Protein Hydration Research Articles

Cold-Set Gelation of Soybean and Amaranth Proteins by Hydration of Freeze-Dried Protein Previously Denatured in the Presence of Calcium

Cold-Set Gelation of Soybean and Amaranth Proteins by Hydration of Freeze-Dried Protein Previously Denatured in the Presence of Calcium

Scrutinizing the protein hydration shell from molecular dynamics simulations against consensus small-angle scattering data

Biological macromolecules in solution are surrounded by a hydration shell, whose structure differs from the structure of bulk solvent. While the importance of the hydration shell for numerous biological functions is widely acknowledged, it remains unknown how the hydration shell is regulated by macromolecular shape and surface composition, mainly because a quantitative probe of the hydration shell structure has been missing. We show that small-angle scattering in solution using X-rays (SAXS) or neutrons (SANS) provide a protein-specific probe of the protein hydration shell that enables quantitative comparison with molecular simulations. Using explicit-solvent SAXS/SANS predictions, we derived the effect of the hydration shell on the radii of gyration Rg of five proteins using 18 combinations of protein force field and water model. By comparing computed Rg values from SAXS relative to SANS in D2O with consensus SAXS/SANS data from a recent worldwide community effort, we found that several but not all force fields yield a hydration shell contrast in remarkable agreement with experiments. The hydration shell contrast captured by Rg values depends strongly on protein charge and geometric shape, thus providing a protein-specific footprint of protein–water interactions and a novel observable for scrutinizing atomistic hydration shell models against experimental data.

Dynamics of protein hydration water at cryogenic temperature revealed by neutron scattering

Dynamics of protein hydration water at cryogenic temperature revealed by neutron scattering

Excessive free radical grafting interferes with the macromolecular association and crystallization of brined porcine myofibrils during heat-set gelatinization

Free radical grafting and oxidative modification show superiority in myofibrillar protein (MP) aggregation patterns during salting process, but their consequent formation mechanisms of protein hydration network require further evaluation. Herein, we explored the effect of salt-curing (0, 1, 3 and 5 %) on MP protein polymer substrate, water-protein interaction, crystallization events and thermal stability under H2O2/ascorbate-based hydroxyl radical (•OH)-generating system (HRGS) (1, 10, 20 mM H2O2). Results showed that moderate salting (≤3%) favored the water binding of MP gels during the oxidation course. Accordingly, the maximum thermal stability (Tm) of MP gels was obtained at 3 % salting could be greatly attributed to the protein chain solubilization and refolding process. However, 5 % salt synergized with •OH oxidation intensified diffraction peak 2 (the most striking diffraction feature). Microstructural analysis validated a maximum compactness of MP gel following brining with 5 % salt at potent oxidation strength (20 mM H2O2). This study maybe promises efficient strategy to the myogenetic fibril products and biomaterials.

Structure, aggregation dynamics and crystallization of superfolder green fluorescent protein: Effect of long alkyl chain imidazolium ionic liquids

Green fluorescent protein (GFP) and its variants are widely used in medical and biological research, especially acting as indicators of protein structural integrity, protein-protein interactions and as biosensors. This study employs superfolder GFP (sfGFP) to investigate the impact of varying alkyl chain length of 1-Cn-3-methylimidazolium chloride ionic liquid (IL) series ([Cnmim]Cl, n = 2, 4, 6, 8, 10, 12) on the protein fluorescence, structure, hydration, aggregation dynamics and crystallization behaviour. The results revealed a concentration-dependent decrease in the sfGFP chromophore fluorescence, particularly in long alkyl chain ILs ([C10mim]Cl and [C12mim]Cl). Tryptophan (Trp) fluorescence showed the quenching rate increased with longer alkyl chains indicating a nonpolar interaction between Trp57 and the alkyl chain. Secondary structural changes were observed at the high IL concentration of 1.5 M in [C10mim]Cl and [C12mim]Cl. Small-angle X-ray scattering (SAXS) indicated relatively stable protein sizes, but with IL aggregates present in [C10mim]Cl and [C12mim]Cl solutions. Dynamic light scattering (DLS) data showed increased protein size and aggregation with longer alkyl chain ILs. Notably, ILs and salts, excluding [C2mim]Cl, promoted sfGFP crystallization. This study emphasizes the influence of the cation alkyl chain length and concentration on protein stability and aggregation, providing insights into utilizing IL solvents for protein stabilization and crystallization purposes.

Sugar Molecules Inhibit Insulin Aggregation: A Decisive Role Being Played by the Protein Solvation Energetics.

Insulin plays vital roles in controlling blood sugar level in the human body. However, it sometimes aggregates during the storage, and its efficacy (on the treatment of diabetes II disease) reduces significantly. So, understanding the insulin aggregation could help in long-term storage. Here we investigate the amyloid growth of human insulin protein in the presence of sugar molecules and observe that glucose and sucrose delay the insulin aggregation, the effect being systematically sugar dependent. We then investigate protein hydration during the aggregation process using terahertz spectroscopy, as the hydration plays a pioneering role in maintaining biological systems. Our study infers that the water network changes systematically with protein conformations and solvation entropy-enthalpy balance plays a decisive role in the aggregation process.

Functionality of pea protein isolate solutions is affected by reconstitution conditions.

Pea protein isolate (PPI), a high-concentration protein ingredient derived from peas, is increasingly utilized in food applications, including beverages, meat or dairy alternatives, and baked goods. The protein extraction process typically used to manufacture PPI renders the protein highly denatured, which can have a negative impact on its functionality. Therefore, it is critical to understand how to prepare and utilize PPI to maximize its functionality. The current study evaluates the effect of select reconstitution conditions on the structure and functionality of PPI, across a range of protein concentrations (4%-10%) relevant to a variety of food applications. Temperature during reconstitution with water and hydration time impacted both protein hydration and its functionality. Increasing reconstitution temperature from 20 to 60°C and increasing hydration time from 10 to 40min decreased PPI particle size in solution and increased PPI solubility. Viscosity of PPI solutions also increased with mild heating and longer hydration time, whereas their flow behavior was highly dependent on protein concentration. Experimental data demonstrates that reconstitution conditions have a significant impact on PPI functionality. These findings can help food formulators develop high-quality food products that utilize PPI as a functional ingredient. PRACTICAL APPLICATION: Protein in commercially available pea protein isolates (PPIs) is usually highly denatured, and thus, it is important to find ways to maximize its functionality in practical applications. The findings of this study inform food scientists how to leverage PPI at various protein concentrations with optimal reconstitution conditions to develop high-quality products. Generally, mild heating and longer hydration times improve PPI functional performance.

Climate smart, underutilised, healthful future cereal: Protein content, hydration properties, starch digestibility and consumer liking of pearl millet-based Oyster mushroom crackers

Pearl millet is an underutilised cereal. This study investigated the use of undervalued pearl millet grain in the manufacturing of value-added low sugar crackers that are protein-enhanced by the incorporation of Oyster mushroom. Equal amounts of pearl millet and wheat bread flour were mixed. This mixture was substituted to have 0 % (control), 5, 10 and 15 % Oyster mushroom powder. The moisture, protein, physical characteristics, in vitro starch digestibility of the composite flours and crackers were determined. The 15 % Oyster mushroom incorporation significantly increased the crackers’ protein (8.8 to 10.4 %). The inclusion of 15 % Oyster mushroom significantly reduced the in vitro starch digestibility at 20, 60, 120 min from 16 % to 13 %, 26 % to 23 and 37 % to 31 %, respectively. The 5 % Oyster mushroom incorporated cracker was the most liked (7.1 score on a 9-point hedonic scale). These easy to manufacture shelf-stable crackers, using low and affordable technology, from shelf-stable, low water activity flours can be used to alleviate protein malnutrition especially in the rural areas of developing nations where there is no electricity and refrigeration facilities.

Native and nonnative reactions in ethanolamine ammonia-lyase are actuated by different dynamics

We address the contribution of select classes of solvent-coupled configurational fluctuations to the complex choreography involved in configurational and chemical steps in an enzyme by comparing native and nonnative reactions conducted at different protein internal sites. The low temperature, first-order kinetics of covalent bond rearrangement of the cryotrapped substrate radical in coenzyme B12-dependent ethanolamine ammonia-lyase (EAL) from Salmonella enterica display a kink, or increase in slope, of the Arrhenius plot with decreasing temperature. The event is associated with quenching of a select class of reaction-actuating collective fluctuations in the protein hydration layer. For comparison, a nonnative, radical reaction of the protein interior cysteine sulfhydryl group with hydrogen peroxide (H2O2) is introduced by cryotrapping EAL in an aqueous H2O2 eutectic system. The low-temperature aqueous H2O2 protein hydration and mesodomain solvent phases surrounding cryotrapped EAL are characterized by using TEMPOL spin probe electron paramagnetic resonance spectroscopy, including a freezing transition of the eutectic phase that orders the protein hydration layer. Kinetics of the cysteine-H2O2 reaction in the EAL protein interior are monitored by DEPMPO spin trapping of hydroxyl radical product. In contrast to the native reaction, the linear Arrhenius relation for the nonnative cysteine-H2O2 reaction is maintained through the solvent-protein ordering transition. The nonnative reaction is coupled to the generic local, incremental fluctuations that are intrinsic to globular proteins. The comparative approach supports the proposal that select coupled solvent-protein configurational fluctuations actuate the native reaction, and suggests that select dynamical coupling contributes to the degree of catalysis in enzymes.

Osmolytes Modulate Photoactivation of Phytochrome: Probing Protein Hydration.

Phytochromes are bistable red/far-red light-responsive photoreceptor proteins found in plants, fungi, and bacteria. Light-activation of the prototypical phytochrome Cph1 from the cyanobacterium Synechocystis sp. PCC 6803 allows photoisomerization of the bilin chromophore in the photosensory module and a subsequent series of intermediate states leading from the red absorbing Pr to the far-red-absorbing Pfr state. We show here via osmotic and hydrostatic pressure-based measurements that hydration of the photoreceptor modulates the photoconversion kinetics in a controlled manner. While small osmolytes like sucrose accelerate Pfr formation, large polymer osmolytes like PEG 4000 delay the formation of Pfr. Thus, we hypothesize that an influx of mobile water into the photosensory domain is necessary for proceeding to the Pfr state. We suggest that protein hydration changes are a molecular event that occurs during photoconversion to Pfr, in addition to light activation, ultrafast electric field changes, photoisomerization, proton release and uptake, and the major conformational change leading to signal transmission, or simultaneously with one of these events. Moreover, we discuss this finding in light of the use of Cph1-PGP as a hydration sensor, e.g., for the characterization of novel hydrogel biomaterials.

Water State in Hemoglobin Solutions: Microwave Dielectric Spectroscopy Study

In this review, we discuss the dielectric relaxation of water in the Hemoglobin (Hb) aqueous solutions in different states (MetHb, OxyHb, and DeoxyHb)). The interpretation of the results was performed by the 3D trajectory approach, which considers dielectric parameters of water relaxation and protein concentration. It has been shown that the interaction of amino acid dipole residues, located on the surface of a protein macromolecule, with water dipoles determines the protein hydration. In addition, ions of the buffer, in which proteins are dissolved, are also hydrated. The transition from a dipole-ion interaction of water molecules to a dipole-dipole interaction of water with an increase in protein concentration is observed. We proposed a new approach to calculate hemoglobin hydration shells. The theoretical model defines the change in the ratio between the content of free and bound water molecules (BWM) as a function of MetHb concentration in ion-free and ion-containing aqueous solutions. It considers the number of positive and negative charges at the surface of the protein molecule, the number of BWMs in its hydration shells, and the partition of water molecules bound to MetHb and the inorganic ions. The theoretical evaluation of the ratio of free-to-bound water reveals that, in the absence of ions, MetHb binds about 1400 water molecules, which is in good agreement with experimental data. At MetHb concentrations close to physiological, Hb is dominating in binding water over the inorganic ions, and the amount of BWM remains at the level of ~20% of total cytosol water as the concentration of Hb increases above 15 g/dL. These results suggest that in the physiological concentration diapason (29 - 35g/dL), molecules of MetHb are so close together that their hydration shells interact and shrink from four to one layer of water molecules. In contrast, Hb molecules aggregate to neutralize their surface charges mutually.

The effect of calcium removal from skim milk by ion exchange on the properties of the ultrafiltration retentate

New processes are needed to produce concentrated milk feedstocks with tailored calcium content, due to the direct link between calcium concentration and final product texture and functionality. Skim milk treatment with cation exchange resin 1% (w/v) or 2% (w/v) prior to ultrafiltration to a volumetric concentration factor (VCF) of 2.5 or 5 successfully decreased the calcium concentration by 20–30% and produced concentrates with solids content at ∼22–24 g 100 g−1 at a VCF of 5. Calcium reduction partially solubilized the casein micelles, increasing the concentration of soluble protein and individual caseins, leading to decreased turbidity but increased protein hydration and hydrophobicity. Decalcification (2% (w/v) resin treatment) reduced thermal stability, significantly decreasing the denaturation temperature of α-lactalbumin and β-lactoglobulin in the milk by ∼3 °C and ∼1 °C respectively. Filtration was also altered, reducing permeation flux and the gel concentration and increased filtration time. When combined, calcium reduction and filtration altered functional properties including soluble calcium, soluble protein and sedimentable solids, with increased milk protein hydration also contributing to increased viscosity. This study provides a route to produce calcium-reduced milk concentrates with potential for use in retentate-based dairy products with tailored functionality.

Variations in the Protein Hydration and Hydrogen-Bond Network of Water Molecules Induced by the Changes in the Secondary Structures of Proteins Studied through Near-Infrared Spectroscopy.

This study investigated how the secondary structural changes of proteins in aqueous solutions affect their hydration and the hydrogen-bond network of water molecules using near-infrared (NIR) spectroscopy. The aqueous solutions of three types of proteins, i.e., ovalbumin, β-lactoglobulin, and bovine serum albumin, were denatured by heating, and changes in the NIR bands of water reflecting the states of hydrogen bonds induced via protein secondary structural changes were investigated. On heating, the intermolecular hydrogen bonds between water molecules as well as between water and protein molecules were broken, and protein molecules were no longer strongly bound by the surrounding water molecules. Consequently, the denaturation was observed to proceed depending on the thermodynamic properties of the proteins. When the aqueous solutions of proteins were cooled after denaturation, the hydrogen-bond network was reformed. However, the state of protein hydration was changed owing to the secondary structural changes of proteins, and the variation patterns were different depending on the protein species. These changes in protein hydration may be derived from the differences in the surface charges of proteins. The elucidation of the mechanism of protein hydration and the formation of the hydrogen-bond network of water molecules will afford a comprehensive understanding of the protein functioning and dysfunctioning derived from the structural changes in proteins.

Structure and interaction of therapeutic proteins in solution: a combined simulation and experimental study

The aggregation of therapeutic proteins in solution has attracted significant interest, driving efforts to understand the relationship between microscopic structural changes and protein-protein interactions determining aggregation processes in solution. Additionally, there is substantial interest in being able to predict aggregation based on protein structure as part of molecular developability assessments. Molecular Dynamics provides theoretical tools to complement experimental studies and to interrogate and identify the microscopic mechanisms determining aggregation. Here we perform all-atom MD simulations to study the structure and inter-protein interaction of the Fab and Fc fragments of the monoclonal antibody (mAb) COE3. We unravel the role of ion-protein interactions in building the ionic double layer and determining effective inter-protein interaction. Further, we demonstrate, using various state-of-the-art force fields (charmm, gromos, amber, opls/aa), that the protein solvation, ionic structure and protein-protein interaction depend significantly on the force field parameters. We perform SANS and Static Light Scattering experiments to assess the accuracy of the different forcefields. Comparison of the simulated and experimental results reveal significant differences in the forcefields' performance, particularly in their ability to predict the protein size in solution and inter-protein interactions quantified through the second virial coefficients. In addition, the performance of the forcefields is correlated with the protein hydration structure.

Effect of super-chilled preservation on the water-holding properties of fresh beef during storage

In this study, comparison of the effects of chilled (4 °C), super-chilled (-4 °C), and frozen (-18 °C) storage on the water-holding properties was evaluated by drip loss, surface hydrophobicity, and calpain activity of raw beef. These results indicated that storage temperature can significantly affect the water-holding properties. In contrast to chilled storage, super-chilled storage can maintain a higher thiol group content, which can maintain better myofibrillar protein hydration capacity. Furthermore, super-chilled storage effectively slowed down the increase in the rate of protein hydrophobicity compared with chilled and frozen storage. Additionally, the super-chilled sample exhibited the highest glutathione reductase activity, followed by the frozen sample. These results revealed that super-chilled storage is a good way to preserve the water-holding properties of raw beef.

Probing ion-binding at a protein interface: Modulation of protein properties by ionic liquids

Ions are important to modulate protein properties, including solubility and stability, through specific ion effects. Ionic liquids (ILs) are designer salts with versatile ion combinations with great potential to control protein properties. Although protein-ion binding of common metals is well-known, the IL effect on proteins is not well understood. Here, we employ the model protein lysozyme in dilute and concentrated IL solutions to determine the specific ion binding effect on protein phase behaviour, activity, size and conformational change, aggregation and intermolecular interactions. A combination of spectroscopic techniques, activity assays, small-angle X-ray scattering, and crystallography highlights that ILs, particularly their anions, bind to specific sites in the protein hydration layer via polar contacts on charged, polar and aromatic residues. The specific ion binding can induce more flexible loop regions in lysozyme, while the ion binding in the bulk phase can be more dynamic in solution. Overall, the protein behaviour in ILs depends on the net effect of nonspecific interactions and specific ion binding. Compared to formate, the nitrate anion induced high protein solubility, low activity, elongated shape and aggregation, which is largely owing to its higher propensity for ion binding. These findings provide new insights into protein-IL binding interactions and using ILs to modulate protein properties.

Structural and gelling properties of whey proteins influenced by various acids: Experimental and computational approaches

Five different acids, i.e., hydrochloric acid (HCl), acetic acid, lactic acid, γ-aminobutyric acid (GABA) and taurine were applied to investigated their effects on heat-induced gelation properties of whey protein isolate (WPI) at pH = 5.5. All WPI gels were found to have self-similar structures with the fractal dimension, df = 2.40. Those denatured WPI formed strong physical gels that were susceptible to shear and strain. Of all the acids, HCl, GABA and taurine significantly strengthened WPI gels. Moreover, denser packing of protein aggregates was observed from the gels with GABA and taurine. Besides experimental approaches, molecular dynamics (MD) simulations were also applied to investigate the effects of various acids on denaturation of β-lactoglobulin (β-LG). GABA and taurine were found to protect β-LG from denaturation and delayed the molecular heat-induced unfolding. Moreover, presence of HCl, GABA and taurine conserved the rigidity of unfolded β-LG and localized the residue –SH121, which was believed to result in promoted reaction-limited clustering aggregations (RLCA) of β-LG molecules and strengthened WPI gels. Further studies suggested that there was no strong binding or aggregation between cations (Na+) or anions/zwitterions and β-LG molecule. Therefore, the effects from various acids (anions/zwitterions) on β-LG and WPI were supposed to result from long-distanced electrostatic interactions and/or their effects on the protein hydration shells.

Volumetric analysis of the energetics of protein hydration

Water plays a dominant role in all biomacromolecular folding and binding events. For decades, volumetric observables have been used to characterize the hydration properties of solutes. These studies aimed at gaining insights into the number of water molecules solvating solutes or changes in these numbers upon folding and binding events involving biopolymers. However, the link between the volumetric properties of solutes and the energetics of their hydration has been missing. This deficiency limits the potential benefits of the use of volumetric measurements in hydration studies. In this contribution, we derive a possible link between the free energy of hydration of amino acid side chains and a select set of other organic compounds and their volumetric characteristics. Potentially, this approach can be used to estimate the contribution of hydration to the energetics of protein folding and binding reactions.

Modeling of protein hydration dynamics is supported by THz spectroscopy of highly diluted solutions.

In this investigation, we report the effect on the microscopic dynamics and interactions of the cytokine interferon gamma (IFN-γ) and antibodies to IFN-γ (anti-IFN-γ) and to the interferon gamma receptor 1 (anti-IFNGR1) prepared in highly dilute (HD) solutions of initial proteins. THz spectroscopy measurements have been conducted as a means to analyze and characterize the collective dynamics of the HD samples. MD simulations have also been performed that have successfully reproduced the observed signatures from experimental measurement. Using this joint experimental-computational approach we determine that the HD process associated with the preparation of the highly diluted samples used in this investigation induces a dynamical transition that results in collective changes in the hydrogen-bond network of the solvent. The dynamical transition in the solvent is triggered by changes in the mobility and hydrogen-bonding interactions of the surface molecules in the HD samples and is characterized by dynamical heterogeneity. We have uncovered that the reorganization of the sample surface residue dynamics at the solvent-protein interface leads to both structural and kinetic heterogeneous dynamics that ultimately create interactions that enhance the binding probability of the antigen binding site. Our results indicate that the modified interfacial dynamics of anti-IFN-γ and anti-IFGNR1 that we probe experimentally are directly associated with alterations in the complementarity regions of the distinct antibodies that designate both antigen-antibody affinity and recognition.

Molecular insight on hydration of protein tyrosine phosphatase 1B and its complexes with ligands

Knowledge of hydration and the role of solvent in protein–ligand (P-L) binding is essential for rational drug design. In this direction, a complete description of P-L binding requires a clear understanding of the change in protein and ligand hydration states when they form a complex. In the present work, we investigated the 3D-hydration of protein tyrosine phosphatase 1B (PTP1B) in the unbound and bound states and changes in this process occurring upon the complex formation with 2HB1 and 2QBP inhibitors as an example. To this end, we have used a simplified version of the site density functional theory - the three-dimensional reference interaction site model. The obtained data indicate that both the ligands and PTP1B in unbound state are well-hydrated. Upon complex formation, the ligands experience significant dehydration, while the level of PTP1B hydration changes little. Moreover, the hydrated state of P-L complex remains practically the same as for liganded PTP1B itself. In this case, some of the waters are partly “collectivized” by the protein and the ligand, i.e., solvent molecules will be shared by PTP1B and 2HB1/2QBP. The active site pocket of the protein was also found to be well-hydrated. P–L binding results in partial dehydration of the pocket, which is connected with the displacement of part of the water from the pocket by the ligand. The results show the presence of two/seven waters inside the pocket for PTP1B-2HB1/2QBP. Location of the most tightly bound two/three waters in the PTP1B-2HB1/2QBP complex is coinsiding with the PDB data. Among tightly bound waters, only two molecules are “bridging” ones, i.e., forming simultaneously H-bonds with pocket residues and ligands. In addition, all possible H-bonds between the ligand, pocket amino acid residues and water are discussed for both complexes. The data obtained are necessary for understanding the molecular mechanisms of the PTP1B functioning and the creation of selective regulators of its activity.