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Role of solvent properties of water in crowding effects induced by macromolecular agents and osmolytes a

Accepted 00th January 20xx DOI: 10.1039/x0xx00000x www.rsc.org/

b

a

L. A. Ferreira, V. N. Uversky and B. Y. Zaslavsky * Solvent properties of water in aqueous solutions of polyethylene glycols of various molecular weights, L-proline, betaine, and a series of chlorides of varied concentrations are assayed using three solvatochromic dyes. The properties include solvent dipolarity/polarizability, hydrogen bond donor acidity, and hydrogen bond acceptor basicity. These properties are also evaluated in mixtures of two polymers, polymer and osmolyte, and two osmolytes. It is shown that linear combinations of the solvent dipolarity/polarizability and hydrogen bond donor acidity assayed in individual solutions of crowders strongly correlate with effects of the crowders on stability of various proteins and nucleic acids reported in the literature. The solvent properties of water in aqueous mixtures of two macromolecular crowders, two osmolytes, or mixtures of osmolyte and macromolecular crowder vary differently for various solvent properties. The overall effects of the two components in the mixture on a given solvent property of water may be additive, reduced or enhanced depending on the particular composition of the mixture. It is hypothesized that changes in the solvent properties of water are related to changes in the water hydrogen-bonding structuring. It is suggested that the observed crowder-induced changes in the solvent properties of water should be taken into account in theoretical considerations of crowding effects in biological systems.

Introduction It is well established that the high overall concentration of biological macromolecules, which may occupy up to 40% of the cellular volume, is typical for different biological systems.111 Macromolecular crowding is the term generally used to describe an effect of relatively high content of various biological macromolecular components on different properties of proteins and nucleic acids in vivo, such as conformational stability, folding mechanisms, aggregation propensity, interactions with partners, etc.12-23 This effect was initially ascribed to the restriction of the volume accessible to a query protein or nucleic acid by the excluded volume effects induced by macromolecular components, but later additional weak or “soft” nonspecific interactions between the target protein or nucleic acid and surrounding macromolecules have been introduced to explain numerous experimental observations.1223 Two new terms “crowding homeostasis” and “homeocrowding” have been suggested to describe the ability of microorganisms to maintain relatively constant levels of macromolecules.22

Multiple examples of protein-specific responses to 24-26 macromolecular crowding are presented in refs. As an example, it has been demonstrated that stability of a small protein SH3 is decreased in E. coli cells and is identical to that in buffer contrary to the expected protein stabilization in 26 crowded environment. It has been reported that the effects of mixtures of Ficoll-70 and dextran-6 on stability of protein FKBP were non additive 27 relative to those of individual crowding agents. Synergistic effects of mixtures of crowding agents, such as Ficoll-70, dextran-70, and PEG-2000, with calf thymus DNA on refolding of rabbit muscle creatinine kinase have been reported as 28 well. The individual crowding effects of dextran and sucrose on refolding of human muscle creatine kinase were reported to cancel each other in the mixture, in contrast to the 29 expected increase of excluded volume effect. It has been suggested that small compounds may display volume exclusion effects exceeding those of 30 macromolecules. This suggestion apparently agrees with the multiple studies of the effects of small organic osmolytes, such as glucose, sucrose, and trimethylamine N-oxide (TMAO), which, being present at high concentrations, may display crowding effects and hence are sometimes called molecular 31-34 crowders. Therefore, osmolytes are often considered as molecular crowders in the literature due to the fact that any compound, including osmolytes, occupies definite volume in the solution, thereby restricting space that might be occupied by biomacromolecule, such as protein or nucleic acid. We would like to emphasize also that the applicability of the

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excluded volume theory to the description of the effect of 35 osmolytes on protein behaviour was considered in ref., where the effects of high concentrations of three osmolytes, such as glucose, sucrose, and raffinose on the reversible homodimerization of α-chymotrypsin were investigated by sedimentation equilibrium. The authors used two models to explain the osmolyte-mediated stabilization of the alphachymotrypsin homodimer and considered both binding interactions (transfer-free energy analysis) and steric interactions (excluded volume theory). Their analysis revealed that the observed stabilization of the alpha-chymotrypsin homodimer can be reasonably described by the thermodynamic nonideality arising from molecular crowding 35 by osmolytes. These compounds, including osmolytes, may affect stability of proteins similarly to common macromolecular crowding agents, and it is well known that many of them do not interact with many proteins directly. It gradually becomes unavoidable to admit that the excluded volume effect, being a cornerstone of the theoretical views on crowding phenomena, is oversimplified, and its role in the crowding effect is likely overestimated. In fact, the representation of macromolecular crowders and proteins as hard spheres is obviously a very crude approximation. The view that all proteins interact with typical synthetic macromolecular agents, such as polyethylene glycol (PEG), dextran, polyvinylpyrrolidone, or Ficoll, contradicts all the experimental data reported so far in the studies of protein partitioning in aqueous two-phase systems 36, 37 formed by different pairs of these polymers. It should be also emphasized here that the results showing the presence of interactions between the protein and the macromolecular crowding agent are typically based on the models used for the interpretation of the experimental data (this issue is discussed 38 in detail in ref. ). Verma et al. used three different IR-probes to show that the water dynamics remains unchanged in aqueous solutions of PEG dimethyl ether, while there are significant perturbations 39 in in the hydrogen-bonded network of water. Kilburn et al. reported changes in water activity in aqueous solution of RNA induced by polymer crowder PEG-1000 and suggested that 40 these changes might be related to the RNA stabilization. Changes in water activity in the presence of macromolecular crowders are considered also in a work by Nakano and 41 Sugimoto. Results of molecular dynamics simulations demonstrate that different polyols affect the water structure 42 in their aqueous solutions. It was shown that TMAO makes 43 the water hydrogen-bond network relatively stronger, and that TMAO reorganizes the hydrogen-bond network of water 44 45 in a specific way, whereas the data reported in ref. did not confirm this observation. It has been shown, however, that TMAO interactions with water do lead to a more structured 46 hydrogen-bond network. It has been reported also that TMAO and urea in their aqueous mixtures cooperatively 47 enhance the hydrogen-bond structure of water. These effects of various solutes on water structure in their aqueous solutions, however, are not generally taken into account in 48 studies of crowding effects.

We have shown that various water soluble polymers commonly used as macromolecular crowding agents, e.g., PEG, dextran, Ficoll, polyvinylpyrrolidone, etc., affect the solvent 38 features of water in their solutions. These solvent features may be assayed with the solvatochromic dyes responding to changes in their environment by alterations in their UV/Vis 49 spectra. The solvatochromic comparison method pioneered by Kamlet, Taft, and their co-workers is based on using dyes responsive to changes in the solvent properties by shifting their maximum 50-52 wavelength positions. Solute–solvent interactions include a multitude of various interactions of different physicochemical nature, such as dipole–dipole and electrostatic interactions, as well as hydrogen bonding. In order to characterize the ability of various solvents to participate in these interactions, Kamlet, Taft, and their co-workers developed three different scales of solvent features important for their interactions with any given 50-52 One of these scales was suggested to characterize solute. the relative ability of various solvents to participate in the 50 dipole–dipole interactions. The other two scales were constructed to describe the relative abilities of different solvents to participate in hydrogen bonding as acceptors of hydrogen bonds (HBA basicity scale), and in hydrogen bonding 51, 52 as donors of the hydrogen bonds (HBD acidity scale). Various solvents are positioned on each of these scales according to the response they induce in a particular solvatochromic dye (or set of dyes). The response of a dye to its environment is represented by the position of the longest wavelength UV/Vis absorption band of the dye in a given solvent. Various solvent-dependent physicochemical properties of a solute in a given solvent, such as its solubility, equilibrium constant, the logarithm of a gas/solvent or solvent/solvent partition coefficient, may be described as functions of linear combinations of the above solvent features. 49 It should be also noted that according to ref. the position of a given solvent on the chosen scale is not the fundamental property of a solvent, but a guide to the effect of the solvent on solute species that are sensitive to the particular type of interactions with the solvent. Therefore, the solvatochromic comparison method is an arbitrary approach, and the precise values of the responses of the dyes studied have no fundamental physical meaning. Different dyes respond to changes in different properties of the solvent – ability to participate in dipole-dipole interactions (dipolarity/polarizability, π*), ability to donate hydrogen bond (solvent hydrogen bond donor acidity, α), and ability to accept hydrogen bond (solvent hydrogen bond acceptor basicity, β). These solvent features of water change in polymer solutions 38 depending on the polymer nature and concentration. Qualitatively similar changes in the solvent features of water have been also observed in aqueous solutions of various small 53 54 organic osmolytes and inorganic salts. Importantly, it has been shown that various physicochemical properties of aqueous solutions of polymers, salts, and small organic compounds, such as water activity, osmotic coefficient, surface tension, relative permittivity, and viscosity, may be described

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Experimental

PEG8000

Materials and methods

KCl CsCl

LiCl

Osmolytes. L-proline, betaine, D-sorbitol, sarcosine and trimethylamine N-oxide (TMAO) dihydrate were obtained from Sigma-Aldrich and used without further purification. Urea was obtained from USB Corporation. HPLC grade water was used for preparation of all solutions.

MgCl2

Inorganic salts. Potassium chloride, litium chloride, cesium chloride, and magnesium chloride were obtained from SigmaAldrich and used without further purification. All salts were of analytical-reagent grade. HPLC grade water was used for preparation of all solutions. Solvatochromic dyes. The solvatochromic probe 4-nitrophenol (spectrophotometric grade) was purchased from SigmaAldrich and 4-nitroanisole (GC, > 99%) was supplied by Acros Organic (New Jersey, USA). Reichardt’s carboxylated betaine dye, sodium 2,6-diphenyl-4-[4-(4-carboxylato-phenyl)-2,6diphenylpyridinium-1-yl]phenolate, was synthesized according 56 to the procedure reported previously.

Betaine

L-Proline

Polymers. Polyethylene glycol (PEG-300) with molecular weight (MW) of 300, PEG-1000 with MW of 1,000, PEG-8000 with MW of 8,000, Ficoll-70 with an average MW of 70,000, and polyvinylpyrrolidone 40 (PVP-40) with MW of 40,000 were purchased from Sigma-Aldrich (St. Louis, MO, USA). Polyethylene glycol 600 (PEG-600) with MW of 600 was purchased from EMD (Billerica, MA, USA). Dextran-75 with an average MW of 75,000 by light scattering was obtained from USB Corporation (Cleveland, OH, USA). Ucon 50-HB-5100 (Ucon-4000), a random copolymer of 50 % ethylene oxide and 50 % propylene oxide, with MW 3930 was purchased from Dow-Chemical (Midland, MI, USA). HPLC grade water was used for preparation of all solutions.

Conc.

π*

α

β

10 %wt.

1.096 ± 0.003 1.107± 0.001

1.237± 0.003 1.112± 0.001

0.596± 0.001 0.603± 0.001

20 %wt.

1.111± 0.001

1.015± 0.001

0.617± 0.002

30 %wt.

1.115± 0.001

0.923± 0.002

0.635± 0.002

40 %wt.

1.120± 0.001

0.830± 0.001

0.655± 0.001

10 %wt.

1.107± 0.002

1.085± 0.003

0.611± 0.002

20 %wt.

1.110± 0.002

0.987± 0.003

0.627± 0.002

30 %wt.

1.116± 0.002

0.889± 0.002

0.646± 0.002

40 %wt.

1.121± 0.003

0.789± 0.003

0.669± 0.003

10 %wt.

1.107± 0.001

1.072 ± 0.002

0.608± 0.002

20 %wt.

1.110± 0.001

0.974 ± 0.003

0.625± 0.002

30 %wt.

1.114± 0.002

0.875 ± 0.002

0.646± 0.001

40 %wt.

1.120± 0.002

0.772 ± 0.001

0.670± 0.001

0.5 M

1.111± 0.001

1.265± 0.002

0.607± 0.002

1.0 M

1.121± 0.001

1.269± 0.002

0.617± 0.001

1.5 M

1.129± 0.001

1.263± 0.002

0.629± 0.001

2.0 M

1.138± 0.001

1.254± 0.001

0.641± 0.001

3.0 M

1.151± 0.002

1.231± 0.004

0.669± 0.002

0.5 M

1.103± 0.002

1.211± 0.001

0.602± 0.001

1.0 M

1.100± 0.003

1.185± 0.002

0.608± 0.002

1.5 M

1.098± 0.002

1.157± 0.001

0.616± 0.001

2.0 M

1.096± 0.002

1.126± 0.001

0.629± 0.001

3.0 M

1.091± 0.001

1.065± 0.001

0.652± 0.001

0.25 M

1.120± 0.002

1.223± 0.003

0.597± 0.003

0.50 M

1.136± 0.001

1.219± 0.004

0.597± 0.002

0.75 M

1.152± 0.002

1.215± 0.004

0.598± 0.002

1.00 M

1.167± 0.002

1.213± 0.003

0.599± 0.002

0.25 M

1.112± 0.002

1.236± 0.003

0.594± 0.002

0.50 M

1.121± 0.001

1.236± 0.002

0.593± 0.003

0.75 M

1.130± 0.002

1.231± 0.002

0.591± 0.002

1.00 M

1.138± 0.001

1.223± 0.001

0.589± 0.002

0.25 M

1.119± 0.002

1.212± 0.002

0.597± 0.003

0.50 M

1.134± 0.001

1.220± 0.004

0.598± 0.002

0.75 M

1.148± 0.002

1.232± 0.008

0.599± 0.003

1.00 M

1.163± 0.002

1.246± 0.009

0.600± 0.002

0.05 M

1.105± 0.001

1.213± 0.001

0.595± 0.001

0.10 M

1.110± 0.001

1.217± 0.002

0.595± 0.001

0.20 M

1.119± 0.002

1.226± 0.002

0.594± 0.002

0.25 M

1.124± 0.002

1.228± 0.002

0.593± 0.002

0.40 M

1.137± 0.002

1.234± 0.001

0.592± 0.002

0.50 M

1.145± 0.001

1.238± 0.002

0.591± 0.001

0.75 M

1.163± 0.002

1.246± 0.002

0.589± 0.001

a

π* - solvent dipolarity/polarizability; α – solvent H-bond donor acidity; β – solvent H-bond acceptor basicity.

The detailed description of the solvatochromic analysis is presented in the ESI.

Results and Discussion

Methods

Solvent properties of water in solutions of individual compounds

Solvatochromic studies. The solvatochromic probes 4nitroanisole, 4-nitrophenol, and Reichardt’s carboxylated betaine dye were used to determine the solvent dipolarity/polarizability π*, H-bond acceptor (HBA) basicity β, and H-bond donor (HBD) acidity α of the media in the solutions of polymers, osmolytes, and salts as described 38, 53, 54 previously.

The solvent features of water in aqueous solutions of PEGs of different molecular weights, proline, betaine, and several chlorides are listed in Table 1. It should be noted that the PEG molecular weight (from 300 to 10,000) has no impact on the polymer effect on the water dipolarity/polarizability, π*; i.e., the ability of water to participate in dipole-dipole and dipoleinduced dipole interactions (additional data see in ref.38).

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PEG1000

PEG-300

Table 1. Solvent featuresa of water in aqueous solutions of different compounds and inorganic salts at different concentrations.

Compounds

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in terms of the solvent dipolarity/polarizability, π*, and solvent 55 H-bond donor acidity, α. The purpose of this study was to examine the solvent features of water in solutions of some additional crowding agents, inorganic salts, and osmolytes as well as in aqueous mixtures of different macromolecular crowding agents, in mixtures of these agents with TMAO, and in mixtures of TMAO and other osmolytes. The applicability of these data to description of crowding effects reported in the literature is considered.

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13.0

1.13

1.12

Solvent di po

1.23 1.11

larity/polar

1.10

1.09

1.24

330 325

1.20 1.18

320

1.16

315 1.20

Figure 1. Interrelationship between the stability of ribonuclease A expressed as the free energy change for protein unfolding in the 57 presence of guanidinium chloride, ∆Gd, (data from ref. ) and the water dipolarity/polarizability, π*, and water HBD acidity, α, in solutions of TMAO, sorbitol, sarcosine, and betaine at concentrations 0.25 M, 0.50 M, 0.75 M, and 1.0 M (see all the data used in Table S1).

It should be mentioned also that the same polymers affect the water solvent hydrogen bond donor acidity much more significantly than organic osmolytes or inorganic salts (at 53, 54 concentrations of ~1.0 M). The data listed in Table 1 agree with those reported 38, 53, 54 previously and show that nonionic polymers often used as macromolecular crowding agents, organic osmolytes, and inorganic salts affect solvent properties of aqueous media in their solutions in a compound-specific and concentrationdependent manner. Using these data we can now explore if various effects of macromolecular crowding agents and osmolytes often considered as low molecular weight 30 crowders may be described in terms of the solvent properties of water. The guanidinium chloride-induced unfolding of ribonuclease A was examined in solutions of various individual osmolytes (TMAO, sorbitol, sarcosine, and betaine) and their binary 57 mixtures. To see how these data can be interpreted in terms of changes in the solvent properties of water, the free energy 57 change for the protein unfolding ∆Gd values reported in ref. are plotted against the solvent dipolarity/polarizability, π*, and solvent HBD acidity, α, of water in aqueous solutions of the above individual compounds (see Figure 1). It should be mentioned that the π* and α values at the particular 57 concentrations used in ref. were interpolated from the concentration dependences of these properties listed in Table 1 for betaine and for sarcosine, TMAO, and sorbitol reported 53 previously. The relationship shown in Figure 1 may be described as: 2

1.15

1.10

Solve n

izability, π *

∆Gd = 22.7±3.9 + 21.9±2.7π* - 29.4±1.7α

1.14

(1)

N = 17; r = 0.9694; SD = 0.11; F = 221 where ∆Gd is the free energy change for the ribonuclease A unfolding in the presence of different concentrations of guanidinium chloride; π* and α are the solvent dipolarity/polarizability and HBD acidity of water as defined above; N is the number of experimental conditions used;

1.05

t HBD

1.12

1.00

acidit

0.95

y, α

0.90

1.10

Figure 2. Interrelationship between the stability of ubiquitin expressed as the melting temperature, Tm, (data from ref.34) and the water dipolarity/polarizability, π*, and water HBD acidity, α, in aqueous solutions of glucose, dextran-40, and PEG-20,000 at different concentrations (see all the data used in Table S1).

r – correlation coefficient; SD – standard deviation; F – ratio of variance. It should be noted that the ∆Gd values for the binary 57 mixtures of osmolytes estimated in ref. from the incomplete denaturation profiles do not fit the above relationship. The relationship described by Eq. 1 seems to indicate that the ribonuclease A stability in aqueous solutions of various osmolytes is increased due to the osmolytes influence on the aforementioned solvent properties of water because there is a smooth gradual changes of the ribonuclease A stability as a function of the bulk solvent properties, independent of the identity of the solvent additive. The thermal unfolding of ubiquitin was studied in aqueous solutions of glucose, dextran-40, and PEG-20,000 at their 34 different concentrations. The melting temperatures reported 34 ref. at pH 2.0 are plotted in Figure 2 against the solvent dipolarity/polarizability, π*, and HBD acidity, α, of water in aqueous solutions of the aforementioned compounds. It should be mentioned that the π* and α values at the particular 34 concentrations used in ref. were interpolated from the concentration dependences of these properties reported 38, 53 earlier, and for solutions of PEG-20,000, we used the 38 concentration dependences reported for PEG-10,000. The relationship observed in Figure 2 may be described as: Tm = -72.5±16 + 338.8±12.6π* + 16.4±4.4α

(2)

2

N = 10; r = 0.9904; SD = 1.01; F = 359 where Tm is the melting temperature of ubiquitin; all the other parameters are as defined above. 34

It should be noted that the Tm values reported in ref. for the co-solute free solution, pH 2.0 and for the solutions of 0.1 M and 0.2 M KCl do not fit the relationship. It should be 34 mentioned also that solutions of dextran-40 in ref. have been prepared in grams per litre of water, and from our experience it is a questionable procedure for preparation of aqueous solutions of highly hydrated polymers, such as dextran or Ficoll, and should always be followed by checking the final concentration of the solution by gravimetrical analysis using

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Molecular BioSystems Accepted Manuscript

1.22

10.0

335

ab ilit y, π*

10.5

340

Ubiquitin meltin

11.0

HB Da cidit

1.17 1.18 1.19 1.20 1.21

y, α

11.5

345

So lve nt dip ola rity /p ola riz

12.0

Solv ent

Free energy of

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unfolding, ∆G

o

Tm, K g temperature,

350

12.5

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Table 2. Coefficients of equation: Y = ko + k1π* + k2α (where Y is the property of proteins or nucleic acid in aqueous solution); N – 2 a number of experimental data; r – correlation coefficient; SD- standard deviation; F – ratio of variance). ko 22.7±3.9

k1 21.9±2.7

k2 -29.4±1.7

N 17

r2 0.9694

SD 0.11

F 221

Tm

-72.5±16

338.8±12.6

16.4±4.4

10

0.9904

1.01

359

∆G ∆GN-U

-5±1.96 2731±531

10.4±1.4 -1986±422

-0.99±0.35 -395±77.5

4 4

0.9977 0.9668

0.03 7.77

218 15

G-actin α/β-Tubulin

Tm kapp

347±36 579± 65

-171±23 -332 ±45

-89±12 -167 ±19

9 13

0.9225 0.8994

1.6 2.0

35.7 44.7

gqDNA dsDNA RNA

Tm Tm Tm

455±21.2 230± 7.8 189± 36

-245 ± 10.3 -122 ±4.3 -108± 31

-96±10.1 -23±3.9 -20± 4.65

8 8 9

0.9918 0.9941 0.8170

0.83 0.42 1.8

304 421 13

CMgCl2m CNaClm CMgCl2m

0 -2711±1313 69.7± 19.6

-6.7±5.6 2093±1128 -60.5± 16.8

3.2±0.35 428± 61.9 -2.3±0.95

5 8 12

0.9815 0.9943 0.8984

0.05 2.5 0.03

53 437 39.8

wt-tRNAPhe

Tm

194±49.4

-87±43.3

-38.4±7.8

8

0.8459

2.23

13.7

RNA

Tm

2048 ± 69

0

-1408 ± 57

3

0.9984

0.32

620

Ubiquitin

RNA RNA

a

PEG-4000, PEG-8000; Dex-70, Ficoll-70, proline, TMAO, betaine KCl (0.025-0.300 M)

Ref. 57 34 32 58 59 33 61 61 62 40 64 65

63

All the values of property Y and solvent properties π* and a used in the provided relationships are listed in Table S1.

72

90

a

70

b

80

68 gqDNA

64

Tm

Tm

dsDNA

66

62

70

60

60 58

50

56 54

ac

,α i ty id

π* ility, 1.16 izab 1.18 olar 1.26 /p 1.20 larity dipo ent Solv

BD H

1.08

1.10 1.12

1.14

40 1.12 1.14 1.16 1.18 1.20 1.22 1.24 1.26

nt

So 1.12 lve 1.14 nt HB 1.16 D 1.18 ac 1.20 id 1.22 ity , α 1.24

e lv So

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Chymotrypsin inhibitor 2 Alkaline phosphatase

Crowders, osmolytes TMAO, sorbitol, sarcosine, betaine Dex-40, PEG-20,000; glucose Ficoll-70, sucrose PEG-4000; Dex-70, Ficoll-70 Ficoll-70, TMAO, urea TMAO, urea, sucrose, Ficoll-70 TMAO, urea TMAO, urea Ficoll-70, sucrose, PEG-6000, PEG-20,000 PEG-1000 (0-20 %wt) PEG-1000, PEG-8000

1.14

1.12

1.10

1.08

y, π* 1.16 abilit 1.18 lariz ty/po ri 1.20 la ipo ent d Solv

Figure 3. Interrelationship between the stability of DNA of two different sequences expressed as the melting temperature, Tm, (see all the data used in Table S1) and the water dipolarity/polarizability, π*, and water HBD acidity, α, in aqueous solutions of urea and TMAO at different 53 concentrations (data from ref. ): (a) dsDNA sequence forming canonical DNA duplex; (b) gqDNA sequence forming a four-stranded Gquadruplex.

lyophylization of aliquots or measuring optical rotation or other properties of solution, since the “dry” polymer may contain rather significant amount of water. It is surprising that the data for dextran-40 fit the above relationship so well. The relationship described by Eq. 2 implies that the ubiquitin stability in aqueous solutions of different crowders, such as dextran and PEG and small organic osmolyte, such as glucose, is increased due to these compounds influence on the solvent properties of water. As shown in Table 2, similar relationships were also found for chymotrypsin inhibitor 2 in the presence 32 of Ficoll-70 and sucrose, alkaline phosphatase in aqueous 58 solutions of PEG-4000, Ficoll-70, and dextran-70, and globular actin in the presence of Ficoll-70, TMAO, urea (up to 59 2.0 M) and TMAO-urea mixture. An additional example worth mentioning is the effects of Ficoll, sucrose, TMAO, and

urea on the polymerization kinetics of α/β-tubulin reported by 33 Schummel et al. The polymerization kinetics was characterized by the apparent growth rate, kapp value, assayed o 33 at 37 C in the buffer containing 3.26 M glycerol, pH 6.8; i.e., under conditions drastically different from those used to estimate the solvent properties of aqueous media in the 38, 53 polymers and osmolytes solutions. Nevertheless the reasonably good relationship with coefficients listed in Table 2 was established implying that protein-water interactions are important to the protein aggregation as well as for the protein stability. (All the values for the stability of proteins and nucleic acids and solvent properties of aqueous media are listed in Table S1). It should be mentioned, however, that we could not 60 find any relationship between the data reported in ref. on the effects of polymers and osmolytes on the equilibrium

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Protein/Nucleic Acid RNase A

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TmdsDNA = 222±22 - 120±10.7π* - 19.3±10.5α

(3a)

2

N = 8; r = 0.9710; SD = 0.86; F = 83.6 dsDNA where Tm is the melting temperature of dsDNA forming canonical DNA duplex; all the other parameters are as defined above. Qualitatively similar relationship is shown in Figure 3b for the DNA sequence forming a four-stranded G-quadruplex. This relationship may be described as: TmgqDNA = 455±21 - 245±10.3π* - 95.7±10.1α

(4a)

2

N = 9; r = 0.8170; SD = 1.79; F = 13.4 RNA where Tm is the melting temperature of the hairpin structured RNA; all the other parameters are as defined above. It is important to note that if the data for Ficoll solutions (prepared in gram per litre of water, and hence questionable due to the reasons discussed above) are omitted, the same relationship is dramatically improved and is described as: TmRNA = 247±24.1 - 162±21.4π* - 18.6±2.5α 2

56 54 52

RNA

50 48 46 44 42 40 38

So lve 0.9 nt H 1.0 BD ac 1.1 id ity 1.2 ,α 1.3

1.14

1.13

1.12

1.11

iza olar ity/p olar ip d ent Solv

1.17

1.16

1.15

1.10

1.09

, π* bility

Figure 4. Interrelationship between the hairpin structured RNA expressed as the melting temperature, Tm, (data from ref.62) and the water dipolarity/polarizability, π*, and water HBD acidity, α, in aqueous solutions of sucrose, PEG-6000, PEG-20,000, and Ficoll-70 at different concentrations (see all the data used in Table S1).

(3b)

2

N = 8; r = 0.9918; SD = 0.83; F = 304 gqDNA where Tm is the melting temperature of the DNA sequence forming a four-stranded G-quadruplex; all the other parameters are as defined above. The quantitative differences between Eq. 3a and 3b seem to imply that differences between the aforementioned DNA structures may lead to significant differences between various types of DNA-water interactions. It has been shown that the RNA hairpin stability is directly related to the water activity in aqueous solutions of sucrose, 62 55 Ficoll-70, and PEG. It has been reported by us that water activity in aqueous solutions of various compounds may be described in terms of solvent dipolarity/polarizability and HBD acidity of water. Therefore, it is unsurprising that the same solvent properties of water describe relatively well the 62 reported in ref. melting temperatures of the RNA in solutions of sucrose, PEG-6000, PEG-20,000, and Ficoll-70. The relationship illustrated in Figure 4 may be described as: TmRNA = 189±36 - 108±31.4π* - 20.1±4.7α

solutions of osmolytes and/or polymers are presented in Table 2.

Tm

stability of side-by-side dimerization of A34F variant of B1 domain of protein G and the solvent properties of aqueous media in solutions of TMAO, urea, sucrose, PEG-8000, and Ficoll-70. The melting temperatures of DNA of two different sequences have been reported in the presence of various 61 concentrations of TMAO and urea. The melting temperatures for the dsDNA forming canonical DNA duplex is plotted against the aforementioned properties of water in solutions of urea and TMAO in Figure 3a. The solvent properties of water were earlier examined in TMAO solutions up to TMAO concentration 53 dsDNA of 2.0 M. Therefore we plotted only Tm values for TMAO solutions not exceeding 15 %wt. The relationship shown in Figure 3a may be described as:

(4b)

N = 6; r = 0.9748; SD = 0.93; F = 58 where all the parameters are as defined above. Several additional relationships for the stability of global or secondary structures of various RNA reported in the 40, 63-65 literature and the solvent properties of water in

It should be noted as an example that the data reported for 40 64 RNA stability in the presence of PEG-1000 in ref. and in ref. are significantly different (for the concentrations of MgCl2 at midpoint of the MgCl2 titration curve in the presence of the MgCl2 crowder; C m, values). These differences lead to quite different relationships shown in Table 2. Different biological macromolecules would respond differently to the same changes in the solvent properties of water. Therefore, if the changes in the solvent properties of aqueous media is the major component of the crowding effect induced by macromolecular and/or low molecular crowders it is readily understandable why the same crowders may affect 25 different proteins or nucleic acids in different ways. Solvent properties of water in mixtures of two different polymers The solvent properties of water in aqueous mixtures of two different polymers could be assayed only over the limited polymers concentration range because these mixtures separate into two aqueous phases at concentrations exceeding certain thresholds. The experimental data for solutions of individual polymers and their mixtures are presented in Table 3 together with the data calculated for the mixtures using the principle of additivity of the individual contributions of each polymer. Each solvent property of the mixture was calculated by addition of the experimentally assayed values in individual solutions of two polymers and subtraction of the experimental value for pure water. The term “calculated value” will be used further in this meaning in each case. The data in Table 3 show that in four pairs of different macromolecular crowders, their overall effects on the solvent HBD acidity, α, is reduced in all cases, while in Ficoll-PEG, and dextran-Ficoll mixtures the overall effects on the solvent dipolarity/polarizability, π*, are additive within the experimental error limits.

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Polymer 2

None

None

Ficoll-70

PEG-600

Exp.

10.0 %wt.

0

Exp.

0

10.0 %wt.

Calc.

10.0 %wt.

10.0 %wt.

Exp.

10.0 %wt.

10.0 %wt.

PEG-8000

Ucon-4000

Exp.

10.0 %wt.

0

Exp.

0

10.0 %wt.

Calc.

10.0 %wt.

10.0 %wt.

10.0 %wt.

10.0 %wt.

Dextran-75

Ficoll-70

Exp.

Exp.

10.0 %wt.

0

Exp.

0

10.0 %wt.

Calc.

10.0 %wt.

10.0 %wt.

Exp.

10.0 %wt.

10.0 %wt.

PVP-40

Ucon-4000

Exp.

10.0 %wt.

0

Exp.

0

10.0 %wt.

Calc.

10.0 %wt.

10.0 %wt.

Exp.

10.0 %wt.

10.0 %wt.

Dextran-75b

Ficoll-70b

Calc.

3.23 %wt.

28.31 %wt.

Exp.

3.23 %wt.

28.31 %wt.

Calc.

11.57 %wt.

9.03 %wt.

Exp.

11.57 %wt.

9.03 %wt.

π* 1.096 ± 0.003 π* 1.128 ± 0.007 1.106 ± 0.001 1.138 ± 0.011 1.126 ± 0.001 π* 1.107 ± 0.002 1.129 ± 0.002 1.140 ± 0.007 1.110 ± 0.001 π* 1.105 ± 0.001 1.128 ± 0.007 1.137 ± 0.011 1.144 ± 0.002 π* 1.140 ± 0.005 1.129 ± 0.002 1.173 ± 0.010 1.134 ± 0.001 π* 1.160 ± 0.012 1.114 ± 0.003 1.142 ± 0.010 1.166 ± 0.002

α 1.237 ± 0.003 α 1.105 ± 0.009 1.113 ± 0.004 0.981 ± 0.016 1.032 ± 0.001 α 1.072 ± 0.002 0.996 ± 0.009 0.831 ± 0.014 0.923 ± 0.003 α 1.172 ± 0.002 1.105 ± 0.009 1.040 ± 0.014 1.075 ± 0.002 α 1.063 ± 0.008 0.996 ± 0.009 0.822 ± 0.020 0.942 ± 0.002 α 0.974 ± 0.010 1.014 ± 0.001 1.041 ± 0.007 1.022 ± 0.002

β 0.596 ± 0.001 β 0.615 ± 0.006 0.611 ± 0.002 0.630 ± 0.009 0.622 ± 0.001 β 0.608 ± 0.002 0.631 ± 0.002 0.643 ± 0.005 0.642 ± 0.002 β 0.609 ± 0.002 0.615 ± 0.006 0.628 ± 0.009 0.620 ± 0.002 β 0.644 ± 0.005 0.631 ± 0.002 0.679 ± 0.008 0.662 ± 0.004 β 0.640 ± 0.009 0.689 ± 0.002 0.629 ± 0.007 0.636 ± 0.001

a

π* - solvent dipolarity/polarizability; α – solvent H-bond donor acidity; β – solvent H-bond acceptor basicity; b – Solvent features experimentally assayed in the coexisting phases of aqueous two-phase system formed in the mixture of 12.9 %wt. dextran-75 and 18.1 %wt. Ficoll-70 in 0.01 M sodium phosphate buffer, pH 7.4 (data from ref.66).

The total effects on the solvent HBA basicity, β, are also additive within the experimental error limits in the mixtures of dextran-Ficoll and PEG-Ucon. The reported effects of mixtures of dextran-6 and Ficoll-70 on the stability of the protein FKBP 27 exceed calculated values. (We attempted to examine effects of dextran-6 on the solvent properties of water but the uncommonly high UV absorption of the particular dextran-6

Change in solvent dipolarity/polarizability, π∗ , relative to water

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Polymer 1

sample at our disposal hindered analysis of the spectra of 27 solvatochromic dyes.) The study by Batra et al. is, to our best knowledge, the only published work reporting crowding effects in mixtures of two synthetic polymers in numerical form. It should be noted that the data presented in Table 3 show that the effects of two polymers on the solvent properties of water in their mixtures are typically non-additive, and these effects may affect various proteins in different manners.

0.10

a

Polymer 1 Polymer 2 Experimental Top phase Bottom phase

0.08

0.06

0.04

0.02

0.00

0.0

Change in solvent HBD acidity, α, relative to water

a

Table 3. Solvent features of aqueous media in mixtures of macromolecular crowding agents (data for solutions of 34 individual macromolecular crowding agents taken from ref. ).

A

B

C

D

E

F

A

B

C

D

E

F

b -0.1

-0.2

-0.3

-0.4

Polymer 1 Polymer 2 Experimental Top Phase Bottom Phase

-0.5

Figure 5. Effects of mixtures of macromolecular crowding agents on (a) the water dipolarity/polarizability, π*, and (b) water HBD acidity, α, in aqueous solutions containing 10 %wt. of each crowding agent. The following mixtures listed in Table 3 were used: A: Polymer 1 – Ficoll-70, polymer 2 – PEG-600; B: Polymer 1 – PEG-8000, polymer 2 – Ucon4000; C: Polymer 1 – PVP-40, polymer 2 – Ucon-4000; D: Polymer 1 – dextran-75, polymer 2 – Ficoll-70; E: top Ficoll-rich phase of aqueous two-phase system; F: bottom dextran-rich phase of aqueous twophase system (composition of both phases see in Table 3). Left column represents the overall effect of two polymers calculated based on the principle of additivity of contributions of each polymer; right column represents the experimentally assayed effect. All effects are shown as changes in the solvent property (a) relative to the solvent dipolarity/polarizability, π*, of water, and (b) solvent water HBD acidity, α, of water. All the numerical data see in Table 3.

These data are graphically illustrated in Figure 5a for changes in the solvent dipolarity/polarizability π* induced by individual polymers and their mixtures relative to water where π* amounts to 1.096±0.003, and in Figure 5b for changes in the solvent HBD acidity α relative to water where α = 1.237±0.003.

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In order to compare these changes to common organic solvents it may be mentioned that the difference between the π* values for methanol and ethanol amounts to 0.06, and the difference between the α values in the same solvents is 67 0.10. The data presented in Table 3 for the mixture of dextran-75 and PEG-8000 may be compared to those reported previously for the coexisting phases in aqueous two-phase system formed by these two polymers in 0.01 M sodium 68 phosphate buffer, pH 7.4. These data are presented in Table 3 together with the calculated values, and they show that the overall effects of the two polymers are quite different in the two phases and do not agree with those for mixtures of individual polymers. The solvent dipolarity/polarizability and HBD acidity of water in the upper Ficoll-rich phase containing 28.31 %wt. Ficoll and 3.23 %wt. dextran-70 are reduced relatively to the calculated values, while the same properties in the lower dextran-rich phase containing 11.57 %wt. dextran and 9.03 %wt. Ficoll exceed the calculated values. The solvent HBA acidity of water exceeds the calculated value in the upper phase and agrees with the calculated value for the lower phase. It should be noted that water exists as a flexible network of hydrogen bonds and changes in the solvent properties of water imply that this network is rearranged. It is currently impossible to particularize structural changes in water corresponding to those in its solvent properties, and it is possible that the identical solvent properties do not imply identical structural arrangement of water. Analysis of effects of temperature, urea, and different salts on phase separation in aqueous mixtures of two nonionic polymers and thermodynamic analysis of phase diagrams indicated that phase separation is due to the incompatibility of the polymer66 modified water structures. The difference between the relative hydrophobicity of the coexisting phases estimated by the free energy of interfacial transfer of a CH2 group may be described by a linear combination of the differences between the solvent features of aqueous media in the two phases in aqueous two-phase systems formed by various pairs of 68 nonionic polymers.

a

Table 4. Solvent features of aqueous media in mixtures of macromolecular crowding agents with trimethylamine N-oxide (TMAO).

Solvent properties of water in mixtures of individual polymers and TMAO The solvent features of water in aqueous mixtures of several individual polymers with TMAO were examined at the polymer concentration of 30 %wt. and TMAO concentrations varied from 1 up to 1.4 M in the solutions of dextran-75, and up to 2.0 M in solutions of other polymers. The TMAO concentration range was chosen to provide reliably measurable changes in the solvent features of water but was limited by TMAO solubility in polymer solutions examined. The experimental data for solutions of individual polymers and TMAO and their mixtures are presented in Table 4 together with the “additive” values calculated for the mixtures as described above. The data obtained for mixtures of TMAO and Ficoll are illustrated graphically in Figure 6.

π* 1.096

α 1.237

β 0.596

± 0.003

± 0.003

± 0.001

0

π* 1.164

α 0.997

β 0.642

± 0.001

± 0.002

± 0.003

0

1.0 M

1.096

1.190

0.633

± 0.003

± 0.003

± 0.003

Calc.

30.0 %wt.

1.0 M

1.158

0.950

0.679

± 0.007

± 0.008

± 0.007

Exp.

30.0 %wt.

1.0 M

1.148

0.941

0.670

± 0.003

± 0.003

± 0.003

Exp.

0

1.5 M

1.096

1.163

0.650

± 0.003

± 0.005

± 0.002

Calc.

30.0 %wt.

1.5 M

1.158

0.923

0.696

± 0.007

± 0.011

± 0.006

Exp.

30.0 %wt.

1.5 M

1.143

0.904

0.705

± 0.002

± 0.002

± 0.003

Extrap.

0

1.96 M

1.096

1.140

0.666

± 0.003

± 0.004

± 0.004

Calc.

30.0 %wt.

1.96 M

1.158

0.897

0.713

± 0.007

± 0.009

± 0.008

Exp.

30.0 %wt.

1.96 M

1.140

0.869

0.734

± 0.002

± 0.003

± 0.004

Dextran-75

TMAO

Exp.

30.0 %wt.

0

π* 1.151

α 1.062

β 0.630

± 0.001

± 0.002

± 0.002

Exp.

0

1.0 M

1.096

0.996

0.631

± 0.003

± 0.009

± 0.002

Calc.

30.0 %wt.

1.0 M

1.145

1.015

0.667

± 0.007

± 0.014

± 0.005

Exp.

30.0 %wt.

1.0 M

1.145

1.016

0.664

± 0.003

± 0.003

± 0.005

Interp.

0

1.4 M

1.096

1.168

0.647

± 0.003

± 0.003

± 0.005

Calc.

30.0 %wt.

1.4 M

1.145

0.993

0.681

± 0.009

± 0.009

± 0.011

Exp.

30.0 %wt.

1.4 M

1.143

0.983

0.676

± 0.002

± 0.002

± 0.004

PEG-600

TMAO

Exp.

30.0 %wt.

0

π* 1.109

α 0.899

β 0.669

± 0.005

± 0.004

± 0.001

Exp.

0

1.0 M

1.096

0.996

0.631

± 0.003

± 0.009

± 0.002

Calc.

30.0 %wt.

1.0 M

1.109

Exp.

30.0 %wt.

1.0 M

1.113

Exp.

0

1.5 M

Calc.

30.0 %wt.

1.5 M

1.113

Exp.

30.0 %wt.

1.5 M

1.113

Exp.

0

2.0 M

Calc.

30.0 %wt.

2.0 M

1.113

Exp.

10.0 %wt.

2.0 M

1.113

TMAO

Polymer

TMAO

None

None

Ficoll-70

TMAO

Exp.

30.0 %wt.

Exp.

b

± 0.005

-

0.679 ± 0.004

Nd

0.704

1.096

1.163

0.650

± 0.003

± 0.005

± 0.002

± 0.004

± 0.005

-

± 0.005

0.696 ± 0.008

Nd

0.749

1.096

1.137

0.667

± 0.003

± 0.004

± 0.004

± 0.004

± 0.005 ± 0.004

-

± 0.003

0.713 ± 0.008

Nd

0.803

α 1.039

β 0.618

± 0.005

Exp.

Nd

0

π* 1.101 ± 0.001

± 0.003

± 0.001

Interp.

0

0.56 M

1.096

1.210

0.617

± 0.003

± 0.004

± 0.002

Calc.

Nd

0.56 M

1.101

1.012

0.639

± 0.007

± 0.010

± 0.007

Exp.

Nd

0.56 M

1.132

1.072

0.634

± 0.001

± 0.002

± 0.001

Dextran-rich phase

a

π* - solvent dipolarity/polarizability; α – solvent H-bond donor acidity; β – solvent H-bond acceptor basicity; b – dextran-rich bottom phase of aqueous two-phase system formed by 12.0 %wt. dextran-75, 6.0 %wt. PEG-8000, and 0.01 M sodium/potassium phosphate buffer, pH 7.4.

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ARTICLE 1.20

1.2

1.18

π*calc

π*calc

π*exp

1.16

1.0

αcalc

0.9

αexp

1.14

π*exp

1.12 1.10 1.08

0.8

αexp

Published on 20 September 2017. Downloaded by University of Windsor on 25/09/2017 11:21:05.

1.06

βexp 0.7

1.04

βcalc

αcalc 1.02

0.6

0.0

0.0

0.5

1.0

1.5

2.0

2.5

0.5

1.0

1.5

2.0

2.5

Concentration of TMAO in urea solution, M

Concentration of TMAO, M in 30 %wt. Ficoll-70 solution

Figure 6. Solvent dipolarity/polarizability, π*, HBD acidity, α, and HBA basicity, β, experimentally assayed (exp.) and calculated based on the assumption of additivity of contributions of the components (calc.) in aqueous mixtures of Ficoll-70 and TMAO as functions of TMAO concentration in the mixtures containing 30 %wt. Ficoll-70. All the numerical data see in Table 4.

It may be seen from the data in Figure 6 that in the mixtures of TMAO and Ficoll-70, the solvent HBD acidity α and HBA basicity β exceed calculated values, while the solvent dipolarity/polarizability π* is either equal or slightly decreases relative to the calculated values. However, in mixtures of TMAO and dextran-75, the experimental values of all the water solvent properties agree with the calculated values within the experimental errors values. It was found that the solvatochromic band of the Reichardts’ dye, due to unclear as of yet reason, was unstable in aqueous mixtures of PEG-600 and TMAO, and hence the water solvent HBD acidity could not be assayed in these mixtures. Solvent dipolarity/polarizability in these mixtures agrees with the calculated values, while the experimentally measured solvent HBA basicity exceeds the calculated values over the whole range of TMAO concentrations examined. Overall, the data in Table 4 show that the solvent properties of water depend on the particular composition of the polymerosmolyte mixture. This observation may explain why the overall effect of dextran-70 and sucrose on refolding of human muscle creatine kinase was shown to be less than the sum of 29 their individual effects. Additional data presented in Table 4 show how the solvent properties of the Dextran-rich bottom phase change with addition of 0.56 M TMAO concentration in the phase of aqueous two-phase system formed by dextran-75 and PEG69 8000 and 0.5 M TMAO. Comparison of the solvent properties of water in the dextran-rich phase in the presence of 0.56 M TMAO with those assayed in the same phase in the same TMAO free system shows that the solvent dipolarity/polarizability of water exceeds the calculated value, and the HBD acidity decreases relatively to the calculated value. The HBA basicity agrees with the calculated value. In this case again the overall effect of TMAO and polymer in the phase of aqueous two-phase system differs from the one observed in the aqueous mixtures of individual dextran-75 and TMAO.

Figure 7. Solvent dipolarity/polarizability, π*, and HBD acidity, α, experimentally assayed (exp.) and calculated based on the assumption of additivity of contributions of the components (calc.) in aqueous mixtures of urea and TMAO as functions of TMAO concentration in the mixtures containing 2.0 M urea. All the numerical data see in Table 5.

This finding may be explained as due to the aforementioned rearrangement of the water structure in the phases relative to those in aqueous solutions. Solvent properties of water in mixtures of two different osmolytes The experimental and calculated values of the different solvent properties of water in mixtures of TMAO and urea, betaine, and sarcosine and mixtures of betaine with sorbitol and with sarcosine are presented in Table 5. The experimentally assayed solvent dipolarity/polaizability, π*, and HBD acidity, α, values in the mixtures decrease relative to the calculated values in mixtures of TMAO and 2.0 M urea over the whole range of TMAO concentrations varied from 1.0 M to 2.0 M TMAO, and in the mixtures of 1.0 M TMAO with 1.0 M sarcosine and 1.0 M betaine with 1.0 M sarcosine. In the other mixtures examined here these solvent properties of water agree with the calculated values. The experimentally measured solvent HBA basicity, β, values, agree with the calculated values in all the mixtures except in mixture of 2.0 M urea and 2.0 M TMAO. The data obtained for the solvent properties in mixtures of TMAO and urea are illustrated graphically in Figure 7. The data presented in Table 5 show that the overall effects of mixtures of osmolytes on the solvent properties of water depend on the particular osmolyte composition of the mixture in agreement with the data observed in mixtures of two polymers. Essentially all the authors of the studies of water in solutions of various osmolytes conclude that these compounds impose extensive changes in structure of their aqueous 42-47, 70-73 environment. The information provided by the commonly used techniques of neutron or X-ray scattering of 74 water in solutions of complex molecular solutes is limited. Whereas the results of computer simulation of solvent structuring depend on the water model used in the 74 calculations.

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Solvent features

1.1

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Molecular BioSystems

a

Exp. Exp. Calc. Exp. Exp. Calc. Exp. Exp. Calc. Exp. Exp. Exp. Calc. Exp. Exp. Exp. Calc. Exp. Exp. Exp. Calc. Exp. Exp. Exp. Calc. Exp. Exp. Exp. Calc. Exp. Exp. Exp. Calc. Exp. Exp. Exp. Calc. Exp. Exp. Exp. Calc. Exp.

Urea None 2.0 M 0 2.0 M 2.0 M 0 2.0 M 2.0 M 0 2.0 M 2.0 M Sarcosine 1.0 M 0 1.0 M 1.0 M 0 0.5 M 0.5 M 0.5 M Betaine 1.0 M 0 1.0 M 1.0 M 0 0.5 M 0.5 M 0.5 M Betaine 1.0 M 0 1.0 M 1.0 M 0.5 M 0 0.5 M 0.5 M Betaine 1.0 M 0 1.0 M 1.0 M 0.5 M 0 0.5 M 0.5 M

TMAO None 0 1.0 M 1.0 M 1.0 M 1.5 M 1.5 M 1.5 M 2.0 M 2.0 M 2.0 M TMAO 0 1.0 M 1.0 M 1.0 M 0.5 M 0 0.5 M 0.5 M TMAO 0 1.0 M 1.0 M 1.0 M 0.5 M 0 0.5 M 0.5 M Sorbitol 0 1.0 M 1.0 M 1.0 M 0 0.5 M 0.5 M 0.5 M Sarcosine 0 1.0 M 1.0 M 1.0 M 0 0.5 M 0.5 M 0.5 M

π* 1.096± 0.003 1.166± 0.001 1.096± 0.003 1.169± 0.007 1.154± 0.001 1.096± 0.003 1.169± 0.007 1.146± 0.001 1.096± 0.003 1.169± 0.007 1.139± 0.001 π* 1.120± 0.001 1.096± 0.003 1.114± 0.007 1.102± 0.001 1.096± 0.003 1.112± 0.001 1.106± 0.007 1.101± 0.001 π* 1.100± 0.003 1.096± 0.003 1.100± 0.009 1.090± 0.001 1.096± 0.003 1.103± 0.002 1.103± 0.008 1.095± 0.001 π* 1.100± 0.003 1.132± 0.001 1.136± 0.007 1.130± 0.001 1.103± 0.002 1.111± 0.003 1.118± 0.008 1.116± 0.001 π* 1.100± 0.003 1.120± 0.001 1.124± 0.007 1.110± 0.001 1.103± 0.002 1.112± 0.001 1.119± 0.006 1.115± 0.001

α 1.237± 0.003 1.150± 0.001 1.190± 0.003 1.092± 0.007 1.112± 0.001 1.163± 0.005 1.065± 0.009 1.092± 0.002 1.137± 0.004 1.039± 0.008 1.068± 0.001 α 1.230± 0.003 1.190± 0.003 1.183± 0.009 1.232± 0.002 1.213± 0.004 1.212± 0.004 1.188± 0.011 1.207± 0.002 α 1.185± 0.002 1.190± 0.003 1.138± 0.008 1.136± 0.001 1.213± 0.004 1.211± 0.001 1.187± 0.008 1.192± 0.001 α 1.185± 0.002 1.210± 0.004 1.158± 0.009 1.158± 0.002 1.211± 0.001 1.225± 0.005 1.199± 0.009 1.200± 0.002 α 1.185± 0.002 1.230± 0.003 1.178± 0.008 1.202± 0.002 1.211± 0.001 1.212± 0.004 1.186± 0.008 1.194± 0.002

β 0.596± 0.001 0.622± 0.001 0.633± 0.003 0.655± 0.005 0.649± 0.002 0.650± 0.002 0.672± 0.004 0.672± 0.002 0.667± 0.004 0.689± 0.006 0.702± 0.003 β 0.616± 0.001 0.633± 0.003 0.653± 0.005 0.652± 0.001 0.615± 0.002 0.606± 0.001 0.625± 0.005 0.621± 0.001 β 0.608± 0.002 0.633± 0.003 0.645± 0.006 0.653± 0.001 0.615± 0.002 0.602± 0.001 0.621± 0.004 0.616± 0.002 β 0.608± 0.002 0.609± 0.004 0.621± 0.007 0.621± 0.001 0.602± 0.001 0.604± 0.005 0.610± 0.007 0.607± 0.002 β 0.608± 0.002 0.616± 0.001 0.628± 0.004 0.629± 0.001 0.602± 0.001 0.606± 0.001 0.612± 0.003 0.611± 0.002

a – π* - solvent dipolarity/polarizability; α – solvent H-bond donor acidity; β – solvent H-bond acceptor basicity It has been shown previously that contributions of various types of protein-water interactions (dipole-dipole, hydrogen bonding with water donating hydrogen bond and accepting hydrogen bond) are highly protein specific and may vary 36 significantly with small changes in the protein environment. Therefore, it might be expected that the protein crowders may display very significant effects on the solvent properties of water. In agreement with this hypothesis, the effects of human heat shock protein HspB6 on the solvent

dipolarity/polarizability and HBA basicity were shown to be similar to those of nonionic polymers, whereas the effects of this protein on the solvent HBD acidity of water exceeded 75 those of nonionic polymers and osmolytes quite significantly. Furthermore, it is known that similar to synthetic polymers, many proteins and polysaccharides are capable of forming 76 aqueous two-phase systems. The capability of the protein crowders to show significant effects on the solvent properties of water is also in agreement with the estimates of polarity for

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Table 5. Solvent features of aqueous media in mixtures of urea with trimethylamine N-oxide (TMAO), sarcosine with TMAO, betaine with TMAO, betaine with sorbitol, and sarcosine with betaine.

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the aqueous media in cytoplasm and nucleus that were 77 reported to be very different. Distribution of proteins between cytoplasm and cytoplasmic membrane-less organelles (or nucleoplasm and nuclear membrane-less organelles) was suggested to be controlled by microenvironment (and likely different solvent properties of 78-81 aqueous media) in these phases. It should be emphasized that water exists as a flexible network of hydrogen bonds and changes in the solvent properties of water imply that this network is rearranged. It is currently impossible to particularize structural changes in water corresponding to those in its solvent properties, and it is likely that identical solvent properties do not imply identical structural arrangement of water. Summing up, it should be noted that the presence of the described in this study relationships between stability of different biological macromolecules (proteins and nucleic acids) determined in the presence of various solutes (macromolecular crowders, osmolytes, and their mixtures) and solvent properties of aqueous media cannot be viewed as an evidence of the solvent properties of aqueous media in crowded solutions being the only dominant factor in crowding effect on various biochemical processes, but it clearly shows that this factor should be taken into account when considering situation in vivo.

6. 7. 8.

Conclusions

23.

Analysis of solvent properties of water in aqueous solutions of individual macromolecular crowders and osmolytes shows that linear combinations of the solvent dipolarity/polarizability and hydrogen bond donor acidity of water determined in these solutions strongly correlate with changes in stability of proteins and nucleic acids reported in the literature. The same properties in mixtures of two macromolecular crowders or two osmolytes or mixtures of osmolyte and macromolecular crowder vary differently for various solvent properties, and the overall effects of the two components in the mixture on the solvent properties of water may be additive, reduced or enhanced depending on the particular composition of the mixture. It is hypothesized that changes in the solvent properties of water are related to changes in the water hydrogen-bonding structuring. It is suggested that the observed crowder-induced changes in the solvent properties of water should be taken into account in theoretical considerations of crowding effects in biological systems.

24.

Conflicts of interest

9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22.

25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36.

There are no conflicts of interest to declare. 37.

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Role of solvent properties of water in crowding effects induced by macromolecular agents and osmolytes.

Solvent properties of water in aqueous solutions of polyethylene glycols of various molecular weights, l-proline, betaine, and a series of chlorides o...
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