What Can Be Learned from the Partitioning Behavior of Proteins in Aqueous Two-Phase Systems?

This review covers the analytical applications of protein partitioning in aqueous two-phase systems (ATPSs). We review the advancements in the analytical application of protein partitioning in ATPSs that have been achieved over the last two decades. Multiple examples of different applications, such as the quality control of recombinant proteins, analysis of protein misfolding, characterization of structural changes as small as a single-point mutation, conformational changes upon binding of different ligands, detection of protein-protein interactions, and analysis of structurally different isoforms of a protein are presented. The new approach to discovering new drugs for a known target (e.g., a receptor) is described when one or more previous drugs are already available with well-characterized biological efficacy profiles.

Effect of trimethylamine-N-oxide on the phase separation of aqueous polyethylene glycol-600-Dextran-75 two-phase systems.

The emergence of phase separation in both intracellular biomolecular condensates (membrane-less organelles) and in vitro aqueous two-phase systems (ATPS) relies on the formation of immiscible water-based phases/domains. The solvent properties and arrangement of hydrogen bonds within these domains have been shown to differ and can be modulated with the addition of various inorganic salts and osmolytes. The naturally occuring osmolyte, trimethylamine-N-oxide (TMAO), is well established as a biological condensate stabilizer whose presence results in enhanced phase separation of intracellular membrane-less compartments. Here, we show the unique effect of TMAO on the mechanism of phase separation in model PEG-600-Dextran-75 ATPS using dynamic and static light scattering in conjunction with ATR-FTIR and solvatochromic analysis. We observe that the presence of TMAO may enhance or destabilize phase separation depending on the concentration of phase forming components. Additionally, the behavior and density of mesoscopic polymer agglomerates, which arise prior to macroscopic phase separation, are altered by the presence and concentration of TMAO.

Solvent polarity and hydrophobicity of solutes are two sides of the same coin.

The hydrophobicity of solutes measures the intensity of a solute's interaction with aqueous environment. The aqueous environment may change with its composition, leading to changes in its solvent properties largely characterized by polarity. As a result, the relative hydrophobicity of a solute is a function of the solute structure and the properties of the water-based solvent determined by the total composition of the aqueous phase. This aspect is commonly ignored by medicinal chemists even though it is essential for drug distribution between different biological tissues. Partitioning of solutes in aqueous two-phase systems provides the relative hydrophobicity estimates for any water-soluble compounds that can be used to improve predictions of the toxicity and other biological effects of these compounds.

Biological soft matter: intrinsically disordered proteins in liquid-liquid phase separation and biomolecular condensates.

The facts that many proteins with crucial biological functions do not have unique structures and that many biological processes are compartmentalized into the liquid-like biomolecular condensates, which are formed via liquid-liquid phase separation (LLPS) and are not surrounded by the membrane, are revolutionizing the modern biology. These phenomena are interlinked, as the presence of intrinsic disorder represents an important requirement for a protein to undergo LLPS that drives biogenesis of numerous membrane-less organelles (MLOs). Therefore, one can consider these phenomena as crucial constituents of a new IDP-LLPS-MLO field. Furthermore, intrinsically disordered proteins (IDPs), LLPS, and MLOs represent a clear link between molecular and cellular biology and soft matter and condensed soft matter physics. Both IDP and LLPS/MLO fields are undergoing explosive development and generate the ever-increasing mountain of crucial data. These new data provide answers to so many long-standing questions that it is difficult to imagine that in the very recent past, protein scientists and cellular biologists operated without taking these revolutionary concepts into account. The goal of this essay is not to deliver a comprehensive review of the IDP-LLPS-MLO field but to provide a brief and rather subjective outline of some of the recent developments in these exciting fields.

Mechanism of Phase Separation in Aqueous Two-Phase Systems.

Liquid-liquid phase separation underlies the formation of membrane-less organelles inside living cells. The mechanism of this process can be examined using simple aqueous mixtures of two or more solutes, which are able to phase separate at specific concentration thresholds. This work presents the first experimental evidence that mesoscopic changes precede visually detected macroscopic phase separation in aqueous mixtures of two polymers and a single polymer and salt. Dynamic light scattering (DLS) analysis indicates the formation of mesoscopic polymer agglomerates in these systems. These agglomerates increase in size with increasing polymer concentrations prior to visual phase separation. Such mesoscopic changes are paralleled by changes in water structure as evidenced by Attenuated Total Reflection-Fourier Transform Infrared (ATR-FTIR) spectroscopic analysis of OH-stretch bands. Through OH-stretch band analysis, we obtain quantitative estimates of the relative fractions of four subpopulations of water structures coexisting in aqueous solutions. These estimates indicate that abrupt changes in hydrogen bond arrangement take place at concentrations below the threshold of macroscopic phase separation. We used these experimental observations to develop a model of phase separation in aqueous media.

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.

Hydrogen Bond Arrangement Is Shown to Differ in Coexisting Phases of Aqueous Two-Phase Systems.

Analysis by attenuated total reflection-Fourier transform infrared spectroscopy shows that each coexisting phase in aqueous two-phase systems has a different arrangement of hydrogen bonds. Specific arrangements vary for systems formed by different solutes. The hydrogen bond arrangement is shown to correlate with differences in hydrophobic and electrostatic properties of the different phases of five specific systems, four formed by two polymers and one by a single polymer and salt. The results presented here suggest that the arrangement of hydrogen bonds may be an important factor in phase separation.

Linear Relationships between Partition Coefficients of Different Organic Compounds and Proteins in Aqueous Two-Phase Systems of Various Polymer and Ionic Compositions.

Analysis of the partition coefficients of small organic compounds and proteins in different aqueous two-phase systems under widely varied ionic compositions shows that logarithms of partition coefficients for any three compounds or proteins or two organic compounds and one protein are linearly interrelated, although for protein(s) there are ionic compositions when the linear fit does not hold. It is suggested that the established interrelationships are due to cooperativity of different types of solute-solvent interactions in aqueous media. This assumption is confirmed by analysis of distribution coefficients of various drugs in octanol-buffer systems with varied ionic compositions of the buffer. Analysis of the partition coefficients characterizing distribution of variety of drugs between blood and different tissues of rats in vivo reported in the literature showed that the above assumption is correct and enabled us to identify the tissues with the components of which the drug(s) may engage in presumably direct interactions. It shows that the suggested assumption is valid for even complex biological systems.

Interfacial tension and mechanism of liquid-liquid phase separation in aqueous media.

The organization of multiple subcellular compartments is controlled by liquid-liquid phase separation. Phase separation of this type occurs with the emergence of interfacial tension. Aqueous two-phase systems formed by two non-ionic polymers can be used to separate and analyze biological macromolecules, cells and viruses. Phase separation in these systems may serve as the simple model of phase separation in cells also occurring in aqueous media. To better understand liquid-liquid phase separation mechanisms, interfacial tension was measured in aqueous two-phase systems formed by dextran and polyethylene glycol and by polyethylene glycol and sodium sulfate in the presence of different additives. Interfacial tension values depend on differences between the solvent properties of the coexisting phases, estimated experimentally by parameters representing dipole-dipole, ion-dipole, ion-ion, and hydrogen bonding interactions. Based on both current and literature data, we propose a mechanism for phase separation in aqueous two-phase systems. This mechanism is based on the fundamental role of intermolecular forces. Although it remains to be confirmed, it is possible that these may underlie all liquid-liquid phase separation processes in biology.

Driving Forces of Liquid-Liquid Phase Separation in Biological Systems.

Analysis of liquid-liquid phase separation in biological systems shows that this process is similar to the phase separation observed in aqueous two-phase systems formed by nonionic polymers, proteins, and polysaccharides. The emergence of interfacial tension is a necessary condition of phase separation. The situation in this regard is similar to that of phase separation in mixtures of partially miscible solvents. It is suggested that the evaluation of the effects of biological macromolecules on the solvent properties of aqueous media and the measurement of the interfacial tension as a function of these solvent properties may be more productive for gaining insights into the mechanism of liquid-liquid phase separation than the study of structural details of proteins and RNAs engaged in the process.

Intrinsic Disorder-Based Emergence in Cellular Biology: Physiological and Pathological Liquid-Liquid Phase Transitions in Cells.

The visible outcome of liquid-liquid phase transitions (LLPTs) in cells is the formation and disintegration of various proteinaceous membrane-less organelles (PMLOs). Although LLPTs and related PMLOs have been observed in living cells for over 200 years, the physiological functions of these transitions (also known as liquid-liquid phase separation, LLPS) are just starting to be understood. While unveiling the functionality of these transitions is important, they have come into light more recently due to the association of abnormal LLPTs with various pathological conditions. In fact, several maladies, such as various cancers, different neurodegenerative diseases, and cardiovascular diseases, are known to be associated with either aberrant LLPTs or some pathological transformations within the resultant PMLOs. Here, we will highlight both the physiological functions of cellular liquid-liquid phase transitions as well as the pathological consequences produced through both dysregulated biogenesis of PMLOs and the loss of their dynamics. We will also discuss the potential downstream toxic effects of proteins that are involved in pathological formations.

Stochasticity of Biological Soft Matter: Emerging Concepts in Intrinsically Disordered Proteins and Biological Phase Separation.

At the turn of this century, cardinal changes took place in the perceptions of the structure and function of proteins, as well as in the organizational principles of membrane-less organelles. As a result, the model of the organization of living matter is changing to one described by highly dynamic biological soft matter positioned at the edge of chaos. Intrinsically disordered proteins (IDPs) and membrane-less organelles are key examples of this new outlook and may represent a critical foundation of life, defining its complexity and the evolution of living things.

Effects of sodium chloride and sodium perchlorate on properties and partition behavior of solutes in aqueous dextran-polyethylene glycol and polyethylene glycol-sodium sulfate two-phase systems.

Effects of two salt additives, NaCl and NaClO4, at the fixed concentrations of 0.215 M on the properties of aqueous two-phase systems (ATPSs) formed by dextran (Dex) and polyethylene glycol (PEG), and the effects of NaClO4 at the same concentration on the properties of ATPS formed by PEG and Na2SO4 were examined. The effects of these salt additives on partitioning of 12 small organic compounds and five proteins in the above ATPSs were studied. In each system with a given salt additive, 0.5 M sorbitol, 0.5 M sucrose, and 0.5 M and 1.5 M trimethylamine N-oxide (TMAO) were also used as additives. The results obtained were compared with those reported previously for the Dex-PEG ATPS without salt additives and PEG-Na2SO4 ATPS without salt additives and in the presence of 0.215 M NaCl. It is shown that the differences between the solvent properties of the phases in the systems formed by polymer and salt exceed those observed in the systems formed by two polymers. The three most significant solvent features of the systems are hydrophobic and electrostatic properties and hydrogen bonding donor acidity of the solvent media. Osmolyte additives were found to have a significant effect on the differences between the electrostatic properties of the phases. Analysis of the partition coefficients of 12 organic compounds and five proteins showed that the osmolyte additives may affect the partition behavior of compounds in a compound-specific manner. The relative contributions of different types of interactions of a given compound with aqueous media change in the presence of salt and osmolyte additives. Analysis of the variability ranges of partition coefficient, K, in the systems studied showed that for small organic compounds, the ranges of K-values observed in the PEG-Na2SO4 ATPSs exceed those determined in the Dex-PEG ATPSs quite significantly, whereas for proteins, the range of K-values in Dex-PEG ATPSs exceeded those in PEG-Na2SO4 ATPSs for three proteins, and were very similar for two proteins. This observation supported the notion that the ATPSs formed by two polymers are more suitable for protein analysis than those formed by a single polymer and a salt. The single polymer-salt ATPSs have an advantage for protein isolation/separation.

Effect of an Intrinsically Disordered Plant Stress Protein on the Properties of Water.

Dehydrins are plant proteins that are able to protect plants from various forms of dehydrative stress such as drought, cold, and high salinity. Dehydrins can prevent enzymes from losing activity after freeze/thaw treatments. Previous studies had suggested that the dehydrins function by a molecular shield effect, essentially preventing a denatured enzyme from aggregating with another enzyme. Therefore, the larger the dehydrin, the larger the shield and theoretically the more effective the protection. Although this relationship holds for smaller dehydrins, it fails to explain why larger dehydrins are less efficient than would be predicted from their size. Using solvatochromic dyes to probe the solvent features of water, we first confirm that the dehydrins do not bind the dyes, which would interfere with interpretation of the data. We then show that the dehydrins have an effect on three solvent properties of water (dipolarity/polarizability, hydrogen-bond donor acidity and hydrogen-bond acceptor basicity), which can contribute to the protective mechanism of these proteins. Interpretation of these data suggests that although polyethylene glycol and dehydrins have similar protective effects, dehydrins may more efficiently modify the hydrogen-bonding ability of bulk water to prevent enzyme denaturation. This possibly explains why dehydrins recover slightly more enzyme activity than polyethylene glycol.

The solvent side of proteinaceous membrane-less organelles in light of aqueous two-phase systems.

Water represents a common denominator for liquid-liquid phase transitions leading to the formation of the polymer-based aqueous two-phase systems (ATPSs) and a set of the proteinaceous membrane-less organelles (PMLOs). ATPSs have a broad range of biotechnological applications, whereas PMLOs play a number of crucial roles in cellular compartmentalization and often represent a cellular response to the stress. Since ATPSs and PMLOs contain high concentrations of polymers (such as polyethylene glycol (PEG), polypropylene glycol (PPG), Ucon, and polyvinylpyrrolidone (PVP), Dextran, or Ficoll) or biopolymers (peptides, proteins and nucleic acids), it is expected that the separated phases of these systems are characterized by the noticeable changes in the solvent properties of water. These changes in solvent properties can drive partitioning of various compounds (proteins, nucleic acids, organic low-molecular weight molecules, metal ions, etc.) between the phases of ATPSs or between the PMLOs and their surroundings. Although there is a sizable literature on the properties of the ATPS phases, much less is currently known about PMLOs. In this perspective article, we first represent liquid-liquid phase transitions in water, discuss different types of biphasic (or multiphasic) systems in water, and introduce various PMLOs and some of their properties. Then, some basic characteristics of polymer-based ATPSs are presented, with the major focus being on the current understanding of various properties of ATPS phases and solvent properties of water inside them. Finally, similarities and differences between the polymer-based ATPSs and biological PMLOs are discussed.

Phase equilibria, solvent properties, and protein partitioning in aqueous polyethylene glycol-600-trimethylamine N-oxide and polyethylene glycol-600-choline chloride two-phase systems.

The phase diagram of a new aqueous two-phase system (ATPS) formed by polyethylene glycol with molecular weight 600 (PEG-600) and trimethylamine N-oxide (TMAO) in 0.01 M sodium phosphate buffer (NaPB), pH 7.4, is determined and hydrophobic, electrostatic and other solvent properties of the phases are characterized. The same properties are determined for the ATPS formed by PEG-600 and choline chloride in 0.01 M sodium phosphate buffer (NaPB), pH 7.4. Solvent properties of water (dipolarity/polarizability, hydrogen bond donor acidity, and hydrogen bond acceptor basicity) in aqueous solutions of polypropylene glycol-400 (PPG-400), polyethylene glycol dimethyl ether -250 (PEGDME-250), and choline chloride are determined at different concentrations. The concentrations of the aforementioned polymers, as well as PEG-600 and PEG-1000 required for phase separation in mixtures with choline chloride reported in the literature are analyzed. It is found that the concentrations of polymers needed for phase separation in mixtures with 35%wt. choline chloride are linearly related with water hydrogen bond donor acidity or hydrogen bond acceptor basicity in the individual polymer solutions at given concentrations. Partition behavior of nine proteins was examined in both systems. The partition coefficients of proteins in PEG-600-choline chloride ATPS exceeded those observed in PEG-600-TMAO ATPS from ca. 2 to ca. 75-fold possibly due to the larger difference between the composition of the coexisting phases in the former ATPS. Analysis of partition coefficients in the two ATPS were compared to those reported in Dextran-PEG ATPS, and proteins likely engaged in direct interactions with choline chloride were identified.

In Aqua Veritas: The Indispensable yet Mostly Ignored Role of Water in Phase Separation and Membrane-less Organelles.

Despite the common practice of presenting structures of biological molecules on an empty background and the assumption that interactions between biological macromolecules take place within the inert solvent, water represents an active component of various biological processes. This Perspective addresses indispensable, yet mostly ignored, roles of water in biological liquid-liquid phase transitions and in the biogenesis of various proteinaceous membrane-less organelles. We point out that changes in the structure of water reflected in the changes in its abilities to donate and/or accept hydrogen bonds and participate in dipole-dipole and dipole-induced dipole interactions in the presence of various solutes (ranging from small molecules to synthetic polymers and biological macromolecules) might represent a driving force for the liquid-liquid phase separation, define partitioning of various solutes in formed phases, and define the exceptional ability of intrinsically disordered proteins to be engaged in the formation of proteinaceous membrane-less organelles.

The Single-parameter, Structure-based IsoPSA Assay Demonstrates Improved Diagnostic Accuracy for Detection of Any Prostate Cancer and High-grade Prostate Cancer Compared to a Concentration-based Assay of Total Prostate-specific Antigen: A Preliminary Report.

BACKGROUND: IsoPSA is a serum-based assay that predicts prostate cancer (PCa) risk by partitioning isoforms of prostate-specific antigen (PSA) with an aqueous two-phase reagent. OBJECTIVES: To determine the diagnostic accuracy of IsoPSA in identifying the presence or absence of PCa and the presence of high-grade disease in a contemporary biopsy cohort. DESIGN, SETTING, AND PARTICIPANTS: Multicenter prospective study of 261 men scheduled for prostate biopsy at five academic and community centers in the USA enrolled between August 2015 and December 2016. INTERVENTION: Performance of the IsoPSA assay. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Discrimination power was evaluated using receiver operating characteristic (ROC) analysis. The outcome of the IsoPSA assay was transformed into risk probability using logistic regression. Decision curve analysis (DCA) was used to compare the net benefit of IsoPSA against other clinical protocols. RESULTS AND LIMITATIONS: The overall prevalence was 53% for any PCa and 34% for high-grade PCa. The area under the ROC curve was 0.79 for any cancer versus none and 0.81 for high-grade PCa versus low-grade PCa/benign histology. In this preliminary study, DCA revealed a superior net benefit of IsoPSA against no biopsy, all biopsy, and the modified Prostate Cancer Prevention Trial Risk Calculator 2.0. At a cutoff selected to recommend biopsy, IsoPSA demonstrated a 48% reduction in false-positive biopsies; at a cutoff selected to identity men at low risk of high-grade disease, there was a 45% reduction in the false-positive rate. CONCLUSION: The structure-based IsoPSA assay outperformed concentration-based PSA measurement, and provided a net benefit against other protocols. Once validated, clinical use of IsoPSA could significantly reduce unnecessary biopsies while identifying patients needing treatment. PATIENT SUMMARY: The IsoPSA assay outperformed prostate-specific antigen in predicting the overall risk of prostate cancer and the risk of clinically significant cancer in a preliminary study. The IsoPSA assay could assist in determining the need for prostate biopsy for patients.

Effects of osmolytes on solvent features of water in aqueous solutions.

The solvatochromic solvent features of water (dipolarity/polarizability, π*, hydrogen bond donor acidity, α, and hydrogen bond acceptor basicity, ß) of water have been determined in aqueous solutions of erythritol, glucose, inositol, sarcosine, xylitol and urea with concentrations from 0 to ~3 M and higher. The concentration effects of the osmolytes on the solvent features of water were characterized and compared with those reported previously for sorbitol, sucrose, trimethylamine N-oxide (TMAO), and trehalose. The solvent features of water in solutions of all osmolytes except TMAO and sarcosine were established to be linearly interrelated. It is shown that the concentration effects of essentially all nonionic osmolytes depend on osmolytes' lipophilicity, molecular polarizability, and polar surface area. It is demonstrated that solubility of various compounds in aqueous solutions of glucose, sucrose, sorbitol, and urea of varied concentrations may be described in terms of solvent dipolarity/polarizability of water in these solutions. Surface tension of aqueous solutions of sucrose and sorbitol may also be described in the same terms. The relative permittivity of aqueous solutions of glucose and sucrose may be described in terms of the solvent hydrogen bond donor acidity of water. It is suggested that the effects of nonionic osmolytes on behavior of proteins and nucleic acids in aqueous media may be considered in terms of the altered solvent features of water instead of "nano-molecular crowding" effect.