Protein Hydration Research Articles

Diffusion Coefficient of Intracrystalline Water in Intrinsic Hen Egg-White Lysozyme Crystals Determined by Confocal Raman Spectroscopy.

Protein crystals composed of protein molecules are expected as a novel porous material. They have high porosity, and the knowledge of the diffusion of intracrystalline water is important. In this study, the diffusion coefficient of intracrystalline water in intrinsic hen egg-white lysozyme (HEWL) crystals was determined by a method that combines confocal Raman spectroscopy and air convection with controlled relative humidity. Similar to common porous materials, the drying process of the protein crystals includes three periods: constant-rate drying, falling-rate drying, and equilibrium state. During the falling-rate drying period, the drying rate depends on the diffusion of intracrystalline water in the protein crystal. The gradient of the water content was measured using confocal Raman spectroscopy. The diffusion coefficient of the intrinsic HEWL crystals was determined as 3.1 × 10-7 cm2/s with a water content of 36.3 vol %. The estimated diffusion coefficients of the intrinsic HEWL crystals without cross-linking were in close agreement with those of the cross-linked protein crystals. This study is timely as the knowledge of the intrinsic diffusion coefficient is useful not only for understanding the mechanism of hydration of proteins but also in practical applications such as porous materials, drug binding, and cryoprotectant soaks.

Gradients of Orientation, Composition, and Hydration of Proteins for Efficient Light Collection by the Cornea of the Horseshoe Crab.

The lateral eyes of the horseshoe crab, Limulus polyphemus, are the largest compound eyes within recent Arthropoda. The cornea of these eyes contains hundreds of inward projecting elongated cuticular cones and concentrate light onto proximal photoreceptor cells. Although this visual system has been extensively studied before, the precise mechanism allowing vision has remained controversial. Correlating high-resolution quantitative refractive index (RI) mapping and structural analysis, it is demonstrated how gradients of RI in the cornea stem from structural and compositional gradients in the cornea. In particular, these RI variations result from the chitin-protein fibers architecture, heterogeneity in protein composition, and bromine doping, as well as spatial variation in water content resulting from matrix cross-linking on the one hand and cuticle porosity on the other hand. Combining the realistic cornea structure and measured RI gradients with full-wave optical modeling and ray tracing, it is revealed that the light collection mechanism switches from refraction-based graded index (GRIN) optics at normal light incidence to combined GRIN and total internal reflection mechanism at high incident angles. The optical properties of the cornea are governed by different mechanisms at different hierarchical levels, demonstrating the remarkable versatility of arthropod cuticle.

Effects of refrigerated storage on the functional properties of processed cheese analogue with stretchability and its mechanisms

The functionality changes of processed cheese analogue with stretchability during refrigerated storage (0–60 d) at 4 °C were explored and the possible mechanisms were investigated. The results showed that stretchability, meltability, and free oil release of the cheese analogue decreased during refrigerated storage, and stretch length decreased from >270 cm to 130.63 ± 8.24 cm after refrigerated storage. Furthermore, the pH 4.6-soluble nitrogen fractions, soluble calcium and phosphorus, and the fraction of bound water of the cheese analogue significantly increased during refrigerated storage, while the β-casein and the fraction of free water decreased. The microstructure results showed that the whey channel disappeared while the proteins were swollen and squeezed and deformed the fat globules over refrigerated time. In conclusion, the functionality changes of the cheese analogue during refrigerated storage occurred under the compound effects of proteolysis and hydration of protein as well as dissolution of bound calcium.

Effects of direct current magnetic field co-treated with stirring on gel properties of chicken batter: Hydration and textural properties

This study constructed an in-house assembled device that allowed the stirring and direct current magnetic field (DC-MF) to be simultaneously applied to pretreat meat batters. The effects of stirring and DC-MF on the hydration and textural properties of chicken batter gels were investigated, and potential mechanisms were explored. The results demonstrated that the combined treatment (3 mT, 15 min, and 250 rpm) improved the texture and gel properties of meat batter gels, as well as the rheological behavior. It also increased the proportion of immobilized water (PT22) to 97.95% and enhanced protein hydration, decreasing cooking losses by 4.66% (P < 0.05). Microstructural images revealed that the DC-MF was conducive to forming a denser and more homogeneous gel structure with pores ranging from 10 to 100 μm. Specifically, the combined treatment exerted preferable properties in water retention and texture and could be used as a physical modification in emulsified meat products.

Thermodynamic Studies of Aqueous Binary and Ternary Solutions Involving Lysozyme and Urea at 298.15 K

Measurements for osmotic pressure and densities for lysozyme in aqueous and aqueous solutions containing urea have been reported at 298.15 K using a vapor pressure osmometer and vibrating tube digital density meter, respectively. The partial molar volumes and activity coefficients of both the components and the osmotic pressure of the solutions have been calculated. The molar mass and second virial coefficient of lysozyme have been obtained using the McMillan–Mayer theory of solutions and are compared with the values obtained in aqueous salt solutions. The Guggenheim–Stokes equation for osmotic coefficient has been used to obtain the protein molecular diameter in an aqueous medium. The estimated diameter of lysozyme is similar in magnitude to that in the pure state, confirming the retaining of the native state of the protein in aqueous solutions over the studied concentration region. These studies have been extended to aqueous–urea (∼2.0 mol·kg–1) solutions at 298.15 K. Data of water activity and activity coefficient of all the three components in ternary solutions as a function of lysozyme concentration have been determined by the methodology developed by us earlier. The data have been further analyzed to obtain Gibbs free energy changes of transfer of lysozyme as well as that urea from corresponding aqueous binary to aqueous ternary solutions through which information about the binding of denaturant to protein can be derived. The analysis further reveals that the binding of urea to the protein molecules occurs through the breakdown of protein–water H–bonds leading to the opening of a coiled structure of lysozyme. All these results are discussed in terms of protein hydration, protein–protein hydrophobic interactions, and protein denaturant binding equilibria for the solutions of lysozyme.

Triplet States Reveal Slow Local Dynamics in the Solvation Shell of Biomolecules.

Protein hydration shell dynamics plays a pivotal role in biochemical processes such as protein folding, enzyme function, molecular recognition and interaction with biological membranes. Thus, it is crucial to understand the mobility of the solvation shell at the surface of biomolecules. Triplet state solvation dynamics can reveal the slow dynamics of the solvation shell. This is done in the present work without adding separate dye molecules but instead by using a phosphorescent subgroup of the biomolecule itself. In particular, we study a small heptapeptide in a glycerol-water mixture under cryoconservation conditions so that the system can be supercooled without crystallization. We find a significant slowing of molecules in the solvation shell in the millisecond range compared to the bulk. This opens up the possibility to unravel the nature of relaxation processes in the solvation shell usually overlapping at room temperature.

The structural order of protein hydration water

The ability of water to dissolve biomolecules is crucial for our life. It has been shown that protein has a profound effect on the behavior of water in its hydration shell, which in turn affects the structure and function of the protein. However, there is still no consensus on whether protein promotes or destroys the structural order of water in its hydration shell until today, because of the lack of proper structural descriptor incorporating hydrogen-bond (H-bond) information for water at the protein/water interface. Here we performed all-atom molecular dynamics simulations of lysozyme protein in water and analyzed the H-bond structure of protein hydration water by using a newly developed structural descriptor. We find that the protein promotes local structural ordering of the hydration water while having a negligible effect on the strength of individual H-bonds. These findings are fundamental to the structure and function of biomolecules and provide new insights into the hydration of protein in water.

Controlling Amyloid Fibril Properties Via Ionic Liquids: The Representative Case of Ethylammonium Nitrate and Tetramethylguanidinium Acetate on the Amyloidogenesis of Lysozyme.

Protein aggregation into amyloid fibrils has been observed in several pathological conditions and exploited in nanotechnology. It is also key in several biochemical processes. In this work, we show that ionic liquids (ILs), a vast class of organic electrolytes, can finely tune amyloid properties, opening a new landscape in basic science and applications. The representative case of ethylammonium nitrate (EAN) and tetramethyl-guanidinium acetate (TMGA) ILs on lysozyme is considered. First, atomic force microscopy has shown that the addition of EAN and TMGA leads to thicker and thinner amyloid fibrils of greater and lower electric potential, respectively, with diameters finely tunable by IL concentration. Optical tweezers and neutron scattering have shed light on their mechanism of action. TMGA interacts with the protein hydration layer only, making the relaxation dynamics of these water molecules faster. EAN interacts directly with the protein instead, making it mechanically unstable and slowing down its relaxation dynamics.

Probing Local Protein Environments in Adenylate Kinase with 4‐Cyano‐L‐Phenylalanine

Unnatural amino acids (UAAs) containing vibrational reporters, such as nitrile, azide, and nitro groups, can be utilized to effectively probe local protein environments. Specifically, the nitrile symmetric stretching frequency of 4‐cyano‐L‐phenylalanine (pCNF) is sensitive to local protein hydration and occurs in a relatively clear region of the infrared spectrum. In addition, the magnitude of change in this vibrational frequency over a given temperature range is dependent on the local protein environment of the nitrile group of pCNF. Finally, the UAA pCNF can also be site‐specifically incorporated into proteins with high efficiency and fidelity using the Amber codon suppression methodology. Here, pCNF was incorporated into several different local protein environments in the enzyme Adenylate Kinase (AK). The successful incorporation was verified by time‐of‐flight mass spectrometry. These results in addition to the linear temperature‐dependent IR analysis of these constructs to extract the hydration state of the nitrile group of pCNF in each of these AK constructs will be presented.

Expanding the Utility of 4‐cyano‐L‐phenylalanine as a Vibrational Reporter

Unnatural amino acids (UAAs) have become useful tools for understanding protein structure and local environments. One of the most commonly used vibrational reporter UAAs to study local protein hydration environments is 4‐cyano‐L‐phenylalanine (pCNF) due to the position and sensitivity of the nitrile symmetric stretching frequency as well as the ability to efficiently incorporate this UAA site‐specifically into proteins using the Amber codon suppression methodology. Additionally, pCNF provides promise to function as a distance reporter when coupled with two‐dimensional infrared (2D IR) spectroscopy. Here, a number of superfolder green fluorescent protein (sfGFP) constructs have been generated with pCNF site‐specifically incorporated at two unique sites simultaneously. The pairs of sites were selected to permit the nitrile symmetric stretching frequency of pCNF at each site to be spectrally resolved. The temperature dependent linear IR spectra of these sfGFP constructs with two pCNF UAAs incorporated will be presented, illustrating that the nitrile groups are in different local environments. Preliminary X‐ray crystal structures of these sfGFP constructs will be presented to probe if the UAAs resulted in a structural perturbation and to determine the distance between the nitrile groups of the two pCNF UAAs in each structure. These results will serve as the basis for and calibration of employing pCNF in future 2D IR spectroscopic studies to extract the distances between these two groups based upon coupling between them.

INFLUENCE OF PARAMETERS OF MARINATING MEAT SEMI-FINISHED PRODUCTS ON THE QUALITY OF THE FINISHED PRODUCT

Improvement and introduction of modern technologies in the creation of new types of meat semi-finished products, the study of promising components and the development of new recipes for marinades, improving the organoleptic and taste properties of the final product is a relevant and promising area of research. Therefore, this study is focused on the influence of marinating parameters in the technology of meat semi-finished products, namely on the organoleptic, physicochemical, and functional-technological properties of the finished product. To solve the tasks, generalisation, comparison and the following research methods were used: organoleptic evaluation of appearance, cut section, taste, smell, colour, consistency, and juiciness; moisture content – by drying samples in a drying oven at a temperature of 103 ± 2 °C to a constant mass; fat content – by the Soxhlet method; ash content – by the conventional weighing method; determination of protein content – by the Kjeldahl method; the yield of the finished product was determined by weighing before and after heat treatment; moisture binding and moisture retention capacity was determined by pressing. The studies found that the effect of the duration of marinating significantly affects the organoleptic characteristics. Thus, the organoleptic characteristics and taste and aroma properties of the finished product when using honey in marinades improve, and the appearance becomes more attractive. According to the results of the organoleptic evaluation, the finished product, after 24 hours of marinating, received a score of 4.9 points. Also, in these samples were observed greater water retention and water binding capacity, which contributes to increased hydration and solubility of muscle tissue proteins. The influence of marinating parameters on the consumptive properties of the product has been established. Thus, the yield of finished baked beef after 24 hours of ageing in the marinade was the highest in sample No. 1 (marinade based on citrus honey – 10 g) and amounted to 84.96%, while the average weight yield of other samples was in the range of 73.18-77.5%. The obtained research results make it possible to expand the range of semi-finished products, correct organoleptic properties and enrich the finished product with biologically active substances

Влияние электрохимически активированной воды на показатели качества теста и изделий из пшеничной муки

Electrochemically activated water has a high physicochemical and biological activity. It interacts with food ingredients and affects the baking process in a different way than untreated water. The research objective was to study the effect of the anodic and cathodic fractions of electrochemically activated water on the quality indicators of wheat flour, dough, and bread.&#x0D; The study featured electrochemically activated water fractions, wheat flour, yeast, dough, and wheat bread. It employed standard physicochemical and organoleptic methods of analysis to study the redox potential and pH of electrochemically activated water during relaxation.&#x0D; The indicators returned to the initial level, but they changed significantly after 72 h. The experiment included the quantity and quality of gluten, extensibility and hydration, water-retaining capacity of flour, yeast fermentation, titratable acidity, dough fermentation rate, and the quality of finished products. The anolyte did not change the quality of gluten but decreased its amount by 2.0–3.7%, probably due to a weaker protein hydration. As for the effect of catholyte on the quality of gluten, the bonds between proteins became stronger in weak as the measurement of gluten deformation index decreased by 11.3%; they relaxed in stronger as the measurement of gluten deformation index increased by 20%. Catholyte increased the water-retaining capacity of weak flour by 11.7% and that of resistant gluten – by 5.3%. It also activated yeast cells. The samples of bread cooked on catholyte had a greater specific volume by 3.7–5.4% and porosity – by 2.3–4.6%, compared to the samples cooked on anolyte. The research also included a comparative analysis of the shape of the hearth bread samples. It confirmed that the catholyte strengthened the dough when the flour was weak and relaxed it when resistant gluten was used.&#x0D; The research revealed some patterns regarding the effect of electrochemically activated water fractions on the amount and properties of gluten, water-retaining capacity of flour, yeast activity, and bread quality indicators. The results can be used to correct the properties of dough from low-quality flour, as well as for reagent-free control of the properties and behavior of&#x0D; food and biological raw materials.

Conformational Rearrangements in the Redox Cycling of NADPH-Cytochrome P450 Reductase from Sorghum bicolor Explored with FRET and Pressure-Perturbation Spectroscopy.

Simple SummaryNADPH-cytochrome P450 reductase (CPR) enzymes are known to undergo an ample conformational transition between the closed and open states in the process of their redox cycling. To explore the conformational landscape of CPR from the potential biofuel crop Sorghum bicolor (SbCPR), we incorporated a FRET donor/acceptor pair into the enzyme and employed rapid scanning stop-flow and pressure perturbation spectroscopy to characterize the equilibrium between its open and closed states at different stages of the redox cycle. Our results suggest the presence of several open conformational sub-states differing in the system volume change associated with the opening transition (ΔV0). Although the closed conformation always predominates in the conformational landscape, the population of the open conformations increases by order of magnitude upon the two-electron reduction and the formation of the disemiquinone state of the enzyme. In addition to elucidating the functional choreography of plant CPRs, our study demonstrates the high exploratory potential of a combination of the pressure-perturbation approach with the FRET-based monitoring of protein conformational transitions.NADPH-cytochrome P450 reductase (CPR) from Sorghum bicolor (SbCPR) serves as an electron donor for cytochrome P450 essential for monolignol and lignin production in this biofuel crop. The CPR enzymes undergo an ample conformational transition between the closed and open states in their functioning. This transition is triggered by electron transfer between the FAD and FMN and provides access of the partner protein to the electron-donating FMN domain. To characterize the electron transfer mechanisms in the monolignol biosynthetic pathway better, we explore the conformational transitions in SbCPR with rapid scanning stop-flow and pressure-perturbation spectroscopy. We used FRET between a pair of donor and acceptor probes incorporated into the FAD and FMN domains of SbCPR, respectively, to characterize the equilibrium between the open and closed states and explore its modulation in connection with the redox state of the enzyme. We demonstrate that, although the closed conformation always predominates in the conformational landscape, the population of open state increases by order of magnitude upon the formation of the disemiquinone state. Our results are consistent with several open conformation sub-states differing in the volume change (ΔV0) of the opening transition. While the ΔV0 characteristic of the oxidized enzyme is as large as −88 mL/mol, the interaction of the enzyme with the nucleotide cofactor and the formation of the double-semiquinone state of CPR decrease this value to −34 and −18 mL/mol, respectively. This observation suggests that the interdomain electron transfer in CPR increases protein hydration, while promoting more open conformation. In addition to elucidating the functional choreography of plant CPRs, our study demonstrates the high exploratory potential of a combination of the pressure-perturbation approach with the FRET-based monitoring of protein conformational transitions.

Dipolar Interactions and Protein Hydration in Highly Concentrated Antibody Formulations.

Molecular interaction mechanisms in high-concentrated protein systems are of fundamental importance for the rational development of biopharmaceuticals such as monoclonal antibody (mAb) formulations. In such high-concentrated protein systems, the intermolecular distances between mAb molecules are reduced to the size of the protein diameter (approx. 10 nm). Thus, protein-protein interactions are more pronounced at high concentrations; so a direct extrapolation of physicochemical properties obtained from measurements at a low protein concentration of the corresponding properties at a high protein concentration is highly questionable. Besides the charge-charge interaction, the effects of molecular crowding, dipolar interaction, changes in protein hydration, and self-assembling tendency become more relevant. Here, protein hydration, protein dipole moment, and protein-protein interactions were studied in protein concentrations up to 200 mg/mL (= 1.3 mM) in different formulations for selected mAbs using dielectric relaxation spectroscopy (DRS). These data are correlated with the second virial coefficient, A2, the diffusion interaction parameter, kD, the elastic shear modulus, G', and the dynamic viscosity, η. When large contributions of dipolar protein-protein interactions were observed, the tendency of self-assembling and an increase in solution viscosity were detected. These effects were examined using specific buffer conditions. Furthermore, different types of protein-water interactions were identified via DRS, whereby the effect of high protein concentration on protein hydration was investigated for different high-concentrated liquid formulations (HCLFs).

Excipients Do Regulate Phase Separation in Lysozyme and Thus Also Its Hydration.

While the liquid-liquid phase separation (LLPS) process in proteins has been studied in great detail, it has not been widely explored how the associated protein hydration changes during the process and how crucial its role is in the process itself. In this contribution, we experimentally explore the alteration of lysozyme hydration during its LLPS process using attenuated total reflection (ATR)-FTIR spectroscopy in the THz frequency region (1.5-21 THz). Additionally, we explore the role of excipients (l-arginine, sucrose, bovine albumin (BSA), and ubiquitin (Ubi)) in regulating the process and found that, while sucrose stabilizes the LLPS, BSA inhibits it. The effect of Arg in the LLPS is subtle, and that of Ubi is concentration dependent. We made a detailed analysis of the hydration profile of Lys in the presence of these excipients and observe that a change in hydration in terms of H-bond making/breaking is a definite signature regulating the process.

Seeking Solvation: Exploring the Role of Protein Hydration in Silk Gelation.

The mechanism by which arthropods (e.g., spiders and many insects) can produce silk fibres from an aqueous protein (fibroin) solution has remained elusive, despite much scientific investigation. In this work, we used several techniques to explore the role of a hydration shell bound to the fibroin in native silk feedstock (NSF) from Bombyx mori silkworms. Small angle X-ray and dynamic light scattering (SAXS and DLS) revealed a coil size (radius of gyration or hydrodynamic radius) around 12 nm, providing considerable scope for hydration. Aggregation in dilute aqueous solution was observed above 65 °C, matching the gelation temperature of more concentrated solutions and suggesting that the strength of interaction with the solvent (i.e., water) was the dominant factor. Infrared (IR) spectroscopy indicated decreasing hydration as the temperature was raised, with similar changes in hydration following gelation by freezing or heating. It was found that the solubility of fibroin in water or aqueous salt solutions could be described well by a relatively simple thermodynamic model for the stability of the protein hydration shell, which suggests that the affected water is enthalpically favoured but entropically penalised, due to its reduced (vibrational or translational) dynamics. Moreover, while the majority of this investigation used fibroin from B. mori, comparisons with published work on silk proteins from other silkworms and spiders, globular proteins and peptide model systems suggest that our findings may be of much wider significance.

Wheat gluten proteins phosphorylated with sodium tripolyphosphate: Changes in structure to improve functional properties for expanding applications

Poor solubility of wheat gluten proteins (WG) has negative impact on functional attributes such as gelation and emulsification, which limits it use in the food industry. In this study, WG underwent different degrees of phosphorylation using sodium tripolyphosphate (STP). Phosphoric acid groups were successfully incorporated in the WG via covalent bonding (C–N–P and C–O–P) involving hydroxyl and primary amino groups from WG. The introduction of phosphoric acid groups increased the negative charge of phosphorylation-WG, which caused the enhancement of electrostatic repulsion between proteins and reduced the droplet size in emulsions, thereby allowing proteins to be more efficiently dispersed in the solution system. The change of structure induced with phosphorylation improved hydration of protein, making the WG with higher solubility, thereby resulting in the improvement of its emulsification, foaming, thermal stability, and rheological properties. Therefore, WG can be modified by phosphorylation which caused an overall improvement of functional properties, thus facilitating the expansion of WG applications.

Nutritional Considerations for Para-Cycling Athletes: A Narrative Review.

Para-cycling is a sport including athletes with different disabilities competing on the track and on the roads using bicycles, tandems, tricycles, and handbikes. Scientific literature in this special population is scarce, especially in the field of sports nutrition. This review summarizes the physiological aspects and demands of para-cycling. This information together with the existing literature on nutritional interventions in this population, helps to discuss the nutritional considerations. To date, only a limited amount of recommendations are available for this population. In most para-cycling athletes, a reduction in active muscle mass and consequently a reduction in resting energy expenditure occurs, except for visually impaired athletes. Furthermore, carbohydrate and protein intake and hydration, supplementation, heat, and weight loss need to be tailored to the disability-specific adaptations such as the reduced active muscle mass, neurogenic bladder, and bowel, a reduced metabolic cost during exercise, and a higher risk of micronutrient deficiency.

Hydration and its Hydrogen Bonding State on a Protein Surface in the Crystalline State as Revealed by Molecular Dynamics Simulation.

Protein hydration is crucial for the stability and molecular recognition of a protein. Water molecules form a hydration water network on a protein surface via hydrogen bonds. This study examined the hydration structure and hydrogen bonding state of a protein, staphylococcal nuclease, at various hydration levels in its crystalline state by all-atom molecular dynamics (MD) simulation. Hydrophilic residues were more hydrated than hydrophobic residues. As the water content increases, both types of residues were uniformly more hydrated. The number of hydrogen bonds per single water asymptotically approaches 4, the same as bulk water. The distances and angles of hydrogen bonds in hydration water in the protein crystal were almost the same as those in the tetrahedral structure of bulk water regardless of the hydration level. The hydrogen bond structure of hydration water observed by MD simulations of the protein crystalline state was compared to the Hydrogen and Hydration Database for Biomolecule from experimental protein crystals.

Water Thermodynamics and Its Effects on the Protein Stability and Activity

We discuss a phenomenon regarding water that was until recently a subject of scientific interest: i.e., the dynamical crossover, from the fragile to strong glass forming material, for both bulk and protein hydration water. Such crossover is characterized by a temperature TL in which significant dynamical changes like the decoupling (or the violation of the Stokes-Einstein relation) of homologous transport parameters, e.g., the density relaxation time τ and the viscosity η, occur in the system. On this respect we considered the dynamic properties of water-protein systems. More precisely, we focused our study on proteins and their hydration water, as far as bulk and confined water. In order to clarify the effects of the water dynamical crossover on the protein properties we considered and discussed in a comparative way previous and new experimental data, obtained from different techniques and molecular dynamic simulation (MD). We pointed out the reasons for different dynamical findings from the use of different experimental techniques.