Boris Y. Zaslavsky

Intrinsically disordered proteins as crucial constituents of cellular aqueous two phase systems and coacervates

Here, we hypothesize that intrinsically disordered proteins (IDPs) serve as important drivers of the intracellular liquid–liquid phase separations that generate various membrane‐less organelles. This hypothesis is supported by the overwhelming abundance of IDPs in these organelles. Assembly and disassembly of these organelles are controlled by changes in the concentrations of IDPs, their posttranslational modifications, binding of specific partners, and changes in the pH and/or temperature of the solution. Each resulting phase provides a distinct solvent environment for other solutes leading to their unequal distribution within phases. The specificity and efficiency of such partitioning is determined by the nature of the IDP(s) and defines “targeted” enrichment of specific molecules in the resulting membrane‐less organelles that determines their specific activities.

Beyond the Excluded Volume Effects: Mechanistic Complexity of the Crowded Milieu

Macromolecular crowding is known to affect protein folding, binding of small molecules, interaction with nucleic acids, enzymatic activity, protein-protein interactions, and protein aggregation. Although for a long time it was believed that the major mechanism of the action of crowded environments on structure, folding, thermodynamics, and function of a protein can be described in terms of the excluded volume effects, it is getting clear now that other factors originating from the presence of high concentrations of “inert” macromolecules in crowded solution should definitely be taken into account to draw a more complete picture of a protein in a crowded milieu. This review shows that in addition to the excluded volume effects important players of the crowded environments are viscosity, perturbed diffusion, direct physical interactions between the crowding agents and proteins, soft interactions, and, most importantly, the effects of crowders on solvent properties.

Solvent Properties Governing Solute Partitioning in Polymer/Polymer Aqueous Two-Phase Systems: Nonionic Compounds

The solvatochromic solvent parameters characterizing the solvent polarity (pi*), solvent hydrogen-bond donor acidity (alpha), and solvent hydrogen-bond acceptor basicity (beta) of aqueous media were measured in the coexisting phases of nine different aqueous polymer/polymer two-phase systems (ATPS), containing 0.15 M NaCl in 0.01 M phosphate buffer, pH 7.4. Partitioning coefficients of six neutral compounds were measured in the nine ATPS at particular polymer concentrations. The solvatochromic equation was used to describe the partitioning of each compound. Three descriptors of the solvent properties of the phases could describe adequately the partitioning of the solutes in all the ATPS employed.

Action of surface-active substances on biological membranes. II. Hemolytic activity of nonionic surfactants

The hemolytic action of commercially available nonionic surfactants and synthesized polyoxyethylene fatty acids and mercaptans on human erythrocytes was measured. It is shown that the hemolytic power of the detergents depends on the mutual effect of the hydrophobic and hydrophylic fragments of the agent molecule and does not depend on the hydrophile-lipophile balance of the compounds. A graphical image of the structure-activity relationship obtained in the study is similar to the one found in the literature when studying the analgesic effect of imidazoline derivatives on rats in vivo. This fact is discussed on the basis of assumption that the mechanism of both processes in vivo and in vitro is related to influence of the agents on cellular membranes. It is suggested that the role of the polyoxyethylene chain is its effect on the dipole moment and the relative lipophilicity of the compound or in the participation of the fragment in the interaction with the surface components when the agent is sorbed on the membrane. It is concluded that when the correlation between the hydrophile-lipophile balance values and a membrane effect the capacity of the surfactants this indicates that the effect is caused not by destruction of the membrane but by some rearrangement of the membrane structure accompanying the surfactant adsorption.

Solvent properties governing protein partitioning in polymer/polymer aqueous two-phase systems

Distribution coefficients of various proteins were measured in aqueous Dextran-Ficoll, Dextran-PES, and Ficoll-PES two-phase systems, containing 0.15M NaCl in 0.01 M phosphate buffer, pH 7.4. The acquired data were combined with data for the same proteins in different systems reported previously and known solvatochromic solvent properties of the systems to characterize the protein-solvent interactions. The relative susceptibilities of proteins to solvent dipolarity/polarizability, solvent hydrogen bond acidity, solvent hydrogen bond basicity, and solvent ability to participate in ion-ion and ion-dipole interactions were characterized. These parameters, which are representative of solute-solvent interactions, adequately described the partitioning of the proteins in each system. It was found that the relative susceptibilities of proteins to solvent dipolarity/polarizability are interrelated with their relative susceptibilities to solvent hydrogen bond acidity and solvent hydrogen bond basicity similarly to those established previously for small nonionic organic compounds.

Parameterization of hydrophobic properties of aqueous polymeric biphasic systems and water-organic solvent systems

Abstract Partition coefficients for a homologous series of dinitrophenylated amino acids with aliphatic side-chains have been determined in two aqueous polymeric Ficoll-dextran 70 and dextran 500-polyethylene glycol 6000 biphasic systems and in the systems formed by n -octanol and the aqueous phases of the above systems. The results afford an estimation of the free energy of transfer of a CH 2 group from one to the other phase of the systems examined. This parameter (Δ g tr CH 2 ) was taken as a measure of the hydrophobic character of an aqueous phase with respect to n -octanol. It was shown that when the partition of a set of homologues in two biphasic systems is correlated according to the known equation ln K i = a ln K o + b , where K i and K o are the partition coefficients for a given solute in the i th system and in the system chosen for reference, respectively, the parameter a is related to the Δ g tr 1 CH 2 and Δ g tr 0 CH 2 by a = Δ g tr 1 CH 2 /Δ g tr 0 CH 2 . This equation was used to determine the hydrophobic character of various organic solvents and that of the aqueous polymeric phases of the aqueous biphasic systems studied, and was found to be valid for comparison of the partition values determined in an aqueous polymeric biphasic system and in the water- n -octanol system. This seems to extend the possibilities of structure-activity relationships studies as the Ficoll-dextran aqueous biphasic system provides as promising a means for their study in biological chemistry as the water- n -octanol system in drug research.

On the Collander equation: Protein partitioning in polymer/polymer aqueous two-phase systems

Distribution coefficients of randomly selected proteins were measured in aqueous two-phase systems (ATPSs) formed by different combinations of Dextran-75 (Dex), Ficoll-70, polyethylene glycol-8000 (PEG), hydroxypropyl starch-100 (PES), and Ucon50HB5100 (Ucon, a random copolymer of ethylene glycol and propylene glycol) at particular polymer concentrations, all containing 0.15M NaCl in 0.01 M phosphate buffer, pH 7.4. Most of the proteins in the PEG-Ucon system precipitated at the interface. In the other ATPSs, namely, PES-PEG, PES-Ucon, Ficoll-PEG, Ficoll-Ucon, and in Dex-PEG and Dex-Ucon described earlier the distribution coefficients for the proteins were correlated according to the solvent regression equation: lnKi=aiolnKo+bio, where Ki and Ko are the distribution coefficients for any protein in the ith and oth two-phase systems. Coefficients aio and bio are constants, the values of which depend upon the particular compositions of the two-phase systems under comparison.

Salt effects on solvent features of coexisting phases in aqueous polymer/polymer two-phase systems☆

The solvatochromic parameters characterizing the solvent dipolarity/polarizability (π*), solvent hydrogen-bond donor acidity (α), and solvent hydrogen-bond acceptor basicity (β) of aqueous media were measured in the coexisting phases of aqueous Dextran-Ficoll, Dextran-Ucon, Dextran-PEG, PEG-Ucon, Ficoll-Ucon, and Ficoll-PEG two-phase systems (ATPS). Ionic composition of each ATPS included 0.15M KCl, 0.15M KBr, 0.15M NaBr, 0.1M Na(2)SO(4), and 0.1M Li(2)SO(4) in 0.01 M sodium phosphate buffer (NaPB), pH 7.4; and 0.01 M and 0.11 M sodium phosphate buffer, pH 7.4. Partition ratios of sodium salts of dinitrophenylated (DNP) amino acids with aliphatic side-chains (glycine, alanine, norvaline, norleucine, and α-amino-n-caprylic acid) were measured in all ATPSs, and the results were evaluated in terms of the differences between the relative hydrophobicity (parameter E) and the electrostatic properties (parameter C) of the aqueous media of the coexisting phases. It was established that parameter E is described by a linear combination of the differences between the solvent dipolarity/polarizability (Δπ*) and between the solvent hydrogen-bond acidity (Δα) of the media in the coexisting phases. Parameter C depends on the phase forming polymer pair and is shown to be described by a linear combination of three parameters: the differences between the solvent hydrogen-bond acidity (Δα) and between the solvent hydrogen-bond basicity (Δβ) of the media in the coexisting phases, and a measure of the effect of a given salt additive on the hydrogen bonds in water. This effect was represented by a parameter (K(b-l)), characterizing the equilibrium between populations of hydrogen bonds with a bent hydrogen bond conformation and with linear hydrogen bond conformation affected by a given salt additive.

Effect of salt additives on partition of nonionic solutes in aqueous PEG–sodium sulfate two-phase system

Partition of 12 nonionic organic compounds in aqueous PEG-8000-Na(2)SO(4) two-phase system was examined. Effects of four salt additives (NaCl, NaSCN, NaClO(4), and NaH(2)PO(4)) in the concentration range from 0.027 up to ca. 1.9 M on binodal curve of PEG-sulfate two-phase system and solute partitioning were explored. It was found that different salt additives at the relatively high concentrations display different effects on both phase separation and partition of various nonionic solutes. Analysis of the results indicates that the PEG-Na(2)SO(4) ATPS with the up to 0.215 M NaCl concentration may be viewed as similar to the ATPS without NaCl in terms of the Collander equations predictive ability of the partitioning behavior of nonionic compounds. All ATPS with each of the salt additive used at the concentration of 0.027 M may be viewed as similar to each other as the Collander equation holds for partition coefficients of nonionic solutes in these ATPS. Collander equation is valid also for the compounds examined in the ATPS with additives of NaSCN and NaClO(4) at the concentrations up to 0.215 M. The observed similarity between these ATPS might be explained by the similar effects of these two salts on the water structure. At concentrations of the salt additives exceeding the aforementioned values, different effects of salt additives on partitioning of various nonionic solutes are displayed. In order to explain these effects of salt additives it is necessary to examine the intensities of different solute-solvent interactions in these ATPS within the framework of the so-called Linear Solvation Energy Relationship (LSER) model.

Relative hydrophobicity of organic compounds measured by partitioning in aqueous two-phase systems

Partitioning of a variety of organic compounds, the majority of which represent therapeutic drugs, was examined in an aqueous dextran-polyethylene glycol (Dex-PEG) two-phase system containing 0.15 M NaCl in 0.01 M sodium phosphate buffer at pH 7.3 and in an octanol-buffer (0.15 M NaCl in 0.01 M sodium phosphate buffer, pH 7.3) system. The possibility of introducing compounds to be partitioned in an aqueous two-phase system with dimethyl sulfoxide, and the effect of this solvent on the solute partitioning was explored. Relative hydrophobicity of the compounds was estimated and expressed in equivalent numbers of methylene units. Comparison of the results obtained for several subsets of compounds in the octanol-buffer and in aqueous Dex-PEG two-phase systems clearly demonstrates the advantage of aqueous two-phase partitioning for the hydrophobicity measurements over partitioning in octanol-buffer system.