Solutions Of Globular Proteins Research Articles

Role of solvent for globular proteins in solution

The properties of the solvent affect the behavior of the solution. We propose a model that accounts for the contribution of the solvent free energy to the free energy of globular proteins in solution. For the case of an attractive square-well potential, we obtain an exact mapping of the phase diagram of this model without solvent to the model that includes the solute-solvent contribution. In particular we find for appropriate choices of parameters upper critical points, lower critical points, and even closed loops with both upper and lower critical points similar to those found before [Macromolecules 36, 5843 (2003)]. In the general case of systems whose interactions are not attractive square wells, this mapping procedure can be a first approximation to understand the phase diagram in the presence of solvent. We also present simulation results for both the square-well model and a modified Lennard-Jones model.

Numerical study of a binary Yukawa model in regimes characteristic of globular proteins in solutions

The main goal of this paper is to assess the limits of validity, in the regime of low concentration and strong Coulomb coupling (high molecular charges), of a simple perturbative approximation to the radial distribution functions (RDF's), based upon a low-density expansion of the potential of mean force and proposed to describe protein-protein interactions in a recent small-angle-scattering (SAS) experimental study. A highly simplified Yukawa (screened Coulomb) model of monomers and dimers of a charged globular protein (beta-lactoglobulin) in solution is considered. We test the accuracy of the RDF approximation, as a necessary complementary part of the previous experimental investigation, by comparison with the fluid structure predicted by approximate integral equations and exact Monte Carlo (MC) simulations. In the MC calculations, an Ewald construction for Yukawa potentials has been used to take into account the long-range part of the interactions in the weakly screened cases. Our results confirm that the perturbative first-order approximation is valid for this system even at strong Coulomb coupling, provided that the screening is not too weak (i.e., for Debye length smaller than monomer radius). A comparison of the MC results with integral equation calculations shows that both the hypernetted-chain (HNC) and Percus-Yevick closures have a satisfactory behavior under these regimes, with the HNC being superior throughout. The relevance of our findings for interpreting SAS results is also discussed.

Influence of the Ionic Strength on the Structure of Heat-Set Globular Protein Gels at pH 7. Ovalbumin

The effect of the NaCl concentration (Cs) on the structure factor and the turbidity of heated ovalbumin solutions was investigated using cross-correlation dynamic light scattering. The heated systems are characterized by a correlation length, which depends on protein and salt concentration, beyond which they are homogeneous. At length scales below this correlation length, the systems have a self-similar, fractal, structure. For Cs ≥ 100 mM, very turbid systems are formed with correlation lengths that show strong concentration dependence, and the correlation length rapidly rises above 1 μm at protein concentrations above 4 g/L. For Cs ≤ 30 mM NaCl the systems remain transparent with relatively small correlation lengths (<20 nm). At 50 mM NaCl the correlation length first increases strongly with increasing protein concentration followed by a decrease above 8 g/L. At both 30 and 50 mM, an unexpected increase in the correlation length and turbidity is observed at high protein concentrations (above 40 and 30 g/L, respectively). From the present work we conclude that the structure of heated globular protein solutions and gels is generally determined by the interplay between the growth of the aggregates and the electrostatic interaction between the aggregates.

Theory and simulation of short-range models of globular protein solutions

We report theoretical and simulation studies of phase coexistence in model globular proteinsolutions, based on short-range, central, pair potential representations of the interactionamong macro-particles. After reviewing our previous investigations of hard-core Yukawaand generalized Lennard-Jones potentials, we report more recent results obtainedwithin a DLVO-like description of lysozyme solutions in water and added salt. Weshow that a one-parameter fit of this model, based on static light scattering andself-interaction chromatography data in the dilute protein regime, yields demixing andcrystallization curves in good agreement with experimental protein-rich–protein-poor andsolubility envelopes. The dependence of cloud and solubility point temperatures ofthe model on the ionic strength is also investigated. Our findings highlight theminimal assumptions on the properties of the microscopic interaction sufficient for asatisfactory reproduction of the phase diagram topology of globular protein solutions.

Microscopic Determination of the Phase Diagrams of Lysozyme and γ-Crystallin Solutions

We determine the phase diagrams of two widely studied protein solutions, namely, lysozyme and γ-crystallin in water and added salt, within a short-range, pair-potential representation of macroparticle interactions. A one-parameter fit of the model is performed on the basis of static or dynamic light scattering and self-interaction chromatography data. Demixing and crystallization curves, as calculated through numerical approaches, turn out to be in good agreement with experimental protein-rich−protein-poor and solubility envelopes. Our findings highlight the minimal assumptions about the nature of the microscopic potential allowing a satisfactory reproduction of the phase-diagram topology of globular protein solutions.

Phase behavior of aqueous solutions containing dipolar proteins from second-order perturbation theory.

Due to the interplay of Coulombic repulsion and attractive dipolar and van der Waals interactions, solutions of globular proteins display a rich variety of phase behavior featuring fluid-fluid and fluid-solid transitions that strongly depend on solution pH and salt concentration. Using a simple model for charge, dispersion and dipole-related contributions to the interprotein potential, we calculate phase diagrams for protein solutions within the framework of second-order perturbation theory. For each phase, we determine the Helmholtz energy as the sum of a hard-sphere reference term and a perturbation term that reflects both the electrostatic and dispersion interactions. Dipolar effects can induce fluid-fluid phase separation or crystallization even in the absence of any significant dispersion attraction. Because dissolved electrolytes screen the charge-charge repulsion more strongly than the dipolar attraction, the ionic strength dependence of the potential of mean force can feature a minimum at intermediate ionic strengths offering an explanation for the observed nonmonotonic dependence of the phase behavior on salt concentration. Inclusion of correlations between charge-dipole and dipole-dipole interactions is essential for a reliable calculation of phase diagrams for systems containing charged dipolar proteins and colloids.

SPECTROSCOPY WITH ELECTRONIC TERAHERTZ TECHNIQUES FOR CHEMICAL AND BIOLOGICAL SENSING

By illuminating the sample with a broadband 10-500 GHz stimulus and coherently detecting the response, we obtain reflection and transmission spectra of common powdered substances, and compare them as a starting point for distinguishing concealed threats in envelopes and on personnel. Because these samples are irregular and their dielectric properties cannot be modulated, the spectral information we obtain is largely qualitative. To show how to gain quantitative information on biological species at micro- and millimeter-wave frequencies, we introduce thermal modulation of a globular protein in solution, and show that changes in microwave reflections coincide with accepted visible absorption spectra, pointing the way toward gaining quantitative chemical and biological spectra from broadband terahertz systems.

Low-Frequency Modes of Peptides and Globular Proteins in Solution Observed by Ultrafast OHD-RIKES Spectroscopy

The low-frequency (1–200cm−1) vibrational spectra of peptides and proteins in solution have been investigated with ultrafast optical heterodyne-detected Raman-induced Kerr-effect spectroscopy (OHD-RIKES). Spectra have been obtained for di-L-alanine (ALA(2)) and the α-helical peptide poly-L-alanine (PLA) in dichloroacetic acid solution. The poly-L-alanine spectrum shows extra amplitude compared to the di-L-alanine spectrum, which can be explained by the secondary structure of the former. The globular proteins lysozyme, α-lactalbumin, pepsin, and β-lactoglobulin in aqueous solution have been studied to determine the possible influence of secondary or tertiary structure on the low-frequency spectra. The spectra of the globular proteins have been analyzed in terms of three nondiffusive Brownian oscillators. The lowest frequency oscillator corresponds to the so-called Boson peak observed in inelastic neutron scattering (INS). The remaining two oscillators are not observed in inelastic neutron scattering, do therefore not involve significant motion of hydrogen atoms, and may be associated with delocalized backbone torsions.

On the behavior of electrokinetic streaming potential during protein filtration with fully and partially retentive nanopores

An experimental investigation of the electrokinetic streaming potentials of both fully and partially retentive nanopores as compared with the filtration progress of dilute globular protein solution under different surface charge conditions was performed using hollow fibers. The streaming potential is generated by the electrokinetic flow effect within the electric double layer of the charged surface. Depending on the solution pH, both the protein and the pore wall can be either repulsive or attractive due to the long-range electrostatic interaction. The repulsive electrostatic interaction allows the protein particles to stay in a suspended state above the outer surface of hollow fibers instead of being deposited. The apparent streaming potential value at partially retentive pores is larger than that at fully retentive pores for the oppositely charged case; however, the opposite behavior is shown for the same-charged case. The axial-position-dependent streaming potential was also observed in order to explore the development of a concentration polarization layer during the cross-flow filtration. The time evolution of the streaming potential during the filtration of protein particles is related to the filtrate flux, from which it can be found to provide useful real-time information on particle deposition onto the outer surfaces of hollow fibers.

Nonmonotonic variation with salt concentration of the second virial coefficient in protein solutions.

The osmotic virial coefficient B2 of globular protein solutions is calculated as a function of added salt concentration at fixed pH by computer simulations of the "primitive model." The salt and counterions as well as a discrete charge pattern on the protein surface are explicitly incorporated. For parameters roughly corresponding to lysozyme, we find that B2 first decreases with added salt concentration up to a threshold concentration, then increases to a maximum, and then decreases again upon further raising the ionic strength. Our studies demonstrate that the existence of a discrete charge pattern on the protein surface profoundly influences the effective interactions and that linear and nonlinear Poisson Boltzmann theories fail for large ionic strength. The observed nonmonotonicity of B2 is compared with experiments. Implications for protein crystallization are discussed.

Phase coexistence in a DLVO model of globular protein solutions

Globular protein solutions of lysozyme in water and added salt are modelledaccording to the Derjaguin–Landau–Verwey–Overbeek (DLVO) theory,in order to determine their fluid–fluid and fluid–solid coexistence lines.Calculations are based on both computer simulations and theoreticalapproaches. Our results indicate that, when the potential parametersare obtained by fitting physical properties directly deducible from eitherstatic or dynamic light scattering data, the fluid–fluid phase coexistencepredictions agree quite well with the experiments. Our description of the solidphase allows only a qualitative reproduction of the experimental solubilityboundaries. The overall accuracy of our predictions is discussed in view of thewell known limitations of the DLVO representation of protein solutions.

Broadband 10–300 GHz stimulus-response sensing for chemical and biological entities

By illuminating the sample with a broadband 10–300 GHz stimulus and coherently detecting the response, we obtain reflection and transmission spectra of common powdered substances, and compare them as a starting point for distinguishing concealed threats in envelopes and on personnel. Because these samples are irregular and their dielectric properties cannot be modulated, however, the spectral information we obtain is largely qualitative. To show how to gain quantitative information on biological species at micro- and millimetre-wave frequencies, we introduce thermal modulation of a globular protein in solution, and show that changes in single-wavelength microwave reflections coincide with accepted visible absorption spectra, pointing the way towards gaining quantitative chemical and biological spectra from broadband terahertz systems.

Structural hierarchy of several proteins observed by wide-angle solution scattering.

In the present study using a high-intensity X-ray beam from a third-generation synchrotron radiation source, it is demonstrated that a wide-angle X-ray scattering (WAXS) profile from several globular proteins in solution can reflect not only the overall structures (approximately 300 A distance resolution) but also intramolecular structures ranging to secondary structures (approximately 2.5 A distance resolution). The proteins treated in the present experiments are classified as different types of structure categories, namely, as all-alpha, all-beta and alpha + beta proteins. Here the full-range experimental scattering curves are compared with the theoretical curves, suggesting a further availability of the SR-WAXS method for studies of structure hierarchy and the function of proteins in solutions.

Adhesion and liquid–liquid phase separation in globular protein solutions

We derive a form of adhesive interaction between protein globules in solution, developing from the premise that amino-acid residues are able to rearrange around a globule–globule contact. We show in this approach that globules only become adhesive above a certain threshold temperature, which is sensitive to an osmotic modulus characterizing individual globule elasticity. Above the threshold, adhesive strength increases monotonically with temperature, driving a temperature-reversed liquid–liquid phase separation.

On solid-like rheological behaviors of globular protein solutions

Dynamic viscoelastic and steady flow properties of β-lactoglobulin, bovine serum albumin, ovalbumin, and α-lactalbumin aqueous solutions were investigated at 20°C. When a sinusoidal strain in the linear viscoelastic region was applied, the solutions of the globular proteins except for α-lactalbumin showed typical solid-like rheological behavior: the storage modulus G′ was always larger than the loss modulus G″ in the entire frequency range examined (0.1–100 rad/s). Under a steady shear flow, strong shear thinning behavior was observed with increasing shear rate from 0.001 to 800 s −1, for the globular proteins except for α-lactalbumin. The values of the steady shear viscosity η were lower than those of the dynamic shear viscosity η* at a comparable time scale of observation, violating the Cox–Merz rule, and thus suggesting that a solid-like structure in a globular protein solution was susceptible to a steady shear strain. During isothermal gelation of the protein colloids at 70°C, no crossover between G′ and G″ was observed so that the gelation point was judged by an abrupt increase in the modulus or a sudden decrease in tan δ.

Phase Separation and Association of Globular Protein Aggregates in the Presence of Polysaccharides: 2. Heated Mixtures of Native β-Lactoglobulin and κ-Carrageenan

Aqueous solutions of globular proteins (β-lactoglobulin) at pH 7 and 0.1 M NaCl were heated in the presence of various concentrations of polysaccharide (κ-carrageenan). The fraction of unaggregated proteins was determined as a function of heating time with size exclusion chromatography. The rate at which the proteins aggregate is independent of the polysaccharide concentration at least up to 9 g/L κ-carrageenan. The protein aggregates were characterized using light scattering. At modest concentrations (up to 1 g/L) the presence of κ-carrageenan accelerates the growth of the aggregates and therefore the gel formation, but the structure of the aggregates is not modified. At higher concentrations κ-carrageenan induces phase separation of protein aggregates.

Surface dilational behaviour of aqueous protein solutions: History effects

Aqueous protein solutions have dynamic surface properties that are found to depend on the experimental technique by which they are measured. Especially aqueous solutions of globular proteins have dynamic surface properties that are sensitive for the history of the surface. By means of a Langmuir trough (Lt) technique and an overflowing cylinder (Oc) technique, these differences in surface behaviour can be quantified. It appears that much higher surface dilational viscosities are measured by using the Lt technique than with the Oc technique. These results are interpreted in a qualitative way by taking into account the different histories of the liquid surfaces in the two techniques and the more or less globular nature of the proteins used. The consequence of the observed phenomena for practical application is discussed in view of the behaviour of bubbles in a moving liquid. It is concluded that both techniques, although they give different results, can be applied to different practical situations.

The structure and dipole moment of globular proteins in solution and crystalline states: Use of NMR and X‐ray databases for the numerical calculation of dipole moment

The structure and dipole moment of globular proteins in solution and crystalline states: Use of NMR and X‐ray databases for the numerical calculation of dipole moment

Unraveling double stranded alpha-helical coiled coils: an x-ray diffraction study on hard alpha-keratin fibers.

Transformations of proteins secondary and tertiary structures are generally studied in globular proteins in solution. In fibrous proteins, such as hard alpha-keratin, that contain long and well-defined double stranded alpha-helical coiled coil domains, such study can be directly done on the native fibrous tissue. In order to assess the structural behavior of the coiled coil domains under an axial mechanical stress, wide angle x-ray scattering and small angle x-ray scattering experiments have been carried out on stretched horse hair fibers at relative humidity around 30%. Our observations of the three major axial spacings as a function of the applied macroscopic strain have shown two rates. Up to 4% macroscopic strain the coiled coils were slightly distorted but retained their overall conformation. Above 4% the proportion of coiled coil domains progressively decreased. The main and new result of our study is the observation of the transition from alpha-helical coiled coils to disordered chains instead of the alpha-helical coiled coil to beta-sheet transition that occurs in wet fibers.

The structure and dipole moment of globular proteins in solution and crystalline states: use of NMR and X-ray databases for the numerical calculation of dipole moment.

The large dipole moment of globular proteins has been well known because of the detailed studies using dielectric relaxation and electro-optical methods. The search for the origin of these dipolemoments, however, must be based on the detailed knowledge on protein structure with atomic resolutions. At present, we have two sources of information on the structure of protein molecules: (1) x-ray databases obtained in crystalline state; (2) NMR databases obtained in solution state. While x-ray databases consist of only one model, NMR databases, because of the fluctuation of the protein folding in solution, consist of a number of models, thus enabling the computation of dipole moment repeated for all these models. The aim of this work, using these databases, is the detailed investigation on the interdependence between the structure and dipole moment of protein molecules. The dipole moment of protein molecules has roughly two components: one dipole moment is due to surface charges and the other, core dipole moment, is due to polar groups such as N--H and C==O bonds. The computation of surface charge dipole moment consists of two steps: (A) calculation of the pK shifts of charged groups for electrostatic interactions and (B) calculation of the dipole moment using the pK corrected for electrostatic shifts. The dipole moments of several proteins were computed using both NMR and x-ray databases. The dipole moments of these two sets of calculations are, with a few exceptions, in good agreement with one another and also with measured dipole moments.