Preferential Interactions Of Proteins Research Articles

Osmolyte Effects on Monoclonal Antibody Stability and Concentration-Dependent Protein Interactions with Water and Common Osmolytes.

Preferential interactions of proteins with water and osmolytes play a major role in controlling the thermodynamics of protein solutions. While changes in protein stability and shifts in phase behavior are often reported with the addition of osmolytes, the underlying protein interactions with water and/or osmolytes are typically inferred rather than measured directly. In this work, Kirkwood-Buff integrals for protein-water interactions (G12) and protein-osmolyte interactions (G23) were determined as a function of osmolyte concentration from density measurements of antistreptavidin immunoglobulin gamma-1 (AS-IgG1) in ternary aqueous solutions for a set of common neutral osmolytes: sucrose, trehalose, sorbitol, and poly(ethylene glycol) (PEG). For sucrose and PEG solutions, both protein-water and protein-osmolyte interactions depend strongly on osmolyte concentrations (c3). Strikingly, both osmolytes change from being preferentially excluded to preferentially accumulated with increasing c3. In contrast, sorbitol and trehalose solutions do not show large enough preferential interactions to be detected by densimetry. G12 and G23 values are used to estimate the transfer free energy for native AS-IgG1 (Δμ2N) and compared with existing models. AS-IgG1 unfolding via calorimetry shows a linear increase in midpoint temperatures as a function of trehalose, sucrose, and sorbitol concentrations, but the opposite behavior for PEG. Together, the results highlight limitations of existing models and common assumptions regarding the mechanisms of protein stabilization by osmolytes. Finally, PEG preferential interactions destabilize the Fab regions of AS-IgG1 more so than the CH2 or CH3 domains, illustrating preferential interactions can be specific to different protein domains.

Influence of chemical chaperones on the properties of lysozyme and the reaction center protein from Rhodobacter sphaeroides

The influence of three chemical chaperones: glycerol, 4-hexylresorcinol, and 5-methylresorcinol on the structure, equilibrium fluctuations, and the functional activity of the hydrophilic enzyme lysozyme and the transmembrane reaction center (RC) protein from Rb. sphaeroides in a broad range of concentrations has been studied. Selected chemical chaperones are strongly different by the structure and action on hydrophilic and membrane proteins. The influence of the chemical chaperones (except methylresorcinol) on the structure, dynamics, and functional properties of lysozyme and RC protein are well described within the frames of extended models of preferential hydration and preferential interaction of protein with a chemical chaperone. A molecule of hexylresorcinol consists of a hydrophobic (alkyl radical) and a hydrophilic (aromatic nuclus) moieties. This fact provides additional regulation of functional activity of lysozyme and RC by hexylresorcinol. The influence of methylresorcinol on proteins differs from that of glycerol and hexylresorcinol. Methylresorcinol interacts with the surface of lysozyme directly, not via water hydrogen bonds. This leads to a decrease in denaturation temperature T(d), and an increase in the amplitude of equilibrium fluctuation, which allows him to be a powerful activator. Methylresorcinol interacts with the membrane RC protein only by the condensation of hydration water, which is negligible in the case of methylresorcinol. Therefore, methylresorcinol does not effect the functional properties of the RC protein. It was concluded that various chaperones at one and the same concentration and chaperones at different concentrations form diverse 3D structures of proteins, which differ by dynamic and functional characteristics.

Soft X-ray spectromicroscopy of polymers and bipolymer interfaces.

The status of soft X-ray spectromicroscopy (near edge X-ray absorption, or NEXAFS, microscopy) is summarized, with particular emphasis on recent collaborative studies carried out by the author's group at the scanning transmission X-ray microscopy (STXM) and the photoelectron emission microscopy (PEEM) instruments at the Advanced Light Source. Results described include: characterization of phase segregation and filler particles in polyurethanes; effects of confinement on polymer self-organization; characterization of chemically structured polyurea capsule walls; and preferential interactions of proteins with chemically heterogeneous polymer surfaces. The latter is part of a systematic study of the biocompatibility of polymers used in blood contact applications.

Increased tolerance to large hyperosmotic shocks and compensatory solutes in mammalian cultured cells

L929 and MDCK cells acclimated to sorbitol, proline or taurine media can tolerate a NaCl load that they would be unable to stand when cultured in normal conditions. The protecting effect is observed only at high concentrations, between 100 and 300 mM. In that range, the effect is increasing with increasing concentration. The presence of the compound is not necessary, in the extracellular medium, to induce tolerance. These results are discussed in the framework of the theory considering preferential interactions of proteins with solvent components to explain the protecting effect of such compounds.

Preferential interactions determine protein solubility in three-component solutions: the magnesium chloride system

The correlation between protein solubility and the preferential interactions of proteins with solvent components was critically examined with aqueous MgCl2 as the solvent system. Preferential interaction and solubility measurements with three proteins, beta-lactoglobulin, bovine serum albumin, and lysozyme, resulted in similar patterns of interaction. At acid pH (pH 2-3) and lower salt concentrations (less than 2 M), the proteins were preferentially hydrated, while at higher salt concentrations, the interaction was either that of preferential salt binding or low salt exclusion. At pH 4.5-5, all three proteins exhibited either very low preferential hydration or preferential binding of MgCl2. These results were analyzed in terms of the balance between salt binding and salt exclusion attributed to the increase in the surface tension of water by salts, which is invariant with conditions. It was shown that the increase in salt binding at high salt concentration is a reflection of mass action, while its decrease at acid pH is due to the electrostatic repulsion between Mg2+ ions and the high net positive charge on the protein. The preferential interaction pattern was paralleled by the variation of protein solubility with solvent conditions. Calculation of the transfer free energies from water to the salt solutions for proteins in solution and in the precipitate showed dependencies on salt concentration. This indicates that the nature of interactions between proteins and solvent components is the same in solution and in the solid state, which implies no change in protein structure during precipitation. Analysis of the transfer free energies and preferential interaction parameter in terms of the salting-in, salting-out, and weak ion binding contributions has led to the conclusions that, when the weak ion binding contribution is small, the predominant protein-salt interaction must be that of preferential salt exclusion most probably caused by the increase of the surface tension of water by addition of the salt. A necessary consequence of this is salting-out of the protein, if the protein structure is to remain unaltered.

Mechanism of protein salting in and salting out by divalent cation salts: balance between hydration and salt binding.

The preferential interactions of proteins with solvent components were studied in concentrated aqueous solutions of the sulfate, acetate, and chloride salts of Mg2+, Ba2+, Ca2+, Mn2+ and Ni2+ [except for CaSO4, BaSO4, Mn-(OAc)2, and Ni(OAc)2], and results were compared with those of the Na+ salts. It was found that, for all the salts, the preferential hydration increased in the order of Cl- less than CH3-COO- less than SO42- regardless of the cationic species used, in agreement with the anionic lyotropic series, and that the same parameter exhibited a tendency to increase in the order of Mn2+, Ni2+ less than Ca2+, Ba2+ less than Mg2+ less than Na+. The salting-out and stabilizing or salting-in and destabilizing effectiveness of the salts were interpreted in terms of the observed preferential interactions. The surface tension increment of salts, which is a major factor responsible for the preferential interactions of the Na+ salts, had no correlation with those of the divalent cation salts. It was shown that the binding of divalent cations to the proteins overcomes the salt exclusion due to the surface tension increase, leading to a decrease in the preferential hydration. In conformity with this mechanism, the preferential interaction of MgCl2 was strongly pH dependent, because of the protein charge-dependent affinity of Mg2+ for proteins, while NaCl showed no pH dependence of the preferential interaction. The proposed mechanism was supported by a strong correlation between the preferential interaction results and the interaction of these salts with the model peptide compound acetyltetraglycine ethyl ester, described by Robinson and Jencks.

Preferential interactions of proteins with solvent components in aqueous amino acid solutions

The preferential interactions of proteins with solvent components in concentrated amino acid solutions were measured by high-precision densimetry. Bovine serum albumin and lysozyme were preferentially hydrated in all of the amino acids examined, glycine, α- and β-alanine, and betaine i.e., addition of these amino acids resulted in an unfavorable free energy change. It was shown that, for the former three amino acids, known to have a positive surface tension increment, their perturbation of the surface free energy of water is consistent with their preferential exclusion from the protein surface. In the case of betaine, which does not increase the surface tension of water, preferential exclusion from protein surface must reflect the chemical structure of this cosolvent, which is considerably more hydrophobic than that of the other three amino acids.

Preferential interactions of proteins with salts in concentrated solutions.

The preferential interactions of proteins with solvent components were studied in concentrated salt by densimetric measurements. Proteins were found to be preferentially hydrated in NaCl, NaCH3COO, and Na2SO4. The resulting unfavorable free-energy change was related to the effects of these salts on solubility and stability of the proteins. This unfavorable free-energy change was correlated with the large, positive surface tension increment of these salts, i.e., their perturbation of surface free energy. On the other hand, KSCN, CaCl2, and MgCl2 showed considerable binding to bovine serum albumin, which could be related to their destabilizing and salting-in effects on macromolecules. Since the last two salts have high surface tension increments, it was concluded that this does not necessarily lead to protein preferential hydration and stabilization.

Sedimentation equilibrium study on preferential interaction of protein with solvent components

A sedimentation equilibrium equation was derived in weight-per-volume concentration units to evaluate the degree of preferential binding of one solvent component to a homogeneous neutral macromolecule in a mixture of two components. Using the equation, the preferential binding of chloroethanol to β-lactoglobulin A was calculated from sedimentation equilibrium data, and the result showed good agreement with values obtained by light scattering and refractive index increment experiments. It was also demonstrated that the preferential interaction can in principle be determined by density increment measurements under different conditions: at constant molality and at constant chemical potential of solvent.