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The DinB homolog polymerase (Dbh) is a member of the Y-family of translesion DNA polymerases that can synthesize using a damaged DNA template. Since Dbh comes from the thermophilic archaeon Sulfolobus acidocaldarius, it is capable of functioning over a wide range of temperatures. Existing X-ray structures were determined at temperatures where the protein is least active. Here we use NMR and circular dichroism to understand how the structure and dynamics of Dbh are affected by temperature (2°C-65°C) and metal ion binding in solution. We measured hydrogen exchange protection factors, temperature coefficients, and chemical shift perturbations with and without magnesium and manganese. We report on regions of the protein that become more dynamic as the temperature is increased toward the functional temperature. Hydrogen exchange protection factors and temperature coefficients reveal that both the thumb and finger domains are very dynamic relative to the palm and little-finger (LF) domains. These trends remain true at high temperature with dynamics increasing as temperatures increase from 35°C to 50°C. Notably, NMR spectra show that the Dbh tertiary structure cold denatures beginning at 25°C and increases in denaturation as the temperature is lowered to 5°C with little change observed by CD. Above 35°C, chemical shift perturbation analysis in the presence and absence of magnesium and manganese reveals three ion binding sites, without DNA bound. In contrast, these bound metals are not apparent in any Dbh crystal structures of the protein without DNA. Two ion binding sites are confirmed to be near the active site, as reported in other Y-family polymerases, and we report a novel ion binding site in the LF domain. Thus, the solution-state structure of the Dbh polymerase is distinct from that of the solid-state structures and shows an unusually high cold denaturation temperature.

Freezing is commonly used to extend the shelf life of meat and meat products but may impact the overall quality of those products by inducing structural changes in myofibrillar proteins (MPs) through denaturation, chemical modification, and encouraging protein aggregation. This review covers the effect of freezing on the denaturation of MPs in terms of the effects of ice crystallization on solute concentrations, cold denaturation, and protein oxidation. Freezing-induced denaturation of MPs begins with ice crystallization in extracellular spaces and changes in solute concentrations in the unfrozen water fraction. At typical temperatures for freezing meat (lower than −18 °C), cold denaturation of proteins occurs, accompanied by an alteration in their secondary and tertiary structure. Moreover, the disruption of muscle cells triggers the release of cellular enzymes, accelerating protein degradation and oxidation. To minimize severe deterioration during the freezing and frozen storage of meat, there is a vital need to use an appropriate freezing temperature below the glass transition temperature and to avoid temperature fluctuations during storage to prevent recrystallization. Such an understanding of MP denaturation can be applied to determine the optimum freezing conditions for meat products with highly retained sensory, nutritional, and functional qualities. [ABSTRACT FROM AUTHOR]

[Display omitted] Assessment of cold stability is essential for manufacture and commercialization of biotherapeutics. Storage stability is often estimated by measuring accelerated rates at elevated temperature and using mathematical models (as the Arrhenius equation). Although, this strategy often leads to an underestimation of protein aggregation during storage. In this work, we measured the aggregation rates of two antibodies in a broad temperature range (from 60 °C to −25 °C), using an isochoric cooling method to prevent freezing of the formulations below 0 °C. Both antibodies evidenced increasing aggregation rates when approaching extreme temperatures, because of hot and cold denaturation. This behavior was modelled using Arrhenius and Gibbs-Helmholtz equations, which enabled to deconvolute the contribution of unfolding from the protein association kinetics. This approach made possible to model the aggregation rates at refrigeration temperature (5 °C) in a relatively short timeframe (1–2 weeks) and using standard characterization techniques (SEC-HPLC and DLS). [ABSTRACT FROM AUTHOR]

Thermophilic proteins and enzymes are attractive for use in industrial applications due to their resistance against heat and denaturants. Here, we report on a thermophilic protein that is stable at high temperatures (Ttrs, hot 67 °C) but undergoes significant unfolding at room temperature due to cold denaturation. Little is known about the cold denaturation of thermophilic proteins, although it can significantly limit their applications. We investigated the cold denaturation of thermophilic multidomain protein translation initiation factor 2 (IF2) from Thermus thermophilus. IF2 is a GTPase that binds to ribosomal subunits and initiator fMet-tRNAfMet during the initiation of protein biosynthesis. In the presence of 9 M urea, measurements in the far-UV region by circular dichroism were used to capture details about the secondary structure of full-length IF2 protein and its domains during cold and hot denaturation. Cold denaturation can be suppressed by salt, depending on the type, due to the decreased heat capacity. Thermodynamic analysis and mathematical modeling of the denaturation process showed that salts reduce the cooperativity of denaturation of the IF2 domains, which might be associated with the high frustration between domains. This characteristic of high interdomain frustration may be the key to satisfying numerous diverse contacts with ribosomal subunits, translation factors, and tRNA. [ABSTRACT FROM AUTHOR]

We review the key steps leading to an improved analysis of thermal protein unfolding. Thermal unfolding is a dynamic cooperative process with many short-lived intermediates. Protein unfolding has been measured by various spectroscopic techniques that reveal structural changes, and by differential scanning calorimetry (DSC) that provides the heat capacity change Cp(T). The corresponding temperature profiles of enthalpy ΔH(T), entropy ΔS(T), and free energy ΔG(T) have thus far been evaluated using a chemical equilibrium two-state model. Taking a different approach, we demonstrated that the temperature profiles of enthalpy ΔH(T), entropy ΔS(T), and free energy ΔG(T) can be obtained directly by a numerical integration of the heat capacity profile Cp(T). DSC thus offers the unique possibility to assess these parameters without resorting to a model. These experimental parameters now allow us to examine the predictions of different unfolding models. The standard two-state model fits the experimental heat capacity peak quite well. However, neither the enthalpy nor entropy profiles (predicted to be almost linear) are congruent with the measured sigmoidal temperature profiles, nor is the parabolic free energy profile congruent with the experimentally observed trapezoidal temperature profile. We introduce three new models, an empirical two-state model, a statistical–mechanical two-state model and a cooperative statistical-mechanical multistate model. The empirical model partially corrects for the deficits of the standard model. However, only the two statistical–mechanical models are thermodynamically consistent. The two-state models yield good fits for the enthalpy, entropy and free energy of unfolding of small proteins. The cooperative statistical–mechanical multistate model yields perfect fits, even for the unfolding of large proteins such as antibodies. [ABSTRACT FROM AUTHOR]

When protein crystals are abruptly cooled, the unit-cell, protein and solvent-cavity volumes all contract, but the volume of bulk-like internal solvent may expand. Outflow of this solvent from the unit cell and its accumulation in defective interior crystal regions has been suggested as one cause of the large increase in crystal mosaicity on cooling. It is shown that when apoferritin crystals are abruptly cooled to temperatures between 220 and 260 K, the unit cell contracts, solvent is pushed out and the mosaicity grows. On temperature-dependent timescales of 10 to 200 s, the unit-cell and solvent-cavity volume then expand, solvent flows back in, and the mosaicity and B factor both drop. Expansion and reordering at fixed low temperature are associated with small-amplitude but large-scale changes in the conformation and packing of apoferritin. These results demonstrate that increases in mosaicity on cooling arise due to solvent flows out of or into the unit cell and to incomplete, arrested relaxation of protein conformation. They indicate a critical role for time in variable-temperature crystallographic studies, and the feasibility of probing interactions and cooperative conformational changes that underlie cold denaturation in the presence of liquid solvent at temperatures down to ∼200 K.

The mediation of liquid–liquid phase separation (LLPS) for fused in sarcoma (FUS) protein is generally attributed to the low‐complexity, disordered domains and is enhanced at low temperature. The role of FUS folded domains on the LLPS process remains relatively unknown since most studies are mainly based on fragmented FUS domains. Here, we investigate the effect of metabolites on full‐length (FL) FUS LLPS using turbidity assays and differential interference contrast (DIC) microscopy, and explore the behavior of the folded domains by nuclear magnetic resonance (NMR) spectroscopy. FL FUS LLPS is maximal at low concentrations of glucose and glutamate, moderate concentrations of NaCl, Zn2+, and Ca2+ and at the isoelectric pH. The FUS RNA recognition motif (RRM) and zinc‐finger (ZnF) domains are found to undergo cold denaturation above 0°C at a temperature that is determined by the conformational stability of the ZnF domain. Cold unfolding exposes buried nonpolar residues that can participate in LLPS‐promoting hydrophobic interactions. Therefore, these findings constitute the first evidence that FUS globular domains may have an active role in LLPS under cold stress conditions and in the assembly of stress granules, providing further insight into the environmental regulation of LLPS. [ABSTRACT FROM AUTHOR]

[Display omitted] Through a synergistic collaboration of people with varying backgrounds and expertise, the root-cause of respiratory syncytial virus prefusion (preF) protein aggregation during freezing was identified to be supercooling. This issue was addressed through a comprehensive understanding of the product. Leveraging innovative and unconventional methods, apparatus, and approaches, it was effectively determined that key parameters influencing aggregation were the nucleation temperature and the duration of supercooling. Moreover, additional measurements revealed that a transition from the preF to the postfusion conformation occurs upon supercooling, which is likely caused by cold denaturation. The importance of considering freezing conditions is highlighted supporting analytical sampling and envisioning that better understanding of sample handling/freezing process can be applied to a wide range of protein-based products. [ABSTRACT FROM AUTHOR]

As the majority of marine organisms are water-breathing ectotherms, temperature and dissolved oxygen are key environmental variables that influence their fitness and geographic distribution. In line with the temperature-size rule (TSR), marine ectotherms in warmer temperatures will grow to a smaller maximum body size, yet the extent to which different species experience this temperature-size response varies. Here, we analysed the maximum body length of ten teleost fish species in line with temperature, dissolved oxygen concentration and geographic location (that encompasses multiple latent variables), across a broad (26°) latitudinal gradient throughout Norwegian waters. Our results showed that the two largest study species, spotted wolffish (Anarhichas minor) and cusk (Brosme brosme), display the strongest negative temperature-size response. We also observed smaller maximum body lengths for multiple species within the coldest extent of their temperature range, as well as parabolic relationships between maximum length and temperature for Atlantic wolffish (Anarhichas lupus) and beaked redfish (Sebastes mentella). The smaller maximum body lengths for high latitude species at both warm and cold temperature extremes of species' thermal ranges corroborate the temperature-size mechanisms of the gill-oxygen limitation theory (GOLT), whereby spontaneous protein denaturation limits growth at both warm and cold temperatures. [ABSTRACT FROM AUTHOR]

The effect of temperature on the stability of proteins is well explored above 298 K, but harder to track experimentally below 273 K. Variable‐temperature ion mobility mass spectrometry (VT IM‐MS) allows us to measure the structure of molecules at sub‐ambient temperatures. Here we monitor conformational changes that occur to two isotypes of monoclonal antibodies (mAbs) on cooling by measuring their collision cross sections (CCS) at discrete drift gas temperatures from 295 to 160 K. The CCS at 250 K is larger than predicted from collisional theory and experimental data at 295 K. This restructure is attributed to change in the strength of stabilizing intermolecular interactions. Below 250 K the CCS of the mAbs increases in line with prediction implying no rearrangement. Comparing data from isotypes suggest disulfide bridging influences thermal structural rearrangement. These findings indicate that in vacuo deep‐freezing minimizes denaturation and maintains the native fold and VT IM‐MS measurements at sub ambient temperatures provide new insights to the phenomenon of cold denaturation. [ABSTRACT FROM AUTHOR]

Thermophilic proteins and enzymes are attractive for use in industrial applications due to their resistance against heat and denaturants. Here, we report on a thermophilic protein that is stable at high temperatures (Ttrs, hot 67 °C) but undergoes significant unfolding at room temperature due to cold denaturation. Little is known about the cold denaturation of thermophilic proteins, although it can significantly limit their applications. We investigated the cold denaturation of thermophilic multidomain protein translation initiation factor 2 (IF2) from Thermus thermophilus. IF2 is a GTPase that binds to ribosomal subunits and initiator fMet-tRNAfMet during the initiation of protein biosynthesis. In the presence of 9 M urea, measurements in the far-UV region by circular dichroism were used to capture details about the secondary structure of full-length IF2 protein and its domains during cold and hot denaturation. Cold denaturation can be suppressed by salt, depending on the type, due to the decreased heat capacity. Thermodynamic analysis and mathematical modeling of the denaturation process showed that salts reduce the cooperativity of denaturation of the IF2 domains, which might be associated with the high frustration between domains. This characteristic of high interdomain frustration may be the key to satisfying numerous diverse contacts with ribosomal subunits, translation factors, and tRNA.

We review the key steps leading to an improved analysis of thermal protein unfolding. Thermal unfolding is a dynamic cooperative process with many short-lived intermediates. Protein unfolding has been measured by various spectroscopic techniques that reveal structural changes, and by differential scanning calorimetry (DSC) that provides the heat capacity change Cp(T). The corresponding temperature profiles of enthalpy ΔH(T), entropy ΔS(T), and free energy ΔG(T) have thus far been evaluated using a chemical equilibrium two-state model. Taking a different approach, we demonstrated that the temperature profiles of enthalpy ΔH(T), entropy ΔS(T), and free energy ΔG(T) can be obtained directly by a numerical integration of the heat capacity profile Cp(T). DSC thus offers the unique possibility to assess these parameters without resorting to a model. These experimental parameters now allow us to examine the predictions of different unfolding models. The standard two-state model fits the experimental heat capacity peak quite well. However, neither the enthalpy nor entropy profiles (predicted to be almost linear) are congruent with the measured sigmoidal temperature profiles, nor is the parabolic free energy profile congruent with the experimentally observed trapezoidal temperature profile. We introduce three new models, an empirical two-state model, a statistical–mechanical two-state model and a cooperative statistical-mechanical multistate model. The empirical model partially corrects for the deficits of the standard model. However, only the two statistical–mechanical models are thermodynamically consistent. The two-state models yield good fits for the enthalpy, entropy and free energy of unfolding of small proteins. The cooperative statistical–mechanical multistate model yields perfect fits, even for the unfolding of large proteins such as antibodies.

In a research environment characterized by the five V's of big data, volume, velocity, variety, value, and veracity, the need to develop tools that quickly screen a large number of publications into relevant work is an increasing area of concern, and the data-rich food industry is no exception. Here, a combination of latent Dirichlet allocation and food keyword searches were employed to analyze and filter a dataset of 6102 publications about cold denaturation. After using the Python toolkit generated in this work, the approach yielded 22 topics that provide background and insight on the direction of research in this field, as well as identified the publications in this dataset which are most pertinent to the food industry with precision and recall of 0.419 and 0.949, respectively. Precision is related to the relevance of a paper in the filtered dataset and the recall represents papers which were not identified in the screeningmethod. Lastly, gaps in the literature based on keyword trends are identified to improve the knowledge base of cold denaturation as it relates to the food industry. This approach is generalizable to any similarly organized dataset, and the code is available upon request. Practical Application: A common problem in research is that when you are an expert in one field, learning about another field is difficult, because you may lack the vocabulary and background needed to read cutting edge literature froma new discipline. The Python toolkit developed in this research can be applied by any researcher that is new to a field to identify what the key literature is, what topics they should familiarize themselves with, and what the current trends are in the field. Using this structure, researchers can greatly speed up how they identify new areas to research and find new projects. [ABSTRACT FROM AUTHOR]

The DinB homolog polymerase (Dbh) is a member of the Y-family of translesion DNA polymerases that can synthesize using a damaged DNA template. Since Dbh comes from the thermophilic archaeon Sulfolobus acidocaldarius, it is capable of functioning over a wide range of temperatures. Existing X-ray structures were determined at temperatures where the protein is least active. Here we use NMR and circular dichroism to understand how the structure and dynamics of Dbh are affected by temperature (2°C-65°C) and metal ion binding in solution. We measured hydrogen exchange protection factors, temperature coefficients, and chemical shift perturbations with and without magnesium and manganese. We report on regions of the protein that become more dynamic as the temperature is increased toward the functional temperature. Hydrogen exchange protection factors and temperature coefficients reveal that both the thumb and finger domains are very dynamic relative to the palm and little-finger (LF) domains. These trends remain true at high temperature with dynamics increasing as temperatures increase from 35°C to 50°C. Notably, NMR spectra show that the Dbh tertiary structure cold denatures beginning at 25°C and increases in denaturation as the temperature is lowered to 5°C with little change observed by CD. Above 35°C, chemical shift perturbation analysis in the presence and absence of magnesium and manganese reveals three ion binding sites, without DNA bound. In contrast, these bound metals are not apparent in any Dbh crystal structures of the protein without DNA. Two ion binding sites are confirmed to be near the active site, as reported in other Y-family polymerases, and we report a novel ion binding site in the LF domain. Thus, the solution-state structure of the Dbh polymerase is distinct from that of the solid-state structures and shows an unusually high cold denaturation temperature., 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. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Soto, Moro and Cocco.)

[Display omitted] • Cold denaturation occurs in extrusion at acidic pH values. • Specific thermal energy helps describe textural transformations during extrusion. • Cold extrusion allows for greater oil binding. • Cold extrusion may help in producing fat mimetics. Pea protein is a popular source of plant-based protein, though its application in meat and dairy analog products is still lacking. This is particularly true in the development of products with fatty and creamy textures. Cold denaturation may be a way to induce these types of textures in food since this is a universal phenomenon in protein that occurs due to a weakening of hydrophobic interactions at cold temperatures. This work utilizes a single screw extruder to systematically study the impacts of moisture content (50–65 %) and pH (2,4.5,8) on the outlet temperatures, specific mechanical energy, specific thermal energy, and texture of cold-extruded pea protein. It was found that at pH 2 and moistures of 60 % and greater, the temperature of the product exiting the extruder is <5.5 °C, and also produced 13.7 %–36.5 % more specific thermal energy, indicating the occurrence of cold denaturation in these products. Based on these findings, a comparison of hot and cold extrusion was conducted as a function of pH and oil content. It was found that cold extrusion imparts 43.0 %–56.2 % more mechanical energy into the protein than hot extrusion, and the cold extruded protein had higher values of Young's modulus and breaking stress. The protein extruded at low temperatures was also able to bind 32.93 % more oil than hot extruded proteins when extruded with 10 % added oil, which may aid in the formation of protein-based fat memetics for the food industry. [ABSTRACT FROM AUTHOR]

ABSTRACT The cholesterol-dependent cytolysin (CDC) genes are present in bacterial species that span terrestrial, vertebrate, and invertebrate niches, which suggests that they have evolved to function under widely different environmental conditions. Using a combination of biophysical and crystallographic approaches, we reveal that the relative stability of an intramolecular interface in the archetype CDC perfringolysin O (PFO) plays a central role in regulating its pore-forming properties. The disruption of this interface allows the formation of the membrane spanning β-barrel pore in all CDCs. We show here that the relative strength of the stabilizing forces at this interface directly impacts the energy barrier posed by the transition state for pore formation, as reflected in the Arrhenius activation energy (Ea) for pore formation. This change directly impacts the kinetics and temperature dependence of pore formation. We further show that the interface structure in a CDC from a terrestrial species enables it to function efficiently across a wide range of temperatures by minimizing changes in the strength of the transition state barrier to pore formation. These studies establish a paradigm that CDCs, and possibly other β-barrel pore-forming proteins/toxins, can evolve significantly different pore-forming properties by altering the stability of this transitional interface, which impacts the kinetic parameters and temperature dependence of pore formation. IMPORTANCE The cholesterol-dependent cytolysins (CDCs) are the archetype for the superfamily of oligomeric pore-forming proteins that includes the membrane attack complex/perforin (MACPF) family of immune defense proteins and the stonefish venom toxins (SNTX). The CDC/MACPF/SNTX family exhibits a common protein fold, which forms a membrane-spanning β-barrel pore. We show that changing the relative stability of an extensive intramolecular interface within this fold, which is necessarily disrupted to form the large β-barrel pore, dramatically alters the kinetic and temperature-dependent properties of CDC pore formation. These studies show that the CDCs and other members of the CDC/MACPF/SNTX superfamily have the capacity to significantly alter their pore-forming properties to function under widely different environmental conditions encountered by these species.

This article highlights contributions and the tremendous potential of high pressure (HP) small-angle neutron scattering for our understanding of biopolymer stability and phase behavior, in the context of nutrition and food properties. The use of HP processing as a nonthermal sterilization technique is well established in the food industry, and many other applications are emerging in recent years. Green chemistry, consumer preferences, and nutritional trends push for further developments, which require a database of experimental data. Unbiased studies on pressure-induced effects on colloids and amphiphiles are paramount for the development of new food molecules and methodologies. Biopolymer phase diagrams are described, with an emphasis on proteins. HP small-angle neutron scattering research capabilities and future directions are discussed. Image 1 [ABSTRACT FROM AUTHOR]

• Cold extrusion can be used to selectively modify hydrophobic interactions in protein. • Extrudates may find use in replacing fatty or creamy textures in food products. • Physicochemical data is used to describe textural parameters and extruder responses. • Cold denaturation acts as a novel method of functionalizing pea protein. Pea protein is a popular plant-based protein for mimicking textures in meat and dairy analogues which are more sustainable than their animal-based counterparts. However, precise mechanisms for generating specific textures through different processing methods are still being evaluated. This work utilizes a novel low-temperature extrusion process to selectively alter the chemical structure of pea protein. Changes in secondary structure, surface hydrophobicity, electrostatic interactions, and disulfide bonding are characterized through FTIR, ANS- probes, zeta potential, and SDS-PAGE. Extrudates are further characterized using texture parameter analysis. It was found that a linear combination of physicochemical data, generated with multiple linear regression modelling, led to reasonable estimates of the specific mechanical energy and textural properties. This work offers a new method of reactive extrusion to selectively modify interactions in pea protein using low temperature extrusion, and applications may include fatty textures, since the extrudates are found to be largely stabilized through hydrophobic interactions evaluated with surface hydrophobicity measurements. [ABSTRACT FROM AUTHOR]

Plant-based proteins are commonly used to generate new textures in food by inducing changes in their molecular structures with the application of heat, shear, pressure, and other methods of protein denaturation. One structural transformation that is relatively less understood is cold denaturation, which mainly occurs due to a weakening of hydrophobic forces at low temperatures. In this work, the Gibbs-Helmholtz equation is used to produce stability curves and estimate the temperature of cold denaturation for pea protein. The equation is solved with data obtained from differential scanning calorimetry conducted as a function of the solution pH and salt concentration. It is found that at pH 3, the temperature of cold denaturation is 3.85 °C. The protein functionality is then tested through zeta potential, surface hydrophobicity, solubility, and the emulsion activity/stability index at these low temperatures and compared to results from room temperature. It is found that surface hydrophobicity increases, the magnitude of the zeta potential increases, solubility decreases, and the emulsion activity/stability increases at low temperatures. • Temperature of cold denaturation is estimated in pea protein as a function of pH. • Gibbs-Helmholtz equation is used to create pea protein stability curves. • Functional properties of pea protein changes at low temperatures. • Emulsification properties are improved by cold denaturation. • Bioinformatic modeling helps describe electrostatic interactions at low temperatures. [ABSTRACT FROM AUTHOR]

Abstract Structural and molecular properties extracted from circular dichroism (CD), tryptophan fluorescence and 1-anilino-8-napthalene sulfonate (ANS) binding experiments suggest that the high concentration of synthetic crowding agents (dextran 40, dextran 70 and ficoll 70) stabilizes and refolds the base-denatured ferricytochrome c (Ferricyt c) and lysozyme (Lyz) at pH 12.9 (±0.1) to molten globule (MG) states (C B -states). These results further revealed that the C B -states resemble the generic properties of MG-states. Thermodynamic analysis of thermal denaturation curves of base-denatured Ferricyt c and Lyz at pH 12.9 (±0.1) under variable concentrations of crowding agents (dextran 40, dextran 70 and ficoll 70) revealed that the crowder presence increases the thermal stability of base-denatured proteins and also prevents the cold denaturation of Ferricyt c. The results further showed that the nature, size and shape of crowder influence the crowding-mediated increase in secondary structure stabilization and thermal stability of base-denatured Ferricyt c and Lyz. Analysis of kinetic and thermodynamic parameters measured for CO association reaction of alkaline ferrocytochrome c (Ferrocyt c) at pH 12.9 (±0.1) under variable concentrations of crowding agents (dextran 40, dextran 70 and ficoll 70) revealed that the crowder presence reduces the level of structural fluctuation of M80-containing Ω-loop that control CO association to alkaline Ferrocyt c. Graphical abstract Unlabelled Image Highlights • Crowders transforms the base-denatured Ferricyt c and Lyz to MG-states. • Crowder increases the thermal stability of base-denatured Ferricyt c and Lyz. • Crowders prevents the cold denaturation of Ferricyt c. • Crowder restricted the dynamics of base-denatured Ferrocyt c. • Crowder-induced MG-states are enthalpically stabilized. [ABSTRACT FROM AUTHOR]

Cold denaturation is an intriguing phenomenon that deserves special attention because its rationalization is surely linked to a deeper understanding of the molecular determinants of the marginal stability of small globular proteins dissolved in water and aqueous solutions. It is here reviewed and discussed a theoretical approach that has offered a reliable explanation for the occurrence of both cold and hot denaturations. A cornerstone of the approach is the recognition of the role played by the density of water and its temperature-dependence that are largely determined by the special energetic and geometric features of H-bonds. In fact, the density of water determines the magnitude of the solvent-excluded volume effect, which is a main ingredient of the hydrophobic effect. The relationship between density and solvent-excluded volume effect establishes a strong connection between the physico-chemical properties of water and the conformational stability of small globular proteins. [ABSTRACT FROM AUTHOR]

Proteins tend to adopt a single or a reduced ensemble of configurations at natural conditions [1], but changes in temperature T and pressure P induce their unfolding. Therefore for each protein there is a stability region (SR) in the T – P thermodynamic plane outside which the biomolecule is denaturated. It is known that the extension and shape of the SR depend on i) the specific protein residue-residue interactions in the native state of the amino acids sequence and ii) the water properties at the hydration interface. Here we analyze by Monte Carlo simulations the different coarse-grained protein models in explicit water how changes in i) and ii) affect the SR. We show that the solvent properties ii) are essential to rationalize the SR shape at low T and high P and that our findings are robust with respect to parameter changes and with respect to different protein models, representative of the ordered and disordered proteins. These results can help in developing new strategies for the design of novel synthetic biopolymers. [ABSTRACT FROM AUTHOR]

ABSTRACT The cold denaturation of globular proteins is a process that can be caused by increasing pressure or decreasing the temperature. Currently, the action mechanism of this process has not been clearly understood, raising an interesting debate on the matter. We have studied the process of cold denaturation using molecular dynamics simulations of the frataxin system Yfh1, which has a dynamic experimental characterization of unfolding at low and high temperatures. The frataxin model here studied allows a comparative analysis using experimental data. Furthermore, we monitored the cold denaturation process of frataxin and also investigated the effect under the high-pressure regime. For a better understanding of the dynamics and structural properties of the cold denaturation, we also analyzed the MD trajectories using essentials dynamic. The results indicate that changes in the structure of water by the effect of pressure and low temperatures destabilize the hydrophobic interaction modifying the solvation and the system volume leading to protein denaturation. Proteins 2016; 85:125-136. © 2016 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

We briefly review our studies on the folding/unfolding mechanisms of proteins. In biological self-assembly processes such as protein folding, the number of accessible translational configurations of water in the system increases greatly, leading to a large gain in the water entropy. The usual view looking at only the water in the close vicinity of the protein surface is capable of elucidating neither the large entropic gain upon apoplastocyanin folding, which has recently been found in a novel experimental study, nor the pressure and cold denaturation. With the emphasis on the translational entropy of water, we are presently constructing a reliable method for predicting the native structure of a protein from its amino-acid sequence.

Pea protein is often processed to alter the protein's native structure, enhancing desirable textures in meat and dairy analog products. Cold denaturation is a universal phenomenon in protein that exposes hydrophobic amino acids, though little information exists about this in food science research. In this work, ethanol, shear forces, and low temperatures were used to modify the structure of commercial pea protein isolate, and impacts on gelation are characterized. Treatment at −10 °C in ethanol with applied shear forces led to gels with the lowest tan δ values during temperature ramps and frequency sweeps. A quantitative structure-property relationship (QSPR) method found that hydrogen bonding can be derived from secondary structure (R2 = 0.960), and hydrogen bonding correlated with the temperature dependence of G′ during cooling in treatments. This work suggests cold denaturation forms hydrophobically bound gels that may aid in replacing fatty textures, and a QSPR framework aids in interpreting rheological data. [Display omitted] • Ethanol, shear forces, and low-temperature treatment increase pea protein gel strength. • The number of hydrogen bonds can be estimated from protein secondary structure resulting from FTIR. • Hydrogen bonding correlates to the degree of stiffening during gelation. • Hydrogen bonding correlates to the number of crosslinks estimated using rubber elasticity theory. • Cold denaturation facilitates the design of new cold processing methods to enhance functionality of proteins. [ABSTRACT FROM AUTHOR]

The functional native states of globular proteins become unstable at low temperatures, resulting in cold unfolding and impairment of normal biological function. Fundamental understanding of this phenomenon is essential to rationalizing the evolution of freeze-tolerant organisms and developing improved strategies for long-term preservation of biologicalmaterials. We present fully atomistic simulations of cold denaturation of an α-helical protein, the widely studied Trp-cage miniprotein. In contrast to the significant destabilization of the folded structure at high temperatures, Trp-cage cold denatures at 210 K into a compact, partially folded state; major elements of the secondary structure, including the α-helix, are conserved, but the salt bridge between aspartic acid and arginine is lost. The stability of Trp-cage's α-helix at low temperatures suggests a possible evolutionary explanation for the prevalence of such structures in antifreeze peptides produced by coldweather species, such as Arctic char. Although the 310-helix is observed at cold conditions, its position is shifted toward Trp-cage's C-terminus. This shift is accompanied by intrusion of water into Trp-cage's interior and the hydration of buried hydrophobic residues. However, our calculations also show that the dominant contribution to the favorable energetics of low-temperature unfolding of Trp-cage comes from the hydration of hydrophilic residues. [ABSTRACT FROM AUTHOR]

Protein stability is an important issue for the interpretation of a wide variety of biological problems but its assessment is at times difficult. The most common parameter employed to describe protein stability is the temperature of melting, at which the populations of folded and unfolded species are identical. This parameter may yield ambiguous results. It would always be preferable to measure the whole stability curve. The calculation of this curve is greatly facilitated whenever it is possible to observe cold denaturation. Using Yfh1, one of the few proteins whose cold denaturation occurs at neutral pH and low ionic strength, we could measure the variation of its full stability curve under several environmental conditions. Here we show the advantages of gauging stability as a function of external variables using stability curves. [ABSTRACT FROM AUTHOR]