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In this work, we explored the acid-induced unfolding pathway of non-porin outer membrane protein (OMP), an immunogenic protein from Salmonella Typhi, by monitoring the conformational changes over a pH range of 1.0–7.0 by circular dichroism, intrinsic fluorescence, ANS binding, acrylamide quenching, and dynamic light scattering. The spectroscopic measurements showed that OMP in its native state at pH 7.0 exists in more stable and compact conformation. In contrast, at pH 2.0, OMP retains substantial amount of secondary structure, disrupted side chain interactions, increased hydrodynamic radii, and nearly four-fold increase in ANS fluorescence with respect to the native state, indicating that MG state exists at pH 2.0. Quenching of tryptophan fluorescence by acrylamide further confirmed the accumulation of a partially unfolded state between native and unfolded state. The effect of pH on the conformation and thermostability of OMP points towards its heat resistance at neutral pH (T ₘ ~ 69 °C at pH 7.0, monitored by change in MRE₂₂₂ ₙₘ). Acid unfolded state was also characterized by the lack of a cooperative thermal transition. All these results suggested that acid-induced unfolded state of OMP at pH 2.0 represented the molten globule state. The chemical denaturation studies with GuHCl and urea as denaturants showed dissimilar results. The chemical unfolding experiments showed that in both far-UV CD and fluorescence measurements, GuHCl is more efficient than urea. GuHCl is characterized by low C ₘ (~1 M), while urea is characterized by high C ₘ (~3 M). The fully unfolded states were reached at 2 M GuHCl and 4 M urea concentration, respectively. This study adds to several key considerations of importance in the development of therapeutic agents against typhoid fever for clinical purposes.

Living organisms are very sensitive to temperature, and much of this is attributed to its effect on the structure and function of proteins. Leuenberger et al. explored thermostability on a proteome-wide scale in bacteria, yeast, and human cells by using a combination of limited proteolysis and mass spectrometry (see the Perspective by Vogel). Their results suggest that temperature-induced cell death is caused by the loss of a subset of proteins with key functions. The study also provides insight into the molecular and evolutionary bases of protein and proteome stability. Science , this issue p. eaai7825 ; see also p. 794 Proteomic analysis provides insight into the molecular and evolutionary bases of proteins and proteome thermal stability. Temperature-induced cell death is thought to be due to protein denaturation, but the determinants of thermal sensitivity of proteomes remain largely uncharacterized. We developed a structural proteomic strategy to measure protein thermostability on a proteome-wide scale and with domain-level resolution. We applied it to Escherichia coli , Saccharomyces cerevisiae , Thermus thermophilus , and human cells, yielding thermostability data for more than 8000 proteins. Our results (i) indicate that temperature-induced cellular collapse is due to the loss of a subset of proteins with key functions, (ii) shed light on the evolutionary conservation of protein and domain stability, and (iii) suggest that natively disordered proteins in a cell are less prevalent than predicted and (iv) that highly expressed proteins are stable because they are designed to tolerate translational errors that would lead to the accumulation of toxic misfolded species.

To investigate the effects of traditional high-temperature cooking and sous-vide cooking on the quality of tilapia fillets, muscle microstructure, texture, lipid oxidation, protein structure, and volatile compounds were analyzed. In comparison with samples subjected to traditional high-temperature cooking, sous-vide-treated samples exhibited less protein denaturation, a secondary structure dominated by α-helices, a stable and compact structure, a significantly higher moisture content, and fewer gaps in muscle fibers. The hardness of the sous-vide-treated samples was higher than that of control samples, and the extent of lipid oxidation was significantly reduced. The sous-vide cooking technique resulted in notable changes in the composition and relative content of volatile compounds, notably leading to an increase in the presence of 1-octen-3-ol, α-pinene, and dimethyl sulfide, and a decrease in the levels of hexanal, D-limonene, and methanethiol. Sous-vide treatment significantly enhanced the structural stability, hardness, and springiness of muscle fibers in tilapia fillets and reduced nutrient loss, enriched flavor, and mitigated effects on taste and fishy odor.

Legumes have gained increased dietary importance in recent years due to their recognized health benefits. Recent plant protein revolution has elevated legumes to the forefront from consumers' and food industry's perspective. Unlike cereal proteins and starches, there is a scarcity of information on the structural properties of legume starches. Consumption of legume‐derived dietary fibers have a positive impact on the human health, in particular, gut health, which is a current research focus for nutrition and health professionals. Knowledge of legume ingredients properties (e.g., protein denaturation, starch gelatinization, pasting, and thermal properties) could aid in understanding functionality and potential uses of these materials. The physicochemical, thermal, and the functional properties of legume proteins, starches, and dietary fibers are elucidated. Both the food ingredient manufacturers and research and development professionals in the food industry can benefit from the information provided in this review article.

The effect of temperature fluctuations on the freshness of shrimp in simulated trays was investigated by setting a freeze–thaw (F-T) cycle of 12 h after freezing at −20 °C and thawing at 1 °C under refrigeration. The results showed that the shrimp’s physicochemical properties deteriorated to different extents with the increase in F-T cycles. The total colony count of shrimp was 6.07 lg CFU/g after 21 cycles, and the volatile saline nitrogen content reached 30.36 mg/100 g, which exceeded the edible standard. In addition, the sensory quality and textural properties (hardness, elasticity, chewiness, and adhesion) declined to different degrees with increased F-T cycles. LF-NMR and protein property measurements showed that F-T cycles resulted in reduced water holding capacity and protein denaturation, which were the main factors leading to the deterioration of shrimp quality. Furthermore, flavor changes were analyzed using an electronic nose sensor to establish a freshness model. The W1W, W1S, W2S, and W5S sensors were correlated with the quality changes in shrimp and used as the main sensors for detecting the freshness of Penaeus vannamei. As a result, to better maintain the overall freshness, temperature fluctuations should be minimized in sales and storage, and fewer than 8 F-T cycles should be performed.

Specificity of interactions between two DNA strands, or between protein and DNA, is often achieved by varying bases or side chains coming off the DNA or protein backbone—for example, the bases participating in Watson–Crick pairing in the double helix, or the side chains contacting DNA in TALEN–DNA complexes. By contrast, specificity of protein–protein interactions usually involves backbone shape complementarity 1 , which is less modular and hence harder to generalize. Coiled-coil heterodimers are an exception, but the restricted geometry of interactions across the heterodimer interface (primarily at the heptad a and d positions 2 ) limits the number of orthogonal pairs that can be created simply by varying side-chain interactions 3 , 4 . Here we show that protein–protein interaction specificity can be achieved using extensive and modular side-chain hydrogen-bond networks. We used the Crick generating equations 5 to produce millions of four-helix backbones with varying degrees of supercoiling around a central axis, identified those accommodating extensive hydrogen-bond networks, and used Rosetta to connect pairs of helices with short loops and to optimize the remainder of the sequence. Of 97 such designs expressed in Escherichia coli , 65 formed constitutive heterodimers, and the crystal structures of four designs were in close agreement with the computational models and confirmed the designed hydrogen-bond networks. In cells, six heterodimers were fully orthogonal, and in vitro—following mixing of 32 chains from 16 heterodimer designs, denaturation in 5 M guanidine hydrochloride and reannealing—almost all of the interactions observed by native mass spectrometry were between the designed cognate pairs. The ability to design orthogonal protein heterodimers should enable sophisticated protein-based control logic for synthetic biology, and illustrates that nature has not fully explored the possibilities for programmable biomolecular interaction modalities. Computational design incorporating modular buried hydrogen networks produces highly orthogonal protein heterodimers.

The well-established temperature-dependence of growth parameters and maximum sizes of fish and other water-breathing ectotherms (WBEs) form the basis for various “temperature-size rules” for fish and WBEs. Numerous adaptationist interpretations of these rules exist, but their biochemical basis is largely ignored. One fundamental, but frequently overlooked component of the mechanism that leads to temperature-size rules, is that proteins only “work” if their native quaternary structure (or native folding) is maintained. However, proteins have half-lives are U-shaped functions of temperature, which means that higher or lower than optimal temperatures increase their rates of spontaneous denaturation in aqueous solutions, i.e., within body cells. Proteins that lose their quaternary structures cease to function and, in most cases, need to be resynthesized. Thus, protein denaturation may explain why the metabolic rates of fish and other ectotherms increase with temperatures, both above 4 °C, the temperature at which hydrogen bonding in water is the strongest and hydration of protein nonpolar groups the weakest, and below 4 °C, the regime of “cold denaturation.” Considering the biochemical basis of temperature-size rules for fish and other WBEs would enable biologists to better understand adverse consequences of climate warming for marine and freshwater biodiversity.

Introduction: Inflammation is a fundamental response of the immune system during tissue damage or pathogen infection to protect and maintain tissue homeostasis. However, inflammation may lead to life-threatening conditions. The most common treatment of inflammation is non-steroidal anti-inflammatory drugs (NSAIDs). Nowadays, the development of safer new NSAIDs is critical as most of the existing NSAIDs have serious adverse effects, such as gastrointestinal (GI) toxicity and cardiotoxicity. In the present study, four compounds as Schiff base derivatives of 7-hydroxy-4-formyl coumarin and 7-methoxy-4-formyl coumarin were designed and synthesized aiming to develop a lead compound that exhibits anti-inflammatory activity and circumvents the side effects of NSAIDs, especially GI toxicity. Materials and Methods: Lipinski's rule of five was applied for each designed molecule to evaluate the drug-likeness properties. Molecular docking studies were performed using the ligands and the cyclooxygenase-2 (COX-2) protein to select the best-scored molecule using AutoDock 4.2.6. The molecules were then synthesized and characterized. An in vitro anti-inflammatory assay of the compounds against the COX-2 receptor was realized through a protein denaturation assay. Results and Discussion: All four synthesized ligands passed Lipinski's rule of five and exhibited higher binding free energy compared to the positive standard control (ibuprofen), and the Ki values of compounds 5, 7, and 8 were in the nanomolar range. However, only compounds 6 and 7 obtained a higher percentage of inhibition of protein denaturation relative to ibuprofen. Conclusion: The present study suggested that compound 7 may be a lead molecule because this ligand not only exhibited the best computational and experimental results but also exhibited the strongest correlation between the concentration and percentage of protein denaturation (R = 0.986 and [R.sup.2] = 0.972) with the lowest P-value (0.014). Keywords: coumarin, non-steroidal anti-inflammatory drugs, ibuprofen, lead compound, cyclooxygenase, binding free energy, Schiff base derivatives

Selective denaturation of meat proteins - essential to reach desired textures - requires cooking temperatures corresponding to their different structure and interactions. Sous-vide cooking allows precise control over the denaturation state of meat proteins (and thus the cooking state of meat products) due to the possibility to cook at very well defined temperatures. Additionally, kinetic effects also play an important role. Differential scanning calorimetry (DSC) has been used here to follow the denaturation state of proteins in pork filet ( Musculus psoas major ), which had been heat treated at different time (10–2880 min) and temperature (45–74 °C) combinations. Additionally, the water loss (cooking loss) occurring during heat treatments has been determined. Four endothermic peaks have been observed in the DSC curves. Their individual time and temperature dependent enthalpies show that proteins become denatured at temperatures well below the peak temperatures if kept there for long times. This observation is underlined by statistical arguments. Cooking loss increases with time and temperature, while the main water loss occurs during the first 240 min and at temperatures above 60 °C. Due to the different kinetics found for protein denaturation and cooking loss, it is not possible to directly correlate the two quantities.

Dry molten globular (DMG) intermediates, an expanded form of the native protein with a dry core, have been observed during denaturant-induced unfolding of many proteins. These observations are counterintuitive because traditional models of chemical denaturation rely on changes in solvent-accessible surface area, and there is no notable change in solvent-accessible surface area during the formation of the DMG. Here we show, using multisite fluorescence resonance energy transfer, far-UV CD, and kinetic thiol-labeling experiments, that the guanidinium chloride (GdmCl)-induced unfolding of RNase H also begins with the formation of the DMG. Population of the DMG occurs within the 5-ms dead time of our measurements. We observe that the size and/or population of the DMG is linearly dependent on [GdmCl], although not as strongly as the second and major step of unfolding, which is accompanied by core solvation and global unfolding. This rapid GdmCl-dependent population of the DMG indicates that GdmCl can interact with the protein before disrupting the hydrophobic core. These results imply that the effect of chemical denaturants cannot be interpreted solely as a disruption of the hydrophobic effect and strongly support recent computational studies, which hypothesize that chemical denaturants first interact directly with the protein surface before completely unfolding the protein in the second step (direct interaction mechanism).