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.

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.

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.

This study investigates the impact of dry heat pretreatment on the functionality of soy, chickpea, and pea protein ingredients for use in texturized vegetable protein (TVP) production via low moisture extrusion. The protein powders were heat-treated at temperatures ranging from 80 °C to 160 °C to modulate the extent of protein denaturation and assess their effects on RVA pasting behavior, water absorption capacity (WAC), and color attributes. The results indicate that the pretreatment temperature significantly influenced the proteins\' functional properties, with an optimal temperature of 120 °C enhancing pasting properties and maintaining WAC, while a higher pretreatment temperature of 160 °C led to diminished ingredient functionality. Different protein sources exhibited distinct responses to heat pretreatment. The subsequent extrusion processing revealed significant changes in extrudate density and color, with increased density and darkness observed at higher pretreatment temperatures. This research provides insights into the interplay between protein sources, pretreatment conditions, and extrusion outcomes, highlighting the importance of controlled protein denaturation for developing high-quality, plant-based meat analogues. The findings have broad implications for the optimization of meat analogue manufacturing, with the aim of enhancing the sensory experience and sustainability of plant-based foods.

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.

The quality of surimi, widely used in processed seafood, is compromised by freeze-thaw cycles, leading to protein denaturation and oxidative degradation. The objective of this study is to explore the effects of adding natural whey peptide hydrolysate (WPH) on the myofibrillar proteins of repeatedly freeze-thawed surimi. Results indicated surimi treated with 15% WPH exhibited only a 128% increase in surface hydrophobicity and a maximum peroxide value of 7.84 μg/kg, significantly lower than the control group. Additionally, salt-soluble protein content, emulsification activity, and stability decreased with the increase in freeze-thaw cycles. With a 15% WPH offering the most significant protective effect, evidenced by reductions of only 25.02%, 42.52% and 37.02% in salt-soluble protein content, emulsification activity, and stability, respectively. These outcomes demonstrate that WPH effectively reduces protein denaturation during repeated freeze-thaw processes. Future research should explore the molecular mechanisms underlying WPH\'s protective effects and evaluate their applicability in other food systems.

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.

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.

RNA bacteriophage MS2-derived virus-like particles (VLPs) have been widely used in biomedical research as model systems to study virus assembly, structure-function relationships, vaccine development, and drug delivery. Considering the diverse utility of these VLPs, a systemic engineering approach has been utilized to generate smaller particles with optimal serum stability and tissue penetrance. Additionally, it is crucial to demonstrate the overall stability of these mini MS2 VLPs, ensuring cargo protection until they reach their target cell/organ. However, no detailed analysis of the thermal stability and heat-induced disassembly of MS2 VLPs has yet been attempted. In this work, we investigated the thermal stability of both wild-type (WT) MS2 VLP and its \"mini\" variant containing S37P mutation (mini MS2 VLP). The mini MS2 VLP exhibits a higher capsid melting temperature (Tm) when compared to its WT MS2 VLP counterpart, possibly attributed to its smaller interdimer angle. Our study presents that the thermal unfolding of MS2 VLPs follows a sequential process involving particle destabilization, nucleic acid exposure/melting, and disassembly of VLP. This observation underscores the disruption of cooperative intersubunit interactions and protein-nucleic acid interactions, shedding light on the mechanism of heat-induced VLP disassembly.

Drying, as a critical step in the production of air-dried beef, has a direct impact on the quality of the final product. Innovatively, a composite system incorporating contact ultrasound (CU) and infrared radiation (IR) as auxiliary measures within a hot air drying (HAD) framework was built in this research, and the effects of these techniques on the drying kinetics, protein denaturation, and moisture transformation of air-dried beef were investigated. In comparison to HAD treatment, the integrated CU and IR (CU-IRD) system displayed marked enhancements in heat and moisture transport efficiency, thereby saving 36.84% of time expenditure and contributing favorably to the improved moisture distribution of the end-product. This was mainly ascribed to the denaturation of myosin induced by IR thermal effect and the micro-channel produced by CU sponge effect, thus increasing T2 relaxation time and the proportion of free water. In conclusion, the composite system solved the problem of surface hardening and reduces hardness and chewiness of air-dried beef by 40.42% and 45.25% respectively, but inevitably increased the energy burden by 41.60%.