Solutions Of Globular Proteins Research Articles

Surface Properties of Protein-Polyelectrolyte Solutions. Impact of Polyelectrolyte Hydrophobicity.

The strong influence of an amphiphilic polyelectrolyte, poly(N,N-diallyl-N-hexyl-N-methylammonium chloride), on the surface properties of solutions of globular proteins (lysozyme, β-lactoglobulin, bovine serum albumin, and green fluorescent protein) depends on the protein structure and allows elucidation of the contribution of hydrophobic interactions in the protein-polyelectrolyte complex formation at the liquid-gas interface. At the beginning of adsorption, the surface properties are determined by the unbound amphiphilic component, but the influence of the protein-polyelectrolyte complexes of high surface activity increases at the approach to equilibrium. The kinetic dependencies of the dilational dynamic surface elasticity with one or two local maxima give a possibility to distinguish clearly between different steps of the adsorption process and to trace the formation of the distal region of the adsorption layer. The conclusions from the surface rheological data are corroborated by ellipsometric and tensiometric results.

How does dextran sulfate promote the egg white protein to form transparent hydrogel?the gelation mechanism and molecular force changes

Most globular protein solutions will form turbid gels when heated and exhibit unsatisfactory mechanical strength. Our previous study found that highly charged dextran sulfate (DS) has the potential to induce egg white protein (EWP) to form transparent hydrogel with excellent gelation properties. However, the mechanism is not clear. Therefore, the effect of DS on the gelation characteristics of EWP was investigated, including rheological properties, microstructures, water distributions, molecule conformation, and intermolecular forces. Results showed that DS addition significantly prevented the formation of large insoluble aggregates of EWP during heating, which is the prerequisite for forming a transparent gel. The EWP/DS hydrogel possesses remarkably higher gel strength and water holding capacity compared to EWP. The microstructure analysis showed that EWP/DS has a highly ordered fibrous mesh structure after heat treatment. Fourier transform infrared spectroscopy (FT-IR) revealed that DS addition promotes the denaturation of EWP (decrease in α-helices and increase in β-sheets). In addition, hydrophobic interactions are confirmed to be the major intermolecular force in EWP/DS, rather than disulfide bonds in EWP and EWP/dextran gels. Overall, combining the data from turbidity, FT-IR, and microstructure analysis, we found that the EWP/DS complex formation effectively suppresses the further disordered aggregation process of denatured protein by electrostatic repulsion. This work will be beneficial to understand the mechanism of DS on the gelation of EWP after thermal treatment, which will expand the application of sulfated polysaccharides on food structure design. • Dextran sulfate (DS) addition induced egg white protein (EWP) to form a transparent hydrogel under heat treatment. • The EWP/DS composite hydrogel possesses excellent gelation properties compared to that of the native EWP. • The improvement was largely due to high electrostatic repulsion of EWP/DS coacervates which suppress the formation of insoluble protein aggregates. • Hydrophobic interactions were the major intermolecular force in EWP/DS composite hydrogel.

Molecular rotors in haemoglobin and bovine serum albumin proteins.

Molecular rotors are fluorescent viscosity probes and their response in simple fluids is known to be a Förster-Hoffman power law, allowing the viscosity of the medium to be quantified by its fluorescence intensity. They are attractive probes in biological media, usually consisting of proteins, but how does a molecular rotor behave in a protein solution? The response of the DASPI molecular rotor is compared in two globular protein solutions of similar size, haemoglobin (Hb) and bovine serum albumin, one absorbent, the other not. In absorbent Hb, a model validated by experiments in triangular geometry allows one to correct the absorbing effect and to compare the rotor response in both proteins. With concomitant microrheology measurements, we investigate the relation between the DASPI fluorescence intensity and solution viscosity. In protein solutions, we show that viscosity is no longer the parameter determining the rotor response in contrast to simple fluids. Varying the viscosity by concentration or temperature is not equivalent, and the Förster-Hoffmann power laws do not apply when the solution concentration varies. We show that the concentration regime of the protein solution, semi-dilute or concentrated, determines the sensitivity of the rotor to its environment.

Arrangement of Hydrogen Bonds in Aqueous Solutions of Different Globular Proteins.

This work presents the first evidence that dissolved globular proteins change the arrangement of hydrogen bonds in water, with different proteins showing quantitatively different effects. Using ATR-FTIR (attenuated total reflection—Fourier transform infrared) spectroscopic analysis of OH-stretch bands, we obtain quantitative estimates of the relative amounts of the previously reported four subpopulations of water structures coexisting in a variety of aqueous solutions. Where solvatochromic dyes can measure the properties of solutions of non-ionic polymers, the results correlate well with ATR-FTIR measurements. In protein solutions to which solvatochromic dye probes cannot be applied, NMR (nuclear magnetic resonance) spectroscopy was used for the first time to estimate the hydrogen bond donor acidity of water. We found strong correlations between the solvent acidity and arrangement of hydrogen bonds in aqueous solutions for several globular proteins. Even quite similar proteins are found to change water properties in dramatically different ways.

Universal effective interactions of globular proteins close to liquid-liquid phase separation: Corresponding-states behavior reflected in the structure factor.

Intermolecular interactions in protein solutions, in general, contain many contributions. If short-range attractions dominate, the state diagram exhibits liquid-liquid phase separation (LLPS) that is metastable with respect to crystallization. In this case, the extended law of corresponding states (ELCS) suggests that thermodynamic properties are insensitive to details of the underlying interaction potential. Using lysozyme solutions, we investigate the applicability of the ELCS to the static structure factor and how far effective colloidal interaction models can help to rationalize the phase behavior and interactions of protein solutions in the vicinity of the LLPS binodal. The (effective) structure factor has been determined by small-angle x-ray scattering. It can be described by Baxter's adhesive hard-sphere model, which implies a single fit parameter from which the normalized second virial coefficient b2 is inferred and found to quantitatively agree with previous results from static light scattering. The b2 values are independent of protein concentration but systematically vary with temperature and solution composition, i.e., salt and additive content. If plotted as a function of temperature normalized by the critical temperature, the values of b2 follow a universal behavior. These findings validate the applicability of the ELCS to globular protein solutions and indicate that the ELCS can also be reflected in the structure factor.

The dynamic surface properties of green fluorescent protein and its mixtures with poly(N,N-diallyl-N-hexyl-N-methylammonium chloride)

Measurements of the dynamic surface elasticity and surface pressure of solutions of green fluorescent protein (GFP) indicate the formation of a dense protein monolayer with a relatively high surface elasticity of approximately 65 mN/m, and are typical for solutions of globular proteins. The mixed solutions of GFP and poly(N,N-diallyl-N-hexyl-N-methylammonium chloride) (PDAHMAC) demonstrate more complex behavior. During the first adsorption step the surface properties are determined by an unbound component, while the next step is mainly the adsorption of the complexes. The dynamic surface elasticity at relatively low polyelectrolyte concentrations (< 2*10−4 g/l) is close to the values for pure protein solutions when the surface pressure is less than 15 mN/m. At higher surface pressures the adsorption of complexes results in an increase of the surface elasticity up to 80 mN/m. If PDAHMAC concentrations are higher than 2*10−4 g/l, the kinetic dependences of the dynamic surface elasticity become non-monotonic and this quantity does not exceed 45 mN/m. The local maximum of the surface elasticity is a result of the formation of the distal region of the surface layer by the polyelectrolyte. Beyond the subsequent local minimum the surface elasticity starts to increase again due to the adsorption of GFP/ PDAHMAC complexes.

Effects of substrate temperature on patterns produced by dried droplets of proteins

Rapid diagnosis provides better clinical management of patients, helps control possible outbreaks, and increases survival. The study of deposits produced by the evaporation of droplets is a useful tool in the diagnosis of some health problems. With the aim to improve diagnostic time in clinical practice where we use the evaporation of droplets, we explored the effects of substrate temperature on pattern formation of dried droplets in globular protein solutions. Three deposit groups were observed: “functional” patterns (from 25 to 37 ∘C), “transition” patterns (from 44 to 50 ∘C), and “eye” patterns (from 58 to 63 ∘C). The dried droplets of the first two groups show a ring structure (“coffee-ring”) that confines a great diversity of aggregates such as needle-like structures, tiny blade-shape crystals, highly symmetrical crystallization patterns, and amorphous salt aggregates. In contrast, the “eye” patterns are deposits with a large inner aggregate surrounded by a coffee ring, and they can appear from the evaporation of droplets in protein binary mixtures and blood serum. Interestingly, the unfolding proteins correlates with the formation of “eye” patterns. We measured stain diameter, “coffee-ring” thickness, radial density profile, and entropy computed by GLCM-statistics to quantify the structural differences among deposit groups. We found that “functional” patterns are structurally indistinguishable among them, but they are clearly different from elements of the other deposit groups. An exponential decay function describes pattern formation time as a function of substrate temperature, which is independent from protein concentration. Patterns formation at 32 ∘C takes place up to 63% less time and preserves the structural characteristics of dried droplets in proteins formed at room temperature. Therefore, we argue that droplet evaporation at this substrate temperature could be an excellent candidate to make a more efficient diagnosis based on droplet evaporation of biofluids.

Impact of denaturing agents on surface properties of myoglobin solutions

The addition of denaturants strongly influences the surface properties of aqueous myoglobin solutions. The effect differs from the results for mixed solutions of the denaturants and other globular proteins, for example, bovine serum albumin (BSA), lysozyme and β-lactoglobulin (BLG), although the surface properties of the solutions of the pure proteins are similar. The kinetic dependencies of the dynamic surface elasticity of myoglobin solutions with guanidine hydrochloride (GuHCl) reveal at least two adsorption steps at denaturant concentrations higher than 1 M: a very fast increase of the dynamic surface elasticity to approximately 30 mN/m at the beginning of adsorption, and a slower growth to abnormally high values of 250–300 mN/m. At the same time, the surface elasticity of BSA/GuHCl, BLG/GuHCl and lysozyme/GuHCl solutions is a non-monotonic function of the surface age, and does not exceed 50 mN/m close to equilibrium. The high surface elasticity of myoglobin/GuHCl solutions may be associated with protein aggregation in the surface layer. The formation of aggregates is confirmed by ellipsometry and Brewster angle microscopy. The addition of ionic surfactants to protein solutions leads to the formation of myoglobin/surfactant complexes, and the kinetic dependencies of the dynamic surface elasticity display local maxima indicating multistep adsorption kinetics, unlike the corresponding results for solutions of other globular proteins mixed with ionic surfactants. Ellipsometry and infrared reflection-absorption spectroscopy allow tracing the adsorption of the complexes and their displacement from the interface at high surfactant concentrations.

Studying the mechanism of phase separation in aqueous solutions of globular proteins via molecular dynamics computer simulations.

Proteins are the most abundant biomacromolecules in living cells, where they perform vital roles in virtually every biological process. To maintain their function, proteins need to remain in a stable (native) state. Inter- and intramolecular interactions in aqueous protein solutions govern the fate of proteins, as they can provoke their unfolding or association into aggregates. The initial steps of protein aggregation are difficult to capture experimentally, therefore we used molecular dynamics simulations in this study. We investigated the initial phase of aggregation of two different lysozymes, hen egg-white (HEWL) and T4 WT* lysozyme and also human lens γ-D crystallin by using atomistic simulations. We monitored the phase stability of their aqueous solutions by calculating time-dependent density fluctuations. We found that all proteins remained in their compact form despite aggregation. With an extensive analysis of intermolecular residue-residue interactions we discovered that arginine is of paramount importance in the initial stage of aggregation of HEWL and γ-D crystallin, meanwhile lysine was found to be the most involved amino acid in forming initial contacts between T4 WT* molecules.

ANS fluorescence: Potential to discriminate hydrophobic sites of proteins in solid states

In the current study, ANS fluorescence was established as a powerful tool to study proteins in solid-state. Silk fibroin from Bombyx mori cocoons was used as a paradigm protein. ANS incorporated into the films of silk fibroin exhibits fluorescence with two-lifetime components that can be assigned to the patches and/or cavities with distinct hydrophobicities. Decay associated spectra (DAS) of ANS fluorescence from both sites could be fit to the single log-normal component indicating their homogeneity. ANS binding sites in the protein film are specific and could be saturated by ANS titration. ANS located in the binding site that exhibits the long-lifetime fluorescence is not accessible to the water molecules and its DAS stays homogeneously broadened upon hydration of the protein film. In contrast, ANS from the sites demonstrating the short-lifetime fluorescence is accessible to water molecules. In the hydrated films, solvent-induced fluctuations produce an ensemble of binding sites with similar characters. Therefore, upon hydration, the short-lifetime DAS becomes significantly red-shifted and inhomogeneously broadened. The similar spectral features have previously been observed for ANS complexed with globular proteins in solution. The data reveal the origin of the short-lifetime fluorescence component of ANS bound to the globular proteins in aqueous solution. Findings from this study indicate that ANS is applicable to characterize dehydrated as well as hydrated protein aggregates, amyloids relevant to amyloid diseases, such as Alzheimer's, Parkinson, and prion diseases.

Enhanced protein adsorption upon bulk phase separation

In all areas related to protein adsorption, from medicine to biotechnology to heterogeneous nucleation, the question about its dominant forces and control arises. In this study, we used ellipsometry and quartz-crystal microbalance with dissipation (QCM-D), as well as density-functional theory (DFT) to obtain insight into the mechanism behind a wetting transition of a protein solution. We established that using multivalent ions in a net negatively charged globular protein solution (BSA) can either cause simple adsorption on a negatively charged interface, or a (diverging) wetting layer when approaching liquid-liquid phase separation (LLPS) by changing protein concentration (cp) or temperature (T). We observed that the water to protein ratio in the wetting layer is substantially larger compared to simple adsorption. In the corresponding theoretical model, we treated the proteins as limited-valence (patchy) particles and identified a wetting transition for this complex system. This wetting is driven by a bulk instability introduced by metastable LLPS exposed to an ion-activated attractive substrate.

Large effects of tiny structural changes on the cluster formation process in model colloidal fluids: an integral equation study

The formation of aggregates is commonly observed in soft matter such as globular protein solutions and colloidal suspensions. A lively debated issue concerns the possibility to discriminate between a generic intermediate-range order taking place in the fluid, as contrasted with the more specific presence of a clustered state. Recently, we have predicted by Monte Carlo simulations of a standard colloidal model — spherical particles interacting via a short-range attraction followed by a screened electrostatic repulsion at larger distances — the existence of a tiny structural change occurring in the pair structure. This change consists in a reversal of trend affecting a portion of the local density as the attractive strength increases, that is shown to take place precisely at the clustering threshold. Here, we address the same issue by refined thermodynamically self-consistent integral equation theories of the liquid state. We document how such theoretical schemes positively account for the observed phenomenology, highlighting their accuracy to finely describe the aggregation processes in model fluids with microscopic competing interactions.

Unification of lower and upper critical solution temperature phase behavior of globular protein solutions in the presence of multivalent cations.

In globular protein systems, upper critical solution temperature (UCST) behavior is common, but lower critical solution temperature (LCST) phase transitions are rare. In addition, the temperature sensitivity of such systems is usually difficult to tune. Here we demonstrate that the charge state of globular proteins in aqueous solutions can alter their temperature-dependent phase behavior. We show a universal way to tune the effective protein interactions and induce both UCST and LCST-type transitions in the system using trivalent salts. We provide a phase diagram identifying LCST and UCST regimes as a function of protein and salt concentrations. We further propose a model based on an entropy-driven cation binding mechanism to explain the experimental observations.

Spinnability and rheological properties of globular soy protein solution

Spinnability and rheological properties of soy protein are studied for potential application in food products. It has been considered impossible to spin globular proteins due to the difficulty in extending the molecules to achieve high degree of intermolecular entanglement. In order to improve the spinnability of globular proteins, other polymers were incorporated. This paper shows a good spinnability of 100% globular soy protein. In this work, aqueous urea/reductant system is selected for efficient stretch and entanglement globular soy proteins for fiber spinning. In this system, hydrogen bonds, hydrophobic and electrostatic interaction were disrupted by urea, while disulfide bonds were cleaved by reductants. Moreover, the backbones of globular soy proteins were not severely destroyed, rendering the stretched globular protein spinnable and the spun fibers mechanically robust. Relationship between rheological properties and spinnability of globular soy protein solutions is quantified. In order to be spinnable, globular soy proteins should be highly stretched, and thus entangled with preserved backbones.

Molecular Dynamics Simulations of Macromolecular Crystals.

The structures of biological macromolecules would not be known to their present extent without X-ray crystallography. Most simulations of globular proteins in solution begin by surrounding the crystal structure of the monomer in a bath of water molecules, but the standard simulation employing periodic boundary conditions is already close to a crystal lattice environment. With simple protocols, the same software and molecular models can perform simulations of the crystal lattice, including all asymmetric units and solvent to fill the box. Throughout the history of molecular dynamics, studies of crystal lattices have served to investigate the quality of the underlying force fields, correlate the simulated ensembles to experimental structure factors, and extrapolate the behavior in lattices to behavior in solution. Powerful new computers are enabling molecular simulations with greater realism and statistical convergence. Meanwhile, the advent of exciting new methods in crystallography, including femtosecond free-electron lasers and image reconstruction for time-resolved crystallography on slurries of small crystals, is expanding the range of structures accessible to X-ray diffraction. We review past fusions of simulations and crystallography, then look ahead to the ways that simulations of crystal structures will enhance structural biology in the future.

Short-time dynamics of lysozyme solutions with competing short-range attraction and long-range repulsion: Experiment and theory.

Recently, atypical static features of microstructural ordering in low-salinity lysozyme protein solutions have been extensively explored experimentally and explained theoretically based on a short-range attractive plus long-range repulsive (SALR) interaction potential. However, the protein dynamics and the relationship to the atypical SALR structure remain to be demonstrated. Here, the applicability of semi-analytic theoretical methods predicting diffusion properties and viscosity in isotropic particle suspensions to low-salinity lysozyme protein solutions is tested. Using the interaction potential parameters previously obtained from static structure factor measurements, our results of Monte Carlo simulations representing seven experimental lysoyzme samples indicate that they exist either in dispersed fluid or random percolated states. The self-consistent Zerah-Hansen scheme is used to describe the static structure factor, S(q), which is the input to our calculation schemes for the short-time hydrodynamic function, H(q), and the zero-frequency viscosity η. The schemes account for hydrodynamic interactions included on an approximate level. Theoretical predictions for H(q) as a function of the wavenumber q quantitatively agree with experimental results at small protein concentrations obtained using neutron spin echo measurements. At higher concentrations, qualitative agreement is preserved although the calculated hydrodynamic functions are overestimated. We attribute the differences for higher concentrations and lower temperatures to translational-rotational diffusion coupling induced by the shape and interaction anisotropy of particles and clusters, patchiness of the lysozyme particle surfaces, and the intra-cluster dynamics, features not included in our simple globular particle model. The theoretical results for the solution viscosity, η, are in qualitative agreement with our experimental data even at higher concentrations. We demonstrate that semi-quantitative predictions of diffusion properties and viscosity of solutions of globular proteins are possible given only the equilibrium structure factor of proteins. Furthermore, we explore the effects of changing the attraction strength on H(q) and η.

Two time scales for self and collective diffusion near the critical point in a simple patchy model for proteins with floating bonds.

Using dynamic Monte Carlo and Brownian dynamics, we investigate a floating bond model in which particles can bind through mobile bonds. The maximum number of bonds (here fixed to 4) can be tuned by appropriately choosing the repulsive, nonadditive interactions among bonds and particles. We compute the static and dynamic structure factor (intermediate scattering function) in the vicinity of the gas-liquid critical point. The static structure exhibits a weak tetrahedral network character. The intermediate scattering function shows a temporal decay deviating from a single exponential, which can be described by a double exponential decay where the two time scales differ approximately by one order of magnitude. This time scale separation is robust over a range of wave numbers. The analysis of clusters in real space indicates the formation of noncompact clusters and shows a considerable stretch in the instantaneous size distribution when approaching the critical point. The average time evolution of the largest subcluster of given initial clusters with 10 or more particles also shows a double exponential decay. The observation of two time scales in the intermediate scattering function at low packing fractions is consistent with similar findings in globular protein solutions with trivalent metal ions that act as bonds between proteins.

Stable, metastable, and supercritical phases in solutions of globular proteins between upper and lower denaturation temperatures

The temperature trends of the standard thermodynamic functions of the native and denatured protein in solution are considered within the concept of excess mixing functions. It is assumed that some protein molecules adopt an intermediate state between native and denatured forms within the temperature range between cold and thermal denaturation and form metastable microphases as a result of a specific interaction with water. A phase diagram in the temperature–standard entropy coordinate plane representing an isobar family is proposed. Two limiting isobars are characterized by an entropy jump, which reflects the first-order phase transition between the native and denatured states. The isobars in the intermediate temperature range are represented as van der Waals curves, which reflect the equilibrium between the main phase of the molecules in native state and microphases. The difference between the phases disappears at critical points. It is assumed that the supercritical range is a macroscopically homogeneous single phase zone of reduced stability, which is represented by a dynamic system of monomers and oligomers of the native protein, monomers and clusters of the protein with partially unfolded structure. The phase diagram is collated with the elliptic phase diagram in the temperature–osmotic pressure plane.

Dielectric Characteristic of a Glycerol and a Globular Protein in Solution

Dielectric Characteristic of a Glycerol and a Globular Protein in Solution

Predicting Protein Interactions of Concentrated Globular Protein Solutions Using Colloidal Models.

Protein interactions of α-chymotrypsinogen A (aCgn) were quantified using light scattering from low to high protein concentrations. Static light scattering (SLS) was used to determine the excess Rayleigh ratio (Rex) and osmotic second virial coefficients (B22) as a function of pH and total ionic strength (TIS). Repulsive (attractive) protein-protein interactions (PPI) were observed at pH 5 (pH 7), with decreasing repulsions (attractions) upon increasing TIS. Simple colloidal potential of mean force models (PMF) that account for short-range nonelectrostatic attractions and screened electrostatic interactions were used to fit model parameters from data for B22 vs TIS at both pH values. The parameters and PMF models from low-concentration conditions were used as the sole input to transition matrix Monte Carlo simulations to predict high concentration Rex behavior. At conditions where PPI are repulsive to slightly attractive, experimental Rex data at high concentrations could be predicted quantitatively by the simulations. However, accurate predictions were challenging when PPI were strongly attractive due to strong sensitivity to changes in PMF parameter values. Additional simulations with higher-resolution coarse-grained molecular models suggest an approach to qualitatively predict cases when anisotropic surface charge distributions will lead to overall attractive PPI at low ionic strength, without assumptions regarding electrostatic "patches" or multipole expansions.