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Perspective

In aqua veritas: Indispensable yet mostly ignored role of water in phase separation and membrane-less organelles Boris Y. Zaslavsky, and Vladimir N. Uversky Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.7b01215 • Publication Date (Web): 05 Jan 2018 Downloaded from http://pubs.acs.org on January 5, 2018

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Biochemistry

Perspective In aqua veritas: Indispensable yet mostly ignored role of water in phase separation and membrane-less organelles

Boris Y. Zaslavsky‚ and Vladimir N. UverskyÁ †

‚

Á

Cleveland Diagnostics, Cleveland, OH 44114, USA;

Department of Molecular Medicine and USF Health Byrd Alzheimer's Research Institute,

Morsani College of Medicine, University of South Florida, Tampa, Florida 33612, USA; §

Laboratory of New Methods in Biology, Institute for Biological Instrumentation of the Russian Academy of Sciences, Pushchino, Moscow region 142290, Russia

*Corresponding

authors: BZ, E-mail: [email protected]; Phone: +1 (216) 432-9050 ×111; Fax:

+1 (216) 432-9050; VNU, E-mail: [email protected]; Phone: +1 (813) 974-5816; Fax: +1 (813) 974-7357

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Abstract Despite a common practice of presenting structures of biological molecules on empty background and 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 liquidliquid phase transitions and in the biogenesis of various proteinaceous membrane-less organelles. It is pointed out that changes in the water structure reflected in the changes of its abilities to donate and/or accept hydrogen bond and participate in dipole-dipole and dipole-induced dipole interactions in the presence of various solutes (ranging from small molecules, to synthetic polymers, and to biological macromolecules) might represent a driving force of the liquid-liquid phase separation, define partition 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.

Keywords: solvent properties of water; proteinaceous membrane-less organelle; liquid-liquid phase transition; phase separation; aqueous two-phase system; intrinsically disordered protein; partitioning


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Introduction There is a large number of reviews on the peculiarities of water, its physical and chemical properties, and its role as an active component of various biological processes.1-8 The website authored by M. Chaplin (www.lbsu.ac.uk/water) provides over 3,600 references to the original publications on the water properties. The vast number of studies of water and aqueous solutions of individual compounds did not succeed in resolving one of the most important questions, however. It is well known and generally accepted that water is highly structured liquid. The conflicting viewpoints are that water forms an effectively continuous three dimensional network of hydrogen bonds or that water consists of a mixture of clusters of water molecules with different degrees of hydrogen bonding in an equilibrium (www.lbsu.ac.uk/water). The hydrogen bonds are known to be distorted from their ideal three dimensional structures, and the strengths of these directional bonds vary with the degrees of distortions. When the hydrogen bond is formed between the two water molecules, the redistribution of electrons changes the ability for further hydrogen bonding. The water molecule donating the hydrogen atom increases its ability to accept hydrogen bond, whereas the accepting water molecule increases its ability to donate hydrogen bond, but reduces its ability to accept hydrogen bonds. This electron redistribution thus results in both the cooperativity (e.g. accepting one hydrogen bond encourages the donation of another) and anti-cooperativity (for example, accepting one hydrogen bond discourages acceptance of another) in hydrogen bond formation in water networks (www.lbsu.ac.uk/water). High dynamics of hydrogen bonds in pure water with an average lifetime of about 1 ps complicates consideration of water structure.3 It should also be mentioned that different physical techniques used to probe aqueous environment in the presence of various salts and organic compounds often provide contradictory information ± from the lack of water hydrogen-bond 3 ACS Paragon Plus Environment

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network perturbations outside the first solvation shells of ions9 to induction by electrolytes of long-range orientational order in the hydrogen-bond network of bulk water.10 We suggest that changes in the abilities of water in aqueous solutions to donate and/or accept hydrogen bond, and its ability to participate in dipole-dipole and dipole-induced dipole interactions are the changes that may be interpreted as indications of rearrangement of the hydrogen-bond network of water in the solution and may be viewed as indicating changes in the water structure. We also hypothesize that such changes in the water structure are crucial for liquid-liquid phase separation and for the biogenesis and properties of proteinaceous membrane-less organelles (PMLOs).

Solvent properties of aqueous solutions It is well-known that various physicochemical properties of aqueous solutions of different compounds differ from those of pure water and are commonly concentration- and compound nature-dependent. These properties include, for example, solubility of various solutes, dielectric properties, surface tension, water activity, osmotic coefficient, etc. It has been found recently that these properties are linearly interrelated for aqueous solutions of the randomly chosen compounds at same concentrations over large concentration range.11 As an example, osmotic coefficients, relative viscosities, and relative permittivities of aqueous solutions of glucose with concentrations ranging from 0 to 2.6 M are perfectly linearly interrelated and are qualitatively similar to the same properties in solutions of NaClO4 over the salt concentrations varied from 0 to 6.0 M. Similar linear interrelationship was established for potentiometric activity of HCl in aqueous solutions of urea and relative viscosities and osmotic coefficients of these solutions over the urea concentrations varied from 0 to 1.8 m (m ± molality).11 The only


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mutual attribute of these solutions of glucose, NaClO4, and urea is the aqueous nature of the solvent. In order to explain the above interrelationships, we need to consider what solvent features of aqueous solutions may be used to describe the interrelated properties. Solvent properties of different organic solvents (and water) may be characterized using the so-called solvatochromic comparison method.12-14 This method is based on using various solvatochromic dyes sensitive to different solvent features. The solvatochromic dyes are those that respond to alterations in the solvent media by changed absorption spectra. There are dyes sensitive to the so-called solvent dipolarity/polarizability, S*, or the ability of the solvent to participate in dipole-dipole and dipole-induced dipole interactions. The other type of dyes are sensitive to the solvent hydrogen bond acceptor basicity, E, and the third type ± sensitive to the solvent hydrogen bond donor acidity, D. The latter solvent feature characterizes the ability of the solvent to serve as the hydrogen bond donor, and the former ± as the hydrogen bond acceptor. It should be stressed that according to Ab Rani et al.,15 the particular value of a given solvent feature has no fundamental physical meaning and may depend on the solvatochromic dye used in the study. However, although it is not a fundamental physical property of a solvent, it serves as a guide to the effect of the solvent on solute species that are sensitive to interactions with the solvent.15 It should also be mentioned that the solvatochromic shift of the spectra of a given dye is related to the difference between the free energy of excited and ground states of the dye, and it is generally unknown if the observed solvent effects are generated by the interactions of the excited or ground states of the dye or both, and hence the given solvent feature is not directly related to the free energy of the dye-solvent interaction. In the studies of a Hofmeister series of sodium salts,16 osmolytes,17 and nonionic polymers,18, 19 it has been shown that all these compounds in their individual solutions induce 5 ACS Paragon Plus Environment

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changes in the aforementioned solvent features of water in the compound natures and concentration specific manner. Furthermore, all physicochemical properties of aqueous solutions of various compounds, such as water activity (which is defined as the ratio of the vapor pressure of water in a material to the vapor pressure of pure water at the same temperature), osmotic coefficient (that characterizes the deviation of a solvent from ideal behavior), relative permittivity (that is the factor by which the electric field between the charges is decreased relative to vacuum), relative viscosity (which is the ratio of the viscosity of a solution to the viscosity of the solvent), surface tension (is the elastic tendency of a fluid surface, which tends to minimize surface area and is caused by the attraction of the molecules in the surface layer by the bulk of the liquid), and solubility (that characterizes the ability of a solute to dissolve in a solvent) of various solutes in these solutions were shown to be described by linear combination of two solvent features of water, dipolarity/polarizability, S*, and hydrogen bond donor acidity, D.11 Figure 1 illustrates this idea by showing a series of plots correlating various properties of aqueous solutions of different compounds with these two solvent features of water, dipolarity/polarizability, S*, and hydrogen bond donor acidity, D It has been demonstrated recently that the crowding effects on stability of proteins and nucleic acids in cells generally simulated by in vitro experiments with nonionic polymers and/or osmolytes may be quantitatively described by the effects of these polymers and osmolytes on the aforementioned solvent features of water.18 This is an important observation, especially in the light of the macromolecular crowding theory introduced to reflect the fact that different biological systems are characterized by the high overall concentrations of biological macromolecules (such as proteins, nucleic acids and polysaccharides) that may occupy up to 40% of the cellular volume.20-30 6 ACS Paragon Plus Environment

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Biochemistry

Figure 1. Relationships between different physicochemical properties of aqueous solutions of various compounds and solvent properties of water in the solutions: A. Relationship between surface tension of sucrose solutions (concentration range 0 ± 1.8 M) and the solvent hydrogen 7 ACS Paragon Plus Environment

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bond donor acidity, ., of water; B. Relationship between the relative permittivity of glucose solutions (concentration range 0 ± 3.0 M) and the solvent dipolarity/polarizability, Œ*, and hydrogen bond donor acidity, ., of water; C. Relationship between the relative viscosity of urea solutions (concentration range 0 ± 4.0 M) and the solvent dipolarity/polarizability, Œ*, and hydrogen bond donor acidity, ., of water; D. Relationship between the osmotic coefficients of glucose solutions (concentration range 0 ± 3.0 M) and the solvent dipolarity/polarizability, Œ*, and hydrogen bond donor acidity, ., of water; E. Relationship between the water activity in solutions of polyethylene glycol (PEG-600) with molecular weight 600 (concentration range ~5.0 ± ~40.0 %wt.) and the solvent dipolarity/polarizability, Œ*, and hydrogen bond donor acidity, ., of water.

It was hypothesized that one of the consequences of crowded environment is the decrease in the space available to a query macromolecule due to the excluded volume effects induced by other biological macromolecules, and that these excluded volume effects might influence different properties of macromolecules.31-42 However, the aforementioned ability of polymers and osmolytes to affect stability of proteins and nucleic acids by introducing changes in the solvent features of water18 indicates that the excluded volume effect, being a cornerstone of the theoretical views on crowding phenomena, is an oversimplification, and that the roles of excluded volume in the crowding effect are likely to be overestimated.18 It seems quite possible that proteins creating crowding conditions in vivo may generate these effects also by altering solvent properties of aqueous media. One might ask a question on how far from the protein surface the aforementioned distortions of the solvent properties can propagate (or, in other words, what types of water (bulk water or hydration shell water) are assessed by the solvatochromic comparison method). To answer this question, one should keep 8 ACS Paragon Plus Environment

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in mind that due to the presence of high concentrations of biological macromolecules (nucleic acids, polysaccharides, proteins, ribonucleoproteins, etc.) inside the cells (which can collectively reach to 80±400 mg/ml26, 27, 43 and can occupy 5±40% of cellular volume21), a highly crowded environment is created characterized by very limited amount of the available space and considerably restricted amounts of free water.20, 22, 43-46 As a result, macromolecules in the cytoplasm or nucleoplasm are located in close proximity to each other, with the average distance between them being around 1 nm.5 This is equivalent to just 3 to 4 molecular layers of water.5 As a result, biological macromolecules inside the cell are characterized by the collective hydration, which, for proteins, occurs over the distance of 3-4 nm.47 In other words, based on the classic solvation theory, water inside the cell cannot be considered as bulk-like,5 being definitely characterized by noticeable changes in its solvent properties. Studies of the effects of proteins on the solvent features of water are complicated by that (a) proteins are generally predisposed to binding of aromatic organic compounds, such as solvatochromic dyes, and (b) proteins with high content of tyrosine, tryptophan and phenylalanine display strong absorption at 280 nm and this peak may mask the solvatochromic bands of some dyes, such as p-nitrophenol and p-nitroanisole with maximum wavelength positions in pure water close to 317 nm. The experimental approach used to verify that a given protein does not directly interact with the solvatochromic dyes is described in refs.48, 49 For example, the data reported for human heat shock protein HSPB6 show that its concentration effects on the solvent features of water are quite significant and noticeably exceed those displayed by the majority of nonionic polymers examined so far.49 Additional data for several proteins supporting these observations have been obtained and are currently in preparation for publication. One should note though that the number of proteins examined in regard to their 9 ACS Paragon Plus Environment

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effects on the solvent features of aqueous media is very limited and clearly insufficient to make any general conclusion. Once the large number of proteins will be examined, however, it may be possible to explore if the function of numerous protein chaperons in regard to polypeptides folding regulation, for example, may be realized not only by specific protein-chaperone interactions but via the effects of chaperons on the solvent features of aqueous media known to be responsible for proper folding of proteins.

Phase separation in aqueous media Phase separation in water is a long known phenomenon. It has attracted attention in biology recently because of the (re)discovery of the presence of numerous and very diverse membrane-less organelles (PMLOs) in living cells.50-58 The diversity of such PMLOs is illustrated by Figure 2 that represents a realm of these cellular compartments found in eukaryotic and bacterial cells. Detailed description of eukaryotic PMLOs and their illustrative examples are given elsewhere.59 In eukaryotic cells, cytoplasm contains stress granules (SGs),60 centrosomes,61 processing or P-bodies,62 neuronal RNA granules,63 and germline P-granules (germ cell granules or nuage).64, 65 In the nucleosome, one can find nucleoli,66 nuclear pores,67 chromatin,68 histone locus bodies (HLBs),69 Cajal bodies (CBs),70 nuclear gems or Gemini of coiled of Cajal bodies,71, 72 nuclear stress bodies (nSBs),73, 74 cleavage bodies,75 nuclear speckles or interchromatin granule clusters,76 Oct1/PTF/ transcription (OPT) domains,77 paraspeckles,78 polycomb bodies (PcG bodies),79 perinucleolar compartment (PNC),80 Sam68 nuclear bodies (SNBs),80 and PML oncogenic domains (PODs).81 In the chloroplasts and mitochondria, there are the chloroplast stress granules (cpSGs)82 and mitochondrial RNA granules,83 respectively.


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Figure 2. Schematic representation of the multitude of cytoplasmic, nuclear, mitochondrial, and chloroplast PMLOs in eukaryotes and bacterial PMLOs.

Finally, the potential candidates for the bacterial PMLOs are given by the cell poles found in the rod-shaped bacteria84 and the bacterial septal ring.85 Despite their remarkable variability, the functional roles of many membrane-less organelles remain an open question. However, the phase separation in water is being considered as an important and general mechanism of regulation of a variety of biological functions,51-53, 56 diseases,50 and aging.57


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Although it is tempting to draw connections between the physical chemistry presented in this perspective and biological systems, it is currently impossible to use the aforementioned physicochemical approaches to the PMLOs discussed here. So far, the corresponding data were obtained for the solutions of just four individual proteins (one reported in ref.49 and three other proteins to be reported soon [manuscript in preparation]). These analyses indicated that the effects of the analyzed proteins on the solvent features of water exceed those already reported for nonionic polymers.18 We hope that it will be possible to make such connections in the future, when the particular macromolecules (proteins, RNA, and DNA) responsible for phase separation will be identified and their effects on the aforementioned solvent features will be investigated. Even when the identity of phase-forming components is known, the challenges are: (a) currently, relatively large quantities of each individual component are needed for the analysis of the concentration effects of such component on the solvent features of water and for the experimental prove that this component does not bind the solvatochromic dyes used in the measurements; and (b) the analysis of phase separation in simple mixture of phase forming components may require the presence of certain additives present in cytoplasm or nucleus, and these additives should also be proven to be inert and not to bind the solvatochromic dyes. The mechanisms of phase separation commonly considered (e.g., see refs.50-58) are based on the Flory-Huggins theory.86, 87 The original Flory-Huggins theory for polymer solutions88 was first applied to polymer mixtures in a common solvent by Scott89 and Tompa.90, 91 Detailed analysis of the theory is beyond the scope of the present discussion, but it is important to note that the Flory-Huggins theory is based on the van der Waals model for the intermolecular interactions. The theory has been developed by Flory for non-polar polymer systems,88 and it was particularly stressed by Tompa90 that the model should not be used to represent mixtures of


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polar components, where the energy of intermolecular interactions may depend on the mutual orientations of the molecules. As an example, about 30 years ago, the Flory-Huggins model was used quite successfully in ref.92 to describe phase diagrams of aqueous two-phase systems formed by two polymers in the presence of various salt additives, urea, etc. The most important result of this analysis was that changes in the temperature or presence of different additives that were altering phase diagram quite significantly did not change the polymer-polymer interaction, but affected each phase-forming polymer-water interactions.92 The major issue, however, is that even when Flory-Huggins model fits the experimental phase diagram very satisfactory, it does not provide any insight into mechanism of phase separation, does not have a predictive power (i.e.; does not enable one to predict the probability of the phase separation occurrence), and does not indicate what independent experimental measurements are necessary for making such a prediction. That is why the attempts to use the Flory-Huggins theory for the explanation of phase separation in aqueous mixtures of macromolecules are doomed to fail. One should keep in mind that the Flory-Huggins theory considers the reason for phase separation to be the repulsive interactions between the two different macromolecules within the inert solvent playing the role of diluent of the number of these interactions. However, in water, macromolecules have particular hydration shells and interact via these shells, and therefore water cannot be viewed as an inert solvent. As a direct experimental evidence of the active role of water in phase separation, it has been shown, for example, that dextran and polyvinyl alcohol are completely miscible in the absences of water and separate into two phases in aqueous mixture (see ref.,93 pp.141-147]. It should be mentioned that the same conclusions may be drawn in regard to other currently existing theoretical treatments of phase separation in water, e.g. based on the virial expansion model (see in ref.93), or excess Gibbs energy model.94 The two possible mechanisms of phase


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separation in aqueous solutions of macromolecules have been briefly discussed by Aumiller and Keating.95 One type of phase separation includes polymer or complex of oppositely charged polymers separating into one polymer-enriched phase and polymer(s) depleted phase. The other, more biologically relevant type, includes separation of the mixture of two non-associative polymers or a single polymer and a salt into two aqueous phases each enriched in one of the two components.93, 96-99 It should be mentioned that in aqueous mixtures of multiple different polymers, phase separation may result in the formation of multiple aqueous phases. The largest number of 18 such separated phases was reported by Albertsson in a mixture of 6 polymers including Dextran sulfate, Dextran, and four different hydroxypropyl dextrans with various degrees of substitution (see ref.,96 pp. 14-15). The list of over 300 phase-separated systems forming from two to six aqueous phases in mixtures of various polymers and surfactants was reported by Mace et al.100 Such systems may be used to form stable step-gradients in density in water with small differences between the density of the phases (about 0.001 g/cm3) and have been demonstrated to separate nanoparticles,101 different forms of erythrocytes,102, 103 and isolate the reticulocyteenriched fraction from blood.104 More often, much simpler aqueous two-phase systems (ATPSs) are studied and applied for various biotechnological purposes. The components in ATPSs formed by two compounds commonly include two polymers, single polymer and salt93, 96-99 or surfactant,105, 106 two different surfactants,107 or ionic liquids.108-110 There are also ATPSs formed by proteins and polysaccharides.111-113 The ATPSs formed in mixtures of two polymers or a single polymer and a salt have been explored and characterized to much better degree. However, regardless of what particular two polymers or a single polymer and a salt is used to form an ATPS, it is commonly 14 ACS Paragon Plus Environment

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characterized by construction of the phase diagram, where the compositions of the two coexisting phases are presented for various overall compositions of mixtures of two polymers (or a single polymer and salt) in water as illustrated in Figure 3.

Figure 3. An example of a typical phase diagram. Ao, A1, and A2 represent the total compositions of three different systems lying on the same tie-line with different volume ratios and the same compositions of the coexisting phases represented by points B (bottom phase) and T (top phase). Point D represents the total composition of the system lying on the different tieline. C ± critical point.


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The concentrations of two phase forming components are plotted on the axes. All compositions below the curved line (binodal line) do not induce phase separation, while those above the binodal line do. Any mixture of polymers with composition above the binodal line separates into two phases. Points representing the compositions of the top (T) and bottom (B) phases lie on the binodal curve. The line connecting the compositions of the two coexisting phases and the overall composition of the system is called a tie line. The length of the tie line decreases as the concentrations of the two polymers in a given ATPS are reduced. At a certain point called the critical point (denoted point C in Figure 3) the compositions of the two coexisting phases are identical. It should be noted that any pair of polymers in water may form a vast variety of two coexisting phases of different compositions and properties. On the other hand, if the overall system composition is varied along a given tie-line (points Ao, A1, A2 in Figure 3), all the twophase systems will have the same compositions of the two phases but varied ratio of volumes of the phases (see in Figure 3). Any overall system composition located off a given tie line (point D in Figure 3) forms an ATPS with compositions of the two phases different from those corresponding, e.g. to the system represented by point Ao. It should be stressed that any ATPS with extremely high (or low) ratio of the volumes of the two phases on a given tie-line would have the overall composition very close to those corresponding to point T or point B. Such composition, being in the vicinity of the binodal line, would be extremely sensitive to any changes in such external factors as concentrations of phase forming polymers and additives (salts, co-solutes), temperature, etc. This situation seems to describe formation of PMLOs of very small volumes formed in a relatively large volume of


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cytoplasm or nucleoplasm in the presence of crowded environment and likely fluctuating concentrations of various large and small co-solutes in vivo. An example of several phase diagrams for ATPSs formed by three different polymers, polypropylene glycol (PPG) with molecular weight 425, polyethylene glycol (PEG) with molecular weight 8000, Ucon (copolymer of ethylene glycol and propylene glycol) with molecular weight 3900, and sodium sulfate reported in refs.114-116 are presented graphically in Figure 4A. The concentrations of different polymers needed for phase separation in the aqueous mixtures with various fixed concentrations of Na2SO4 were determined from phase diagrams in Figure 4A at various salt concentrations: 4.0, 5.0, 6.0, and 8.0 %wt. Na2SO4. The determined concentrations of polymers are plotted in Figure 4B against solvent features of water in the individual polymers solutions18, 19 at the corresponding concentrations, and it may be seen that the concentrations of different polymers needed for phase separation in different aqueous mixtures with Na2SO4 are linearly dependent upon the polymers influenced solvent dipolarity/polarizability, S*, and hydrogen bond donor acidity, D, of water. Other similar examples of relationships between the solvent features of water and polymer concentrations needed for phase separation may be found. These and other experimental data (see in ref.93) show that phase separation in aqueous mixtures of two or more components are related to changes in the solvent properties (and possible structure) of water. Similar conclusion may be made from analysis of distribution of various solutes in ATPS.


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Figure 4. A. Binodal lines of polymer-sodium sulfate systems formed in aqueous mixtures of Na2SO4 and polypropylene glycol (PPG-425) with molecular weight 425 (red circles), Ucon (copolymer of ethylene glycol and propylene glycol) with molecular weight 3900 (green circles), and polyethylene glycol (PEG-8000) with molecular weight of 8000 (blue circles). B. The polymer concentration needed for phase separation in aqueous mixtures with different concentrations of Na2SO4 against the solvent dipolarity/polarizability of water and solvent hydrogen bond donor acidity of water in individual solutions of the polymers at the corresponding concentrations. Data for PPG-425, Ucon, and PEG-8000 are shown by red, green and blue symbols. Measurements were conducted at Na2SO4 concentrations of 4%, 5%, 6%, and 8% shown as differently colored circles, inverse triangles, squares, and diamonds, respectively. 18 ACS Paragon Plus Environment

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Distribution of proteins in ATPSs Distribution of compounds (including proteins and nucleic acids) in a given ATPS is characterized by partition coefficient, K, defined as the ratio of the compound concentration in the upper phase to that in the lower phase. Partition coefficient of any compound is commonly increases or decreases with increasing concentrations of polymers forming an ATPS with increasing distance from the critical point on the phase diagram. Logarithm of the compound partition coefficient is linearly related to the difference between the concentrations of a given phase-forming polymer in the two phases.93 Partition coefficients of nonionic compounds in ATPSs formed by various pairs of nonionic polymers with the same ionic composition reported in ref.117 typically vary. As an example, partition coefficient, K, values for p-nitrophenol are ~1.34 in Ficoll-70-PEG-6000 ATPS and 7.53 in PEG-8000-Ucon (both ATPS with the same ionic composition of 0.15 M NaCl in 0.01 M sodium phosphate buffer, pH 7.4 (NaPB). For 4-hydroxyacetanilide, the partition coefficients vary from ~1.237 in Dextran-75-Ficoll-70 ATPS to 4.20 in Ficoll-70-Ucon ATPS (both ATPS of the aforementioned ionic composition).117 Partition coefficients of ionic compounds may vary even more, e.g., partition coefficients of sodium salt of dinitrophenylamino-n-octanoic acid changes from 0.977 in Ficoll-70-PEG-10,000 ATPS to 14.7 in PEG-8000Ucon ATPS.118 For proteins, the partition coefficients might vary much more,119 e.g., the K-value of ribonuclease B changes from ~0.01 in Dextran-75-PEG-8000 ATPS to 2.71 in Dextran-75PEG-8000 ATPS, and for human transferrin it changes from ca. 0.01 in Dextran-75-PEG-8000 ATPS to 7.145 in PES (hydroxypropyl starch)-100-Dextran-75 ATPS (all ATPS of the above ionic composition).119


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Regardless of the wide variability of the partition coefficients of solutes determined in different ATPSs formed by various pairs of nonionic polymers, analysis of partition coefficients of small organic compounds and proteins reported in refs.117-123 showed that the partition coefficients of all various compounds from individual amino acids to proteins determined in multiple ATPS of the same ionic composition may be described as: logKij = Ssi'S*j + Bsi'Dj + Asi'Ej + Csicj

(1)

where Kij is the i-th solute partition coefficient; 'Œ* is the difference between the solvent dipolarity/ polarizability of the two phases, 'D is the difference between the solvent HBD acidity of the two phases, 'E is the difference between the solvent HBA basicity of the two phases; c is the difference between the electrostatic properties of the two phases; Ssi, Asi, Bsi, and Csi are constants (i-th solute specific coefficients) quantifying the complementary interactions of the solute with the solvent media in the coexisting phases and representing the relative contributions of these interactions into partition coefficient of the solute; the subscript s designates the solute; the subscript j denotes the ATPS used; the difference for each solvent property is determined as the one between the upper and lower phases. It should be noted that partition coefficient of a compound is affected by the polymer concentrations in any given ATPS as well as the presence of the protein-inert additives, such as TMAO, sorbitol, sucrose, trehalose, etc., but the above Equation 1 is applicable to compounds partitioned in an ATPS of any fixed polymer and ionic composition.124, 125 The constancy of the above solute specific coefficients for a given compound across numerous (up to 20) ATPS formed by various pairs of different nonionic polymers is an explicit evidence of the lack of direct interactions between compounds (including proteins) and nonionic 20 ACS Paragon Plus Environment

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polymers used to form ATPS. As an additional evidence in favor of the above conclusion it was reported in ref.126 that increasing concentration of trimethylamine N-oxide (TMAO) in a given ATPS from 0 to ca. 2.0 M results in dramatic changes in distribution of some proteins. For example, the partition coefficients of human albumin and bovine a-chymotrypsinogen A increase from 0.518 and 2.49 in the TMAO-free ATPS to 3.91 and 7.58 in ATPS containing 1.95 M TMAO respectively. For ribonuclease A the partition coefficient under same conditions changes from 0.885 to 1.506, while for concanavalin A it changes just from 0.235 to 0.252.126 TMAO is well known not to be engaged in direct interactions with proteins, and its concentration effect on the protein distribution is clearly caused by the TMAO effect on the solvent properties of the coexisting phases. The effect of TMAO concentration is compound specific, and it agrees with above Equation 1. The above data show additionally that the protein molecular weight is not of primary if any importance for the protein partition behavior governed by the nature and spatial arrangement of the solvent exposed groups and their interactions with the aqueous media in the two phases.126 It is possible that this principle underlies distribution of solutes between various membrane-less compartments in vivo in agreement with the hypothesis suggested by Tanford about 40 years ago.127 An attempt to predict partition coefficients of proteins in ATPSs of various polymer and ionic composition based on the structural features of proteins derived from the proteins crystal structures essentially failed.128 Partition coefficients of 10 different proteins in 29 various ATPSs were analyzed in terms of 57 various structural features.128 It was found that three structural features can provide adequate description of the partition coefficients values for all 10 proteins in any given ATPS.128 These structural features, however, varied in different ATPS, indicating that there is no unique set of protein structural features derived 21 ACS Paragon Plus Environment

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from the proteins crystal structures that may describe partition behavior of proteins in various ATPS.128 In other words, these findings indicated that one can find structural features in all cases, but the specific structural feature differs from case to case. It was suggested that the information gained from the crystal structure of a protein cannot provide sufficient insight about the protein responsiveness to different microenvironments, and that analysis of partition behavior in different ATPS may provide different complimentary information.128 The latter suggestion seems to be in agreement with the different sensitivity of various ATPSs to various structural changes in proteins. It has been shown that in order to detect and characterize differences between structures of closely related proteins it is necessary either to design particular ATPS or to use several ATPS with different ionic compositions.129-131 This approach enables one to detect and monitor changes in the posttranslational modifications in a protein biomarker, such as prostate specific antigen (PSA), for improving clinical detection of prostate cancer,132 detect differences between various batches of a protein,133 etc. Single point mutations may be readily detected with this approach too.134 It should be mentioned also that partition of nucleic acids mostly in polymer-salt ATPS with the purpose of isolation of various forms of nucleic acids has been reported by multiple authors.135-144 The ability of sorting the singlewall DNA-wrapped carbon nanotubes by partition in polymer-polymer ATPS should also be noted here.145-147 It should be mentioned that in the case of the ATPSs formed by the charged polymers as likely is the case in PMLOs, the electrostatic interactions of proteins and nucleic acids with the phase-forming components may play an important role. Hence, it is probable that all types of interactions between biological macromolecules and aqueous media and between the 22 ACS Paragon Plus Environment

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macromolecules themselves are important for their distribution between cytoplasm or nucleus and PMLOs. It was shown that distribution of proteins and drugs in different ATPSs of the same ionic composition and between human blood and various tissues may be described by the similar linear three-dimensional relationship between logarithms of partition (distribution) coefficients of the compounds.148 Analysis of such relationship enables one to readily detect compounds engaging in direct interactions with components of the systems/tissues under comparison. It seems possible to apply this approach to PMLOs in some future, once the information related to the concentrations of various components in these organelles and cytoplasm or nucleoplasm is available.

30/2V LQWULQVLF GLVRUGHU DQG SURSHUWLHV RI ZDWHU Analysis of multiple individual cases of proteins associated with PMLOs149-156 and several systematic studies and reviews59, 152, 153, 157-166 revealed that such proteins typically contain significant levels of intrinsic disorder, suggesting that lack of unique 3D structure might represent an important prerequisite for a protein to undergo biological liquid-liquid phase transition (LLPT) leading to the formation of a PMLO. To illustrate the highly disordered nature of proteins associated with PMLOs, Figure 5 represents the results of the systematic analysis of the overall disorder levels in proteins found in various human PMLOs.166 Several reasons for such critical association between intrinsic disorder and the ability of a protein to be related to the PMLO biogenesis were emphasized. Some of these reasons are listed below:


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Figure 5. Evaluation of the overall disorder levels in human proteins associated with PMLOs. Spread of the protein-average disorder scores in individual PMLOs evaluated by PONDR® VSL2 (black bars), PONDR® VLXT (red bars) and PONDR® FIT (green bars) is shown. Bars represent mean protein-average disorder scores in corresponding PMLOs, whereas error bars reflect the corresponding standard deviations. Plot is based on data reported in ref.166

1) Eukaryotic cells are characterized by high levels of intrinsically disordered proteins (IDPs) or hybrid proteins containing ordered domains and intrinsically disordered 24 ACS Paragon Plus Environment

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protein regions (IDPRs).167-169 As a result, cellular concentrations of some IDPs and hybrid proteins can be high, and many IDPs/IDPRs are characterized by high binding promiscuity.170 This is an important consideration since in aqueous mixtures of polymers, proteins, polysaccharides, and protein-nucleic acid combinations, phase separation typically occurs when a concentration thresholds of each macromolecule is reached;113, 171 2) IDPs/IDPRs are characterized by high conformational flexibility, structural heterogeneity,172, 173 and ability to preserve high levels of disorder in bound forms.174176

These features (especially high conformational flexibility and ability to preserve it in

the bound state) are crucial for the PMLO fluidity; 3) IDPs/IDPRs are able to be involved in multiple specific, but weak interactions; i.e., the type of interactions which is crucial for the creation of signaling complexes and PMLOs. This can be exemplified by the ability of IDPs/IDPRs to be engaged in the polyelectrostatic interaction, where instead of presenting discrete charges, rapidly interconverting and diverse conformers existing in the conformational ensembles of IDPs/IDPRs create mean electrostatic fields that are utilized in polyelectrostatic attraction.177, 178 TKLV IHDWXUH RI ,'3 ,'35V L H WKHLU FDSDELOLW\ WR ³SOD\ VWDFFDWR´ as one of the interaction modes) is important for the PMLO formation, since LLPT is expected to be driven by weak multivalent (see below) interactions;59, 165 4) IDPs/IDPRs often have sequences enriched in repeated elements and in some specific residues. As a result, many IDPs/IDPRs can be engaged in multivalent interactions due to the abundant presence of repetitive units in a form of alternating oppositely charged blocks of residues, or in a form of the repetitive donor-acceptor (or ligand-receptor)


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units connected by flexible linkers.179-188 Again, this structural/sequence modularity and multivalency of IDPs/IDPRs define their ability to undergo LLPTs leading to the formation of a protein-rich phase (PMLOs) and a protein-poor phase;59, 165 5) The lack of fixed structure in IDPs/IDPRs defines an ease with which these proteins and regions can be subjected to various posttranslational modification (PTMs),189-191 which, in their turn, might represent an important means for the regulation of PMLOs;95 6) IDPs/IDPRs are commonly found in various human diseases,192-199 where they are often mutated, with pathological mutations affecting PTM sites and other functionally important regions of these proteins, including their protein-protein interaction sites.200, 201

In application to PMLOs, several recent publications indicated that for some

proteins capable of phase separation, the disease-causing mutations can promote transition of liquid droplets to a more solid (gel-like) phase containing amyloid-like fibrils, suggesting that the mutation-triggered formation of mature solid-like droplets can be related to pathology (reviewed in ref.202); 7) The lack of structure in IDPs/IDPRs involved in LLPTs might also define the stability and resilience of the phase-separated droplets to changes in environment, since ³WDNLQJ a few or even just one brick can lead to the collapse of the wall, whereas a bowl of noodles remains a bowl of noodles even after many noodles are eaten´ 59, 165 8) High sensitivity of IDPs/IDPRs to small changes in their environment (where any changes in the IDP/IDPR surroundings may have very strong effects on the IDP/IDPR structure and function)172 represents important means for regulation of the PMLO biogenesis.


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Biochemistry

The aforementioned reasons for the preferential involvement of IDPs and hybrid proteins with IDPRs in LLPTs and PMLO formation are obviously rooted in the physicochemical properties of their amino acid sequences and easiness of modulation of these properties by various PTMs and mutations, as well as in specific structural features of IDPs/IDPRs, and peculiarities of their conformational behavior and functionality. In addition to these sequence/structure/function-based reasons, one should also consider specificity and uniqueness of the effects of IDPs/IDPRs on properties of water. In fact, because of the lack of specific 3D structure, extremely dynamic nature, and an extended conformation, almost all parts of an IDP/IDPR are highly solvent-exposed. This suggests that hydration of IDPs/IDPRs is principally different from the hydration of ordered globular proteins and domains. The validity of this hypothesis was demonstrated by the solid-state NMR relaxation measurements, which revealed that IDPs were able to bind significantly larger amounts of water than globular proteins.203, 204 Based on the results of the wide-line 1H-NMR intensity and differential scanning calorimetry measurements it has been also concluded that in addition to being characterized by a higher hydration capacity than globular proteins, IDPs can bind larger amounts of charged solute ions.205 Also, wide-line 1H-105 VSHFWURVFRSLF DQDO\VLV RI WKH ³VROLGVWDWH´ O\RSKLOL]HG RU SRO\FU\VWDOOLQH SURWHLQV UHYHDOHG WKDW WKH HQHUJ\ GLVWULEXWLRQ RI WKH potential barriers hindering the motion of the water molecules in IDPs is markedly different from those determined for globular proteins, indicating that globular proteins and IDPs are characterized by different modes of protein±water interactions.206 Finally, transition of an IDP IURP DQ H[WHQGHG WR D PRUH FROODSVHG ³JOREXODU´ IRUP RU IRUPDWLRQ RI DP\ORLGRJHQLF oligomers) might result in dramatic changes of the dynamic behavior of water molecules residing in the vicinity of the polypeptide chain.207-209 In fact, application of the time-resolved 27 ACS Paragon Plus Environment

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fluorescence spectroscopy revealed that water molecules entrapped within such collapsed species of IDPs are characterized by the relaxation time of 1.0-1.4 ns, suggesting profound restrictions in the water mobility within the collapsed IDPs.207-209 In fact, these values are similar to relaxation times measured for the ordered nano-confined water clusters, being more than an order of magnitude slower than the water present on the protein surface and 3 orders of magnitude slower than the relaxation times measured in bulk water.207-209 All these observations indicate that IDPs/IDPRs have profound effects on water properties, and that these effects are different from those conferred by ordered proteins and domains. This suggests that the enhanced capability of IDPs/IDPRs to interact with water and change its solvent properties might represent an additional factor contributing to the exceptional involvement of these proteins/regions in PMLO formation. In other words, changed solvent properties of water in the presence of IDPs/IDPRs might represent a driving force for the biological liquid-liquid phase separation leading to the appearance of various PMLOs. Obviously, further studies are needed to check the validity of this hypothesis.

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Biochemistry

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Biochemistry

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Perspective In aqua veritas: Indispensable yet mostly ignored role of water in phase separation and membrane-less organelles

Boris Y. Zaslavsky‚ and Vladimir N. UverskyÁ †


Perspective - ACS Publications

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