Stabilizing effect of silver carp myofibrillar protein modified by high intensity ultrasound on high internal phase emulsions: Protein denaturation, interfacial adsorption and reconfiguration.

This study evaluated the impact of high intensity ultrasound (HIU) on myofibrillar proteins (MP) from silver carp, and investigated the stabilizing effect of HIU-treated MP (UMP) on high internal phase emulsions (HIPEs). Ultrasonic cavitation induced protein denaturation by decreasing size and unfolding conformation, to expose more hydrophobic groups, particularly UMP at 390 W, showing the smallest particle size (181.71 nm) and most uniform distribution. These structural changes caused that UMP under 390 W exhibited the highest surface hydrophobicity, solubility (92.72 %) and emulsibility (115.98 m2/g and 70.4 min), all of which contributed to fabricating stable HIPEs with oil volume fraction up to 0.8. UMP-based HIPEs possessed tightly packed gel network and self-supporting appearance due to the adsorption of numerous proteins at the oil-water interface and the reduction of interfacial tension by protein reconfiguration. The larger interface coverage reinforced cross-linking between interfacial proteins, thus increasing the viscoelasticity and recoverability of HIPEs, also the resistance to centrifugal force, high temperature (90 °C, 30 min) and freeze-thaw cycles. These findings furnished insightful perspectives for MP deep processing through HIU, expanding the high-value application of UMP-based HIPEs in fat replacer, nutritional delivery system with high encapsulation content and novel 3D printing ink.

Site-specific mutagenesis on Mnemiopsin 2: Calcium coordination and substrate binding properties of new variants.

We used site-specific mutagenesis by targeting E179 and F190 on the structure of photoprotein Mnemiopsin 2 (Mn2) from Mnemiopsis leidyi. The tertiary structure of E179S and F190L mutants was made by the MODELLER program. Far-ultraviolet circular dichroism data showed that the overall secondary structural content of photoprotein is not changed upon mutation, however the helicity and stabilizing interactions in helical structure decreases in mutants as compared with the wild-type (WT) photoprotein. Fluorescence spectra data revealed that the tertiary structure of the mutants is more compact than that of WT Mn2. According to the heat-induced denaturation experiments data, the melting temperature (Tm ) for the unfolding of tertiary structure of the F190L variant increases by 3°C compared with that of the WT and E179S mutant. Interestingly, the conformational enthalpy of the F190L mutant (86 kcal mol-1 ) is considerably lower than those in the WT photoprotein (102 kcal mol-1 ) and E179S mutant (106 kcal mol-1 ). The significant difference in the enthalpy of the thermal unfolding process could be explained by considering that the thermally denatured state of the F190L mutant is structurally less expanded than the WT and E179S variants. Bioluminescence activity data showed that the maximum characteristic wavelengths of the mutants undergo blue shift as compared with the WT protein. Initial intensity of the F190L and E179S variants was recorded to be 137.5% and 55.9% of the WT protein, respectively.

Protective role of chlorogenic acid in preserving cytochrome-c stability against HFIP-induced molten globule state at physiological pH.

Numerous neurodegenerative disorders are characterized by protein misfolding and aggregation. The mechanism of protein aggregation is intricate, and it is very challenging to study at cellular level. Inhibition of protein aggregation by interfering with its pathway is one of the ways to prevent neurodegenerative diseases. In the present work, we have evaluated the protective effect of a polyphenol compound chlorogenic acid (CGA) on the native and molten globule state of horse heart cytochrome c (cyt c). A molten globule state of this heme protein was achieved in the presence of fluorinated alcohol 1,1,1,3,3,3-hexafluoroisopropanol (HFIP) at physiological pH, as studied by UV-Vis absorption, circular dichroism, intrinsic and ANS fluorescence. We found that at 50 % (v/v) HFIP, the native cyt c transformed into a molten globule state. The same techniques were also used to analyze the protective effect of CGA on the molten globule state of cyt c, and the results show that the CGA prevented the molten globular state and retained the protein close to the native state at 1:1 protein:CGA sub molar ratio. Molecular dynamics study also revealed that CGA retains the stability of cyt c in HFIP medium by preserving it in an intermediate state close to native conformation.

Fluorescence-Based Protein Stability Monitoring-A Review.

Proteins are large biomolecules with a specific structure that is composed of one or more long amino acid chains. Correct protein structures are directly linked to their correct function, and many environmental factors can have either positive or negative effects on this structure. Thus, there is a clear need for methods enabling the study of proteins, their correct folding, and components affecting protein stability. There is a significant number of label-free methods to study protein stability. In this review, we provide a general overview of these methods, but the main focus is on fluorescence-based low-instrument and -expertise-demand techniques. Different aspects related to thermal shift assays (TSAs), also called differential scanning fluorimetry (DSF) or ThermoFluor, are introduced and compared to isothermal chemical denaturation (ICD). Finally, we discuss the challenges and comparative aspects related to these methods, as well as future opportunities and assay development directions.

From lab-based to in-line: Analytical tools for the characterization of whey protein denaturation and aggregation-A review.

Whey protein denaturation and aggregation have long been areas of research interest to the dairy industry, having significant implications for process performance and final product functionality and quality. As such, a significant number of analytical techniques have been developed or adapted to assess and characterize levels of whey protein denaturation and aggregation, to either maximize processing efficiency or create products with enhanced functionality (both technological and biological). This review aims to collate and critique these approaches based on their analytical principles and outline their application for the assessment of denaturation and aggregation. This review also provides insights into recent developments in process analytical technologies relating to whey protein denaturation and aggregation, whereby some of the analytical methods have been adapted to enable measurements in-line. Developments in this area will enable more live, in-process data to be generated, which will subsequently allow more adaptive processing, enabling improved product quality and processing efficiency. Along with the applicability of these techniques for the assessment of whey protein denaturation and aggregation, limitations are also presented to help assess the suitability of each analytical technique for specific areas of interest.

Effect of femtosecond laser process parameters on the thermal denaturation degree of skin tissue.

The influence of femtosecond laser parameters on the degree of thermal denaturation was studied experimentally. The relationship between the degree of thermal denaturation and the characteristic parameters of skin microstructure and the secondary structure of skin tissue proteins in characterizing the degree of thermal damage was analyzed. The results showed the interaction of laser power, laser power, and scanning speed had a significant effect on the degree of thermal denaturation; greater degrees of thermal denaturation were associated with larger second-order moments of the texture angle of the skin microtissue and smaller entropy values and contrast, indicating a greater degree of thermal damage; and higher peak temperature, the lower peak intensity of Raman spectra, decrease in the percentage area of α-helix fitted curves and increase in the percentage area of ß-sheet and ß-turn fitted curves indicate that the protein is denatured to a large extent that means thermal damage is large.

Protein Preferential Solvation in (Sucralose + Water) Mixtures.

Addition of sugars such as sucrose to aqueous protein solutions generally stabilizes proteins against thermal denaturation by preferential exclusion of sugars from proteins (preferential hydration of proteins). In this study, we investigated the effect of sucralose, a chlorinated sucrose derivative, on protein stability and preferential solvation. Circular dichroism and small-angle X-ray scattering measurements showed that sucrose increased the denaturation temperature of myoglobin and was preferentially excluded from the protein, whereas sucralose decreased the denaturation temperature of myoglobin and was preferentially adsorbed to the protein. No clear evidence was obtained for the indirect effects of sucralose on protein destabilization via the structure and properties of solvent water from the physicochemical properties (mass density, sound velocity, viscosity, and osmolality) of aqueous sucralose solutions; therefore, we concluded that a direct protein-sucralose interaction induced protein destabilization.

Thermal characterization and separation of whey proteins by differential scanning calorimetry.

Most commercially available whey products contain a mixture of 6-7 whey proteins; however, there is an increased focus on using the individual whey proteins for their unique biological activities. Before extracting individual whey proteins for use, it is important to quantify how much of a particular protein is present in whey mixtures as well as if the protein is still structurally folded. We first characterized the denaturation temperature and enthalpy values for the six purified whey proteins at six pHs (3-9) and under ion chelation using a nano-differential scanning calorimeter (DSC). From the individual protein scans, we determined the optimal condition for detecting all 6 proteins on a single DSC scan was whey in an EDTA MOPs pH 6.7 buffer.

Effect of temperature and pH on the structure and stability of tumor-specific lectin jacalin and insights into the location of its tryptophan residues: CD, DSC and fluorescence studies.

Jacalin, the jackfruit seed lectin, exhibits high specificity for the tumor-specific T-antigen and is used in various biomedical and biotechnological applications. Here, we report biophysical studies on the thermal unfolding of jacalin and the effect of pH and temperature on its secondary structure. Differential scanning calorimetric (DSC) studies revealed that native jacalin unfolds at ∼60 °C and that carbohydrate binding stabilizes the protein structure. Circular dichroism spectroscopic studies indicated that the secondary structure of jacalin remains mostly unaffected over pH 2.0-9.0, whereas considerable changes were observed in the tertiary structure. DSC experiments demonstrated that jacalin exhibits two overlapping transitions between pH 2 and 5, which could be attributed to dissociation of the tetrameric protein into subunits and their unfolding. Interestingly, only one transition between pH 6 and 9 was observed, suggesting that the subunit dissociation and unfolding occur simultaneously. While quenching of the protein intrinsic fluorescence by acrylamide increased significantly upon carbohydrate binding, quenching by succinimide is essentially unaffected. We attribute this difference to increased exposure of Trp-123 in the α-chain as it is involved in carbohydrate binding. Both acrylamide and succinimide gave biphasic Stern-Volmer plots, consistent with differential accessibility of the two tryptophan residues of jacalin to them.

Line Shape Analysis of 19F NMR-Monitored Chemical Denaturation of a Fold-Switching Protein RfaH Reveals Its Slow Folding Dynamics.

The recent discovery of metamorphic proteins, which can switch between multiple conformations under native conditions, has challenged the well-established one sequence-one structure paradigm of protein folding. This is exemplified in the C-terminal domain of the multidomain transcription factor RfaH, which converts from an α-helical coiled-coil conformation in its autoinhibited state to a ß-barrel conformation upon activation. Here, we use multisite line shape analysis of 19F NMR-monitored equilibrium chemical denaturation measurements of two 19F-labeled variants of full-length RfaH, to show that it folds/unfolds slowly on the NMR time scale, in an apparent all-or-none fashion at physiological pH and room temperature in the 3.3-4.8 M urea concentration range. The significant thermodynamic stability and slow unfolding rate (kinetic stability) are likely responsible for maintaining the closed autoinhibited state of RfaH, preventing spurious interactions with RNA polymerase (RNAP) when not functional. Our results provide a quantitative understanding of the folding-function relationship in the model fold-switching protein RfaH.

Impact of cooking temperature on pork longissimus, and muscle fibre type, on quality traits and protein denaturation of four pork muscles.

Variations in pork quality impact consumer acceptance, and fibre type differences between muscles contribute to this variation. The aim was to investigate the influence of variations in muscle fibre types and protein denaturation peaks across four pork muscles and the influence of ageing and cooking temperature on longissimus quality traits. The longissimus, masseter, cutaneous trunci, and psoas major from 13 carcases were removed 1-day postmortem and subjected to 0- or 14-days ageing (d0, d14). Quality traits, protein denaturation peak temperature (DSC), fibre diameter and fibre type proportions were measured. Cook loss for longissimus was similar on d0 and d14, but was higher on d14 for masseter, cutaneous trunci, and psoas major. Warner-Bratzler shear force was highest, and ultimate pH was lowest, for longissimus, and similar among cutaneous trunci, masseter, and psoas major. Masseter had lowest L* and highest a* and longissimus and cutaneous trunci had highest L* and lowest a*. The DSC temperature peaks for longissimus occurred at lower temperatures relative to the other muscles. Fibre diameter was largest for type-IIb fibres relative to type-IIa and type-I. Longissimus and cutaneous trunci had predominantly type-IIb glycolytic (71%, 51% respectively), masseter had predominantly type-IIa intermediate (50%) and psoas major had predominantly type-I oxidative (48%) fibres. The glycolytic longissimus had the lowest DSC temperature peaks and the lowest quality meat. Masseter had the highest proportion of type-I fibres but was generally similar in quality traits to psoas major, and also similar to cutaneous trunci which had more glycolytic fibres than masseter.

Influence of Amino Acid Substitutions in ApoMb on Different Stages of Unfolding of Amyloids.

To date, most research on amyloid aggregation has focused on describing the structure of amyloids and the kinetics of their formation, while the conformational stability of fibrils remains insufficiently explored. The aim of this work was to investigate the effect of amino acid substitutions on the stability of apomyoglobin (ApoMb) amyloids. A study of the amyloid unfolding of ApoMb and its six mutant variants by urea has been carried out. Changes in the structural features of aggregates during unfolding were recorded by far-UV CD and native electrophoresis. It was shown that during the initial stage of denaturation, amyloids' secondary structure partially unfolds. Then, the fibrils undergo dissociation and form intermediate aggregates weighing approximately 1 MDa, which at the last stage of unfolding decompose into 18 kDa monomeric unfolded molecules. The results of unfolding transitions suggest that the stability of the studied amyloids relative to the intermediate aggregates and of the latter relative to unfolded monomers is higher for ApoMb variants with substitutions that increase the hydrophobicity of the residues. The results presented provide a new insight into the mechanism of stabilization of protein aggregates and can serve as a base for further investigations of the amyloids' stability.

Conformational Stability of the N-Terminal Region of MDM2.

MDM2 is an E3 ubiquitin ligase which is crucial for the degradation and inhibition of the key tumor-suppressor protein p53. In this work, we explored the stability and the conformational features of the N-terminal region of MDM2 (N-MDM2), through which it binds to the p53 protein as well as other protein partners. The isolated domain possessed a native-like conformational stability in a narrow pH range (7.0 to 10.0), as shown by intrinsic and 8-anilinonapthalene-1-sulfonic acid (ANS) fluorescence, far-UV circular dichroism (CD), and size exclusion chromatography (SEC). Guanidinium chloride (GdmCl) denaturation followed by intrinsic and ANS fluorescence, far-UV CD and SEC at physiological pH, and differential scanning calorimetry (DSC) and thermo-fluorescence experiments showed that (i) the conformational stability of isolated N-MDM2 was very low; and (ii) unfolding occurred through the presence of several intermediates. The presence of a hierarchy in the unfolding intermediates was also evidenced through DSC and by simulating the unfolding process with the help of computational techniques based on constraint network analysis (CNA). We propose that the low stability of this protein is related to its inherent flexibility and its ability to interact with several molecular partners through different routes.

Isothermal chemical denaturation assay for monitoring protein stability and inhibitor interactions.

Thermal shift assay (TSA) with altered temperature has been the most widely used method for monitoring protein stability for drug research. However, there is a pressing need for isothermal techniques as alternatives. This urgent demand arises from the limitations of TSA, which can sometimes provide misleading ranking of protein stability and fail to accurately reflect protein stability under physiological conditions. Although differential scanning fluorimetry has significantly improved throughput in comparison to differential scanning calorimetry and differential static light scattering throughput, all these methods exhibit moderate sensitivity. In contrast, current isothermal chemical denaturation (ICD) techniques may not offer the same throughput capabilities as TSA, but it provides more precise information about protein stability and interactions. Unfortunately, ICD also suffers from limited sensitivity, typically in micromolar range. We have developed a novel method to overcome these challenges, namely throughput and sensitivity. The novel Förster Resonance Energy Transfer (FRET)-Probe as an external probe is highly applicable to isothermal protein stability monitoring but also to conventional TSA. We have investigated ICD for multiple proteins with focus on KRASG12C with covalent inhibitors and three chemical denaturants performed at nanomolar protein concentration. Data showed corresponding inhibitor-induced stabilization of KRASG12C to those reported by nucleotide exchange assay.

In Situ Observation of Chemically Induced Protein Denaturation at Solvated Interfaces.

Proteins unfold in chaotropic salt solutions, a process that is difficult to observe at the single protein level. The work presented here demonstrates that a liquid-based atomic force microscope and graphene liquid-cell-based scanning transmission electron microscope make it possible to observe chemically induced protein unfolding. To illustrate this capability, ferritin proteins were deposited on a graphene surface, and the concentration-dependent urea- or guanidinium-induced changes of morphology were monitored for holo-ferritin with its ferrihydrite core as well as apo-ferritin without this core. Depending on the chaotropic agent the liquid-based imaging setup captured an unexpected transformation of natively folded holo-ferritin proteins into rings after urea treatment but not after guanidinium treatment. Urea treatment of apo-ferritin did not result in nanorings, confirming that nanorings are a specific signature of denaturation of holo-ferritins after exposture to sufficiently high urea concentrations. Mapping the in situ images with molecular dynamics simulations of ferritin subunits in urea solutions suggests that electrostatic destabilization triggers denaturation of ferritin as urea makes direct contact with the protein and also disrupts the water H-bonding network in the ferritin solvation shell. Our findings deepen the understanding of protein denaturation studied using label-free techniques operating at the solid-liquid interface.

Protein Folding Stability and Kinetics in Alginate Hydrogels.

Proteins are commonly encapsulated in alginate gels for drug delivery and tissue-engineering applications. However, there is limited knowledge of how encapsulation impacts intrinsic protein properties such as folding stability or unfolding kinetics. Here, we use fast relaxation imaging (FReI) to image protein unfolding in situ in alginate hydrogels after applying a temperature jump. Based on changes in the Förster resonance energy transfer (FRET) response of FRET-labeled phosphoglycerate kinase (PGK), we report the quantitative impact of multiple alginate hydrogel concentrations on protein stability and folding dynamics. The gels stabilize PGK by increasing its melting temperature up to 18.4 °C, and the stabilization follows a nonmonotonic dependence on the alginate density. In situ kinetic measurements also reveal that PGK deviates more from two-state folding behavior in denser gels and that the gel decreases the unfolding rate and accelerates the folding rate of PGK, compared to buffer. Phi-value analysis suggests that the folding transition state of an encapsulated protein is structurally similar to that of folded protein. This work reveals both beneficial and negative impacts of gel encapsulation on protein folding, as well as potential mechanisms contributing to altered stability.

Native and Non-Native aggregation pathways of antibodies anticipated by cold-accelerated studies.

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).

A three-state mechanism for trifluoroethanol denaturation of an intrinsically disordered protein (IDP).

Relating the amino acid composition and sequence to chain folding and binding preferences of intrinsically disordered proteins (IDPs) has emerged as a huge challenge. While globular proteins have respective 3D structures that are unique to their individual functions, IDPs violate this structure-function paradigm because rather than having a well-defined structure an ensemble of rapidly interconverting disordered structures characterize an IDP. This work measures 2,2,2-trifluoroethanol (TFE)-induced equilibrium transitions of an IDP called AtPP16-1 (Arabidopsis thaliana phloem protein type 16-1) by using fluorescence, circular dichroism, infrared and nuclear magnetic resonance (NMR) methods at pH 4, 298 K. Low TFE reversibly removes the tertiary structure to produce an ensemble of obligate intermediate ($\mathrm{I}$) retaining the native-state ($\mathrm{N}$) secondary structure. The intermediate $\mathrm{I}$ is preceded by a non-obligate tryptophan-specific intermediate ${\mathrm{I}}_{\mathrm{w}}$ whose population is detectable for AtPP16-1 specifically. Accumulation of such non-obligate intermediates is discriminated according to the sequence composition of the protein. In all cases, however, a tertiary structure-unfolded general obligate intermediate $\mathrm{I}$ is indispensable. The $\mathrm{I}$ ensemble has higher helical propensity conducive to the acquisition of an exceedingly large level of α-helices by a reversible denaturation transition of $\mathrm{I}$ to the denatured state $\mathrm{D}$ as the TFE level is increased. Strikingly, it is the same $\mathrm{N}\rightleftharpoons \mathrm{I}\rightleftharpoons \mathrm{D}$ scheme typifying the TFE transitions of globular proteins. The high-energy state $\mathrm{I}$ characterized by increased helical propensity is called a universal intermediate encountered in both genera of globular and disordered proteins. Neither $\mathrm{I}$ nor $\mathrm{D}$ strictly show molten globule (MG)-like properties, dismissing the belief that TFE promotes MGs.

Counteraction Effects of Ammonium-Based Ionic Liquids on Urea-Induced Denaturation of α-Lactalbumin: A Comprehensive Molecular Simulation Study.

Ionic liquids (ILs) are known to stabilize protein conformations in aqueous medium. Importantly, ILs can also act as refolding additives in urea-driven denaturation of proteins. However, despite the importance of the problem, detailed microscopic understanding of the counteraction effects of ILs on urea-induced protein denaturation remains elusive. In this work, atomistic molecular dynamics (MD) simulations of the protein α-lactalbumin have been carried out in pure aqueous medium, in 8 M binary urea-water solution and in ternary urea-IL-water solutions containing ammonium-based ethyl ammonium acetate (EAA) as the IL at different concentrations (1-4 M). Attempts have been made to quantify detailed molecular-level understanding of the origin behind the counteraction effects of the IL on urea-induced partial unfolding of the protein. The calculations revealed significant conformational changes of the protein with multiple free energy minima due to its partial unfolding in binary urea-water solution. The counteraction effect of the IL was evident from the enhanced structural rigidity of the protein with propensity to transform into a single native free energy minimum state in ternary urea-IL-water solutions. Such an effect has been found to be associated with preferential direct binding of the IL components with the protein and simultaneous expulsion of urea from the interface, thereby providing additional stabilization of the protein in ternary solutions. Most importantly, modified rearrangement of the hydrogen bond network at the interface due to the formation of stronger protein-cation (PC) and protein-anion (PA) hydrogen bonds by breaking relatively weaker protein-urea (PU) and protein-water (PW) hydrogen bonds has been recognized as the microscopic origin behind the counteraction effects of EAA on urea-induced partial unfolding of the protein.

Observation of Protein Unfolding during pH Ramp Evoked by Lipase-Catalyzed Ester Hydrolysis.

Studies of protein folding often involve offline experimental methods such as titrating protein samples with denaturants or equilibrating them in the presence of denaturants. Here, we demonstrate an online analytical approach in which the protein structure is perturbed by a pH ramp evoked by immobilized lipase-catalyzed ester hydrolysis. Changes in the tertiary structure of the protein in response to a pH ramp (from approximately 6.3 to 2.8) are monitored using electrospray ionization mass spectrometry and spectrofluorometry. Interestingly, we discovered a side reaction of ammonium and formate leading to the production of cyanide that occurred during the ionization process. We also found that only certain protein analytes were bound to the formed cyanide species. Nevertheless, this problem was readily overcome by carefully selecting a specific ester substrate. Overall, the alterations in the charge-state distribution and fluorescence intensity─caused by the lipase-induced pH ramp─reveal conformational transitions in different proteins. In line with previous reports, the acid-induced denaturation of holo-myoglobin occurs through a two-step mechanism, which is supported by identification of protein-unfolding intermediates and the loss of noncovalent protein ligand (heme). The results─obtained using the developed catalytic method─are also consistent with the results of equilibrium-based experiments, while sample preparation steps are substantially reduced. The proposed approach simplifies the identification of the pH range that has the greatest impact on the protein structure. Thus, it has the potential to be a useful tool for studying protein conformational transitions in the course of pH changes.