical digestion conditions.

Turmeric-Black Cumin Essential Oils and Their Capacity to Attenuate Free Radicals, Protein Denaturation, and Cancer Proliferation.

Turmeric rhizomes (Curcuma longa) and black cumin seeds (Nigella sativa) are polyherbal ingredients used for the management of cancer and other chronic inflammatory diseases in Nigerian ethnomedicine. Previous studies have shown the antioxidant, anti-inflammatory, and anticancer activities of the individual plant extracts. However, the two spices have not been biologically potentiated in their combined form. Therefore, this study obtained essential oils (EOs) from the combined spices and evaluated their inhibitory effects on free radicals, protein denaturation, and cancer proliferation. The EOs were extracted by hydro-distillation (HD) and characterized by gas chromatography-mass spectrometry (GC-MS). In vitro antioxidant assessment was conducted based on DPPH, hydrogen peroxide (H2O2), nitric oxide (NO), and ferric ion (Fe3+) radical scavenging assays. The cytotoxicity of the oil against non-tumorigenic (HEK293) and cancerous (HepG2 and HeLa) cell lines was determined following the MTT cell viability assay. An in silico molecular docking analysis of the oil constituents was also performed. Six batches of EOs I-VI were afforded, comprising twenty-two major constituents, with aromatic Ar-turmerone being the most prominent compound. There was a marked improvement in the bioactivity of the oils upon repeated HD and as a combination. The batch VI oil exhibited the best activity, with a cytotoxicity (CC50) of 10.16 ± 1.69 µg/100 µL against the HepG2 cell line, which was comparable to 5-fluorouracil (standard, CC50 = 8.59 ± 1.33 µg/100 µL). In silico molecular docking suggested δ-curcumene, Ar-curcumene, Ar-turmerol, and Ar-turmerone among the promising compounds based on their high binding energy scores with NOX2, NF-κB, and mdm2 proteins. In conclusion, the oils from the turmeric-black cumin combined possess a considerable inhibition ability against free radicals, protein denaturation, and cancer proliferation. This study's findings further underscore the effectiveness of turmeric-black cumin as a polyherbal medicinal ingredient.

Interfacial Denaturation at the Droplet Simplifies the Formation of Drug-Loaded Protein Nanocapsules to Enhance Immune Response of Cells.

Nanocapsules enable multicomponent encapsulation of therapeutic cargoes with high encapsulation content and efficiency, which is vital for cancer immunotherapy. In the past, chemical crosslinking is used to synthesize nanocapsules, which can impede the regulatory approval process. Therefore, a new class of protein nanocapsules is developed by eliminating the need for chemical crosslinking by utilizing protein denaturation through a process that is referred to as "baking at the droplet interface". Such protein nanocapsules with antigens incorporated in the shell and a combination of encapsulated drugs showed an enhancement in the immune response of cells.

Factor defining the effects of tetraalkylammonium chloride on stability, folding, and dynamics of horse cytochrome c.

This article describes the molecular mechanism by which tetraalkylammonium chloride ([R4N]Cl: R- = methyl (Me), ethyl (Et), propyl (Pr),butyl (Bu)) modulates the stability, folding, and dynamics of cytochrome c (Cyt c). Analysis of [R4N]Cl effects on thermal/chemical denaturations, millisecond refolding/unfolding kinetics, and slow CO-association kinetics of Cyt c without and with denaturant providing some significant results: (i) [R4N]Cl decreasing the unfolding free energy estimated by thermodynamic and kinetic analysis of thermal/chemical denaturation curves and kinetic chevrons (Log kobs-[GdmCl]) of Cyt c, respectively (ii) hydrophobicity of R-group of [R4N]Cl, preferential inclusion of [R4N]Cl at the protein surface, and destabilizing enthalpic attractive interactions of [Me4N]Cl and steric entropic interactions of [Et4N]Cl,[Pr4N]Cl and [Bu4N]Cl with protein contribute to [R4N]Cl-induced decrease thermodynamic stability of Cyt c (iii) [R4N]Cl exhibits an additive effect with denaturant to decrease thermodynamic stability and refolding rates of Cyt c (iv) low concentrations of [R4N]Cl (≤ 0.5 M) constrain the motional dynamics while the higher concentrations (>0.75 M [R4N]Cl) enhance the structural-fluctuations that denture protein (v) hydrophobicity of R-group of [R4N]Cl alters the [denaturant]-dependent conformational stability, refolding-unfolding kinetics, and CO-association kinetics of Cyt c. Furthermore, the MD simulations depicted that the addition of 1.0 M of [R4N]Cl increased the conformational fluctuations in Cyt c leading to decreased structural stability in the order [Me4N]Cl < [Et4N]Cl < [Pr4N]Cl < [Bu4N]Cl consistent with the experimental results.

The impacts of freeze-drying-induced stresses on the quality of meat and aquatic products: Mechanisms and potential solutions to acquire high-quality products.

Freeze-drying is a preservation method known for its effectiveness in dehydrating food products while minimizing their deterioration. However, protein denaturation and oxidation during freezing and drying can degrade the quality of meat and aquatic products. Therefore, finding the strategies to ensure the dried products' sensory, functional, and nutritional attributes is crucial. This study aimed to summarize protein denaturation mechanisms and overall quality changes in meat and aquatic products during freezing and drying, while also exploring methods for quality control. Different freeze-drying conditions result in varying levels of oxidation and functionality in meat and aquatic products, leading to changes in quality, such as altered fatty and amino acid compositions, protein digestibility, and sensory attributes. To obtain high-quality dried products by freeze-drying, several parameters should be considered, including sample type, freezing and drying temperatures, moisture content, pulverization effects, and storage conditions.

NMR Dynamic View of the Destabilization of WW4 Domain by Chaotropic GdmCl and NaSCN.

GdmCl and NaSCN are two strong chaotropic salts commonly used in protein folding and stability studies, but their microscopic mechanisms remain enigmatic. Here, by CD and NMR, we investigated their effects on conformations, stability, binding and backbone dynamics on ps-ns and µs-ms time scales of a 39-residue but well-folded WW4 domain at salt concentrations ≤200 mM. Up to 200 mM, both denaturants did not alter the tertiary packing of WW4, but GdmCl exerted more severe destabilization than NaSCN. Intriguingly, GdmCl had only weak binding to amide protons, while NaSCN showed extensive binding to both hydrophobic side chains and amide protons. Neither denaturant significantly affected the overall ps-ns backbone dynamics, but they distinctively altered µs-ms backbone dynamics. This study unveils that GdmCl and NaSCN destabilize a protein before the global unfolding occurs with differential binding properties and µs-ms backbone dynamics, implying the absence of a simple correlation between thermodynamic stability and backbone dynamics of WW4 at both ps-ns and µs-ms time scales.

Mechanical release of homogenous proteins from supramolecular gels.

A long-standing challenge is how to formulate proteins and vaccines to retain function during storage and transport and to remove the burdens of cold-chain management. Any solution must be practical to use, with the protein being released or applied using clinically relevant triggers. Advanced biologic therapies are distributed cold, using substantial energy, limiting equitable distribution in low-resource countries and placing responsibility on the user for correct storage and handling. Cold-chain management is the best solution at present for protein transport but requires substantial infrastructure and energy. For example, in research laboratories, a single freezer at -80 °C consumes as much energy per day as a small household1. Of biological (protein or cell) therapies and all vaccines, 75% require cold-chain management; the cost of cold-chain management in clinical trials has increased by about 20% since 2015, reflecting this complexity. Bespoke formulations and excipients are now required, with trehalose2, sucrose or polymers3 widely used, which stabilize proteins by replacing surface water molecules and thereby make denaturation thermodynamically less likely; this has enabled both freeze-dried proteins and frozen proteins. For example, the human papilloma virus vaccine requires aluminium salt adjuvants to function, but these render it unstable against freeze-thaw4, leading to a very complex and expensive supply chain. Other ideas involve ensilication5 and chemical modification of proteins6. In short, protein stabilization is a challenge with no universal solution7,8. Here we designed a stiff hydrogel that stabilizes proteins against thermal denaturation even at 50 °C, and that can, unlike present technologies, deliver pure, excipient-free protein by mechanically releasing it from a syringe. Macromolecules can be loaded at up to 10 wt% without affecting the mechanism of release. This unique stabilization and excipient-free release synergy offers a practical, scalable and versatile solution to enable the low-cost, cold-chain-free and equitable delivery of therapies worldwide.

Whey-based sports supplements: Heat damage and protein breakdown after in vitro gastrointestinal digestion.

This study was aimed to evaluate the effect of heat damage on the release of total amino acids (AA), essential AA (EAA), branched-chain AA (BCAA) and bioactive peptides following in vitro static simulated gastrointestinal digestion (SGID) of four commercial whey-protein based sports supplements. The extent of protein glycation and denaturation was evaluated through the determination of the content of furosine and soluble whey proteins. The strongest protein breakdown (41.3 %) and the highest release of AA, EAA and BCAA (36.20, 27.78, and 11.30 g/100 g protein, respectively) was observed in the sports supplement characterised by the lowest (52.5 %) level of soluble whey proteins; whereas the protein glycation had a negligible impact on the studied parameters. The SGID also led to the release of several peptides with various reported bioactivities that may be beneficial to sports activity.

Light exacerbates local and global effects induced by pH unfolding of Ipilimumab.

Monoclonal antibodies (mAbs) are an essential class of therapeutic proteins for the treatment of cancer, autoimmune and rare diseases. During their production, storage, and administration processes, these proteins encounter various stressors such as temperature fluctuations, vibrations, and light exposure, able to induce chemico-physical modifications to their structure. Viral inactivation is a key step in downstream processes, and it is achieved by titration of the mAb at low pH, followed by neutralization. The changes of the pH pose a significant risk of unfolding and subsequent aggregation to proteins, thereby affecting their manufacturing. This study aims to investigate whether a combined exposure to light during the viral inactivation process can further affect the structural integrity of Ipilimumab, a mAb primarily used in the treatment of metastatic melanoma. The biophysical and biochemical characterization of Ipilimumab revealed that pH variation is a considerable risk for its stability with irreversible unfolding at pH 2. The threshold for Ipilimumab denaturation lies between pH 2 and 3 and is correlated with the loss of the protein structural cooperativity, which is the most critical factor determining the protein refolding. Light has demonstrated to exacerbate some local and global effects making pH-induced exposed regions more vulnerable to structural and chemical changes. Therefore, specific precautions to real-life exposure to ambient light during the sterilization process of mAbs should be considered to avoid loss of the therapeutic activity and to increase the yield of production. Our findings underscore the critical role of pH optimization in preserving the structural integrity and therapeutic efficacy of mAbs. Moreover, a detailed conformational study on the structural modifications of Ipilimumab may improve the chemico-physical knowledge of this effective drug and suggest new production strategies for more stable products under some kind of stress conditions.