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Physical Insights into Materials and Molecular Properties

Ion Complexation Explains Orders of Magnitude Changes in the Equilibrium Constant of Biochemical Reactions in Buffers Crowded by Nonionic Compounds
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Cite this: J. Phys. Chem. Lett. 2022, 13, 1, 112–117
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https://doi.org/10.1021/acs.jpclett.1c03596
Published December 28, 2021

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Abstract

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The equilibrium constant (K) of biochemical complex formation in aqueous buffers with high concentration (>20 wt %) of nonionic compounds can vary by orders of magnitude in comparison with the K in a pure buffer. The precise molecular mechanisms of these profound changes are not known. Herein, we show up to a 1000-fold decrease of the K value of DNA hybridization (at nM concentration) in standard molecular crowder systems such as PEG, dextrans, Ficoll, and glycerol. The effect responsible for the decrease of K is the complexation of positively charged ions from a buffer by nonionic polymers/small molecules. We determined the average equilibrium constant for the complexation of ions per monomer (∼0.49 M–1). We retrieve K’s original value for a pure buffer if we properly increase the ionic strength of the buffer crowded by the polymers, compensating for the loss of complexed ions.

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Biochemical reactions occur in the cytoplasm of living cells crowded by biomolecules. They occupy up to 40 wt % of the cell interior. (1,2) The solutions of nonionic compounds at large concentrations (∼40–50 wt %) are in vitro models of the cell’s cytoplasm. In these solutions, the equilibrium constant of biochemical reactions often decreases by orders of magnitude compared to pure buffers. (3−6) The mechanism of this phenomenon is not known. Herein, we show up to a 1000-fold decrease of the K value of DNA hybridization (at nanomolar concentration) in standard molecular crowder systems, such as PEG, dextrans, Ficoll, and glycerol. We prove that the general mechanism responsible for decreasing K is the complexation of positively charged ions from a buffer by nonionic polymers/small molecules. We confirm the mechanism by restoring K’s original value for a pure buffer by adding a calculated amount of ions to the solution to compensate for the loss of complexed ions. Typically, the buffer’s ionic strength is ∼100 mM, and nonionic compounds often reach ∼1 M concentration. Therefore, even a very weak complexation of ions, say at ∼1 M–1, removes half of all ions from the solution and decrease the equilibrium constant.

The molecular crowding phenomenon is also studied because a high concentration of cosolutes can alter molecules’ structures, diffusion coefficients, and binding rates. (7−11) The crowded environment is frequently recreated artificially to understand biochemical reactions occurring inside living cells. (12,13) Cosolutes used in solutions mimicking cells’ interior have to be chemically inactive. It was shown that cosolutes can increase the K at modest crowder concentrations (∼10 wt %) through one of the crowding mechanisms─depletion forces. (3,14) The origin of those forces is caused by a decrease of the local volume around the biomolecule by crowders, increasing the effective concentration of reactants. Such a mechanism was shown in the example of the formation of DNA hairpins and the reactive structure of proteins/enzymes. (15−17)

The majority of biochemical reactions (e.g., DNA hybridization or protein folding) require well-defined thermodynamic conditions, including pH and ionic strength (IS). Thus, the experiments in crowded systems are frequently performed in fixed buffers at the physiological IS of around 100 mM. However, the living cell’s interior is an active, self-regulating system that can control ion concentration (e.g., through the ionophoric transfer of cations). The most popular crowding polymer, PEG, complexes divalent cations in nonaqueous solutions with the equilibrium association constant below 1 M–1. (20−23) This raises a question: Can crowders being in high concentration (i.e., 10–50 wt %) affect the IS of the standard buffers (e.g., phosphate buffer) and, in consequence, change K of the complex formations? In aqueous solutions, the formation of complexes between nonionic polymers and cations is difficult to observe directly by experiments because of the strong hydration of metal cations. (24) However, some studies explained interactions of PEG moieties with nonionic and anionic surfactants in water-based system as mediated by alkali metal cations, therefore revealing PEG–cation complexation. (25,26) Moreover, Ohki et al. by cooperative application of ion-transfer voltammetry at a liquid/liquid interface together with X-ray absorption fine structure (XAFS) measurements strongly suggested that alkali cations form a complex with PEG in water even in relatively low PEG concentrations (5–16 wt %). (24) In addition, Breton et al. confirmed complexation of (Na+, K+, Rb+, and Cs+) indirectly in the electrophysiology experiments by analyzing partitioning of neutral, flexible PEG 2000 molecules into the α-HL nanopore. In the presence of the mentioned cations, the neutral polymer behaved as if it was charged. Interestingly, for lithium cations (Li+) that effect was not observed. (27) The formation of PEG–sodium complexes was also used in cation template-assisted cyclopolymerization. (28)

In this Letter, we revise molecular crowding’s effect on biochemical reactions at the nanomolar concentration range using a previously validated brightness-based method. (18,19)Figure 1 presents the schematic concept workflow. We studied DNA hybridization in standard molecular crowder systems (i.e., PEG, EG, glycerol, Ficoll, and dextrans) as a noncovalent complex formation model. The DNA hybridization is ionic strength-sensitive and is discouraged in a low ion concentration environment. (29) Thus, it is a good indicator of ions’ potential binding by crowders. We also determined the relationship between K and IS to support our hypothesis. In addition, we confirm binding of sodium by dextran in aqueous solution by independent potentiometric measurements using an ion-selective electrode. Finally, we combined both approaches to show that not the mere physical abundance of crowders, which changes the volume available to reactants, but the complexation of ions by them is responsible for changing the K of reaction. (20−23)

Figure 1

Figure 1. Hybridization of complementary DNA oligonucleotides was used as a model biochemical reaction. (a) The change of the fluorescent properties as a result of the complex formation makes it possible to use the brightness analysis method. (18,19) We investigated two biophysical factors that affect the formation of the noncovalent complex: ionic strength and crowded environment. (b) Ionic strength. The formation of the complex was observed at different ionic strengths. (c) Crowded environment. We used the most common biorelated crowders of different sizes and chemical structures. The influence of cosolute is negligible even at 109 excess over the concentration of substrates (region I). There is no correlation between sizes and structures of different agents (region II). (d) The comparison of the interactions in a crowded environment with respect to different ionic strengths allowed the determination of the sodium cation complexation by crowder molecules.

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First, we checked the influence of crowders on the DNA hybridization equilibrium constant. The formation of a double-stranded DNA backbone is an electrostatic interaction between two complementary, negatively charged strands. We monitor the effect of the crowded environment on the hybridization of complementary strands in the biochemical concentration regime (5 nM). All crowders we were using are known to be chemically inert. Dextrans and ficolls are “similar” in chemical structure as they possess sugar moieties rich in hydroxyl groups, whereas EG and PEGs are alkoxyl-rich. Using the brightness analysis method (described in the Supporting Information, section S3), the effect of crowders was observed by the determination of the equilibrium constant (K) of DNA hybridization.

The crowding effect on the thermodynamic stability of DNA duplexes formation was previously determined in the presence of various crowders. (5,30) Here, at low crowders concentrations (<10 wt %), the change of K is nearly negligible (see Figure 2). The influence of the crowded environment changes the K by 2–3 orders of magnitude for the high concentration of crowders (40 wt %<). In terms of the molar concentration, this value corresponds to values even above 10 M. This means that the crowders are in over 109 times excess over the concentration of substrates in this reaction (∼1 nM). Hence, if the crowding effect were significant, it would be observable at much lower concentrations. Therefore, the effect responsible for changes of K is not secondary interaction with crowder molecule nor effect caused by depletion with it, but rather some weak effect or interaction.

Figure 2

Figure 2. Hybridization reaction was measured at constant ionic strength in the presence of various crowder agents. The effect of crowder molecules on reaction components (presented in nanomolar concentration) is negligible below a few wt % of crowder concentration (∼200 mM). However, when the molar concentration of the crowder is approximately a few molar, the reaction is affected.

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It was reported that crowder molecules, such as polyethers or carbohydrates, form complexes with cations suspended in the nonaqueous solvent. (20−22,24,31−33) The reported values for complexation of the cations by different PEG molecules range from 1 to 102 M–1. These values depend on the charge (e.g., Na+ or Ca2+) and the type of conjugated acidic residual (strong or weak, e.g., phosphate or acetate). (34,35) If we consider a system crowded by 40 wt % PEG 400 (2.68 M) in 0.1 M PB buffer (only sodium cations) and assume no interactions due to depletion forces, then based on reported sodium complexation, κ = 1 M–1, the initial concentration of sodium ions drops by ∼73%.

The concentration of salts regulates the physiological processes of the cell. The changes of IS shift the equilibrium of ions of those biochemical noncovalent complexes, such as complex-forming polypeptides (antibodies), stabilization of membrane-building anionic phospholipids, protein–DNA, DNA–drug, metabolic-substrates with carboxyl or phosphate group, etc. Also, the activity of enzymes or receptors is controlled by the formation of ion-based complexes with specific cations to obtain proper active conformation. (36−39)

Taking all this into consideration, the decrease of the equilibrium constant in a crowded environment (shown in Figure 2) is observed because of ion deficiency caused by complexation of cations in the solution. The decrease of ion concentration in the Debye double layer reduces the screening of negative charges on oligonucleotide backbones. This results in electrostatic repulsion between DNA strands and thus the lower bound fraction. The few nanometer-sized ion-crowder complexes are too large to stabilize DNA hybridization as the average hydrogen bond between complementary nucleotides is 0.2–0.3 nm. (40) DNA duplex formation would require such an object to detach and diffuse. In contrast, angstrom-sized ions can fill the spaces between nucleotides and their proximity without interrupting hybridization. (41)

Following that, we checked the influence of ionic strength on DNA hybridization. The effective ion concentration in the solution is described by IS, and it determines the effective electrostatic repulsion distance (Debye length). During the DNA hybridization, the base pair repulsion (caused by phosphodiester groups) is partially neutralized by cations (e.g., Na+, K+, or Mg2+). We investigated the hybridization of 13 bp complementary DNA strands at different ionic strengths (in a range of ∼100 times lower than physiological concentrations and at a highly saline environment) (see Figure 3). We used PB buffer of various concentrations, where only sodium ions were introduced as positively charged species. At each IS, using the brightness analysis method, we determined the equilibrium constant K. The results were also validated by FRET (see the Supporting Information, section S4).

Figure 3

Figure 3. Influence of ionic strength on the hybridization of complementary DNA oligonucleotide strands (violet points). The data were compared with the observed change of Debye length recorded by Smith et al. (42) The scatters represent data points, and the line (both marked in green) shows dependence of Debye length as a function of ionic strength according to Debye–Hückel theory.

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The increase in salt concentration changes the binding energy between complementary DNA fragments to a significant extent. The presence of sodium cations and phosphate anions with a total IS of 10 mM generates an increase in the constant K by 106 M–1 in relation to pure water, where the reaction does not take place.

The ions in the solution screen the negative charges found on the phosphate groups of DNA backbones. Accordingly, the Debye length and the effective negative charge of the oligonucleotides are reduced. Dispersive interactions start to dominate over the repulsive electrostatic forces between DNA strands. As a result, the probability of forming a DNA complex increases. For IS greater than 700 mM, the K constant decreases. At 2 M ionic strength, the value of K is 10 times lower than the maximum value observed. In this regime, the charge on the oligonucleotides is strongly screened and deviation from Debye–Hückel theory occurs.

We suggest that repulsion due to the densely packed Debye layer prevents effective collisions (interactions between DNA molecules) and hinders formation of the double-stranded DNA. A mutual effect was previously observed on the surface covered by charged nanoparticles at different ionic strengths. (43) Moreover, Smith et al. using a surface force apparatus measured similar anomalous changes in Debye’s screening length at high salt (i.e., NaCl) concentrations. (42) In Figure 3, we show their data and the classical Debye screening length overlaying the K constant determined in our research as a function of IS. The changes in Debye length follow the inverted function of changes in K for DNA hybridization caused by varying IS. Following the surface-oriented Smith et al. experiments, we observed analogous results in bulk solution of reagents, by linking directly the influence of ions on the Debye length, and finally with the K of DNA hybridization.

Finally, we determined the complexation of sodium ions by crowders. The interaction scheme of ion complexation by different crowders is presented schematically in eq 1. The binding site for cation within the crowder structure may differ even between crowders of the same binding moiety (functional group). Therefore, we calculated the interaction with crowder per molecule or monomer (in the case of polymers). This model simplifies the interactions between ions and crowder molecules. Hence, the obtained values of the ion complexation equilibrium constant, κ, can be corrected by a factor dependent on the number of monomers/molecules participating in the binding site.

Na++CW⇌κNa·CW
(1)
The equilibrium constant for this interaction can be written as
κ=[Na·CW]eq[Na+]eq·[CW]eq=[Na·CW]eq([Na+]0−[Na·CW]eq)·([CW]0−[Na·CW]eq)
(2)
where [Na+]0 and [CW]0 are initial molar concentration of sodium ions and crowder molecules, respectively.

The method of buffer preparation enables keeping the pH constant even at different ionic strengths.

To determine the ion complexation, κ, we designed an experiment with the following methodology: we always controlled the total number of sodium ions by preparing a given buffer concentration; with the series of prepared buffers we estimated K of DNA hybridization at each [Na+]0 (see violet points in Figure 4a). Taking into consideration that DNA strands are at 5 nM concentration, great excess of ions in environment (millimolar concentration scale, 6 orders of magnitude difference; thus, [Na+]0 ≫ [DNA]0), we can assume that the initial concentration of sodium ions is almost equal to the concentration at equilibrium [Na+]0 ≈ [Na+]eq. Therefore, we could estimate the relation K = X·[Na+]Y, where X = 9.62 × 1012 and Y = 2.51 (violet dashed line). Next, in a separate series of experiments, we prepared constant concentration of crowders at different concentrations of Na+ and again measured K (see green points in Figure 4a). The obtained K values were transformed to the sodium concentration using the previous relation (K = X·[Na+]Y). At a given concentration of buffer, the difference between calculated concentration of sodium ions without and with the presence of crowders allows us to calculate how many sodium ions got complexed by crowder molecule, [Na·CW]eq. Knowing this value, [Na+]0, and [CW]0 using eq 2 we could estimate κ. This methodology was repeated for a series of multiple buffer concentrations and various crowders at 40 wt %.

Figure 4

Figure 4. (a) Hybridization constant depending on the concentration of sodium ions without the addition of cosolute (violet dots) and in the presence of 40 wt % PEG 400 (green dots). The arrows in the plot correspond to the difference between the K values and thus the number of complexed sodium cations. (b) The sodium ion complexation by PEG 400 at various concentrations of sodium cations in the solution. The violet line represents the average value of the constant κ determined per one PEG 400 monomer (nmer ≈ 8.5). Considering those results, we infer that the complexation of sodium ions by crowders, but not its direct interactions, is in fact mostly responsible for the changes in equilibrium constants. (c) Comparison of determining sodium complexation equilibrium constant by different crowders, excluding depletion force interaction. (d) (left panel) In an ionic solution, the partially charged compound is surrounded by an electrical double layer. Sodium cations screen negative charge of the DNA backbone, which facilities the complex formation. (right panel) After the addition of crowders, sodium cations are getting complexed by weak interaction with crowder molecules. In high concentration of crowders, repulsion of the DNA strands became more pronounced because of reduction of screening charges on the DNA backbone.

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In the example experiment with PEG 400, using the presented methodology we determined ion complexation equilibrium constant, κ (see Figure 4b), and averaged it over a series of data points. The calculated complexation constant estimated with brightness analysis is 0.41 ± 0.10 M–1. We measured cosolutes differentiated in molecular sizes and chemical structure (only oxygen was used as a heteroatom in functional groups). The difference in size is not pronounced, especially in the case of crowders similar in structure (sugar moiety), e.g., big dextran of average molecular weight 70 kDa and ficoll, ∼400 kDa. The determined κ values calculated per crowder molecule or monomer (in the case of polymers) are summarized in Figure 4c. In section S6 of the Supporting Information we discuss how addition of crowders can change other properties of the solution, such as dielectric constant, viscosity, pH, or activity.

All the collected data and calculations together with the proof that adding a certain amount of ions reverse the K constant to the higher value suggest complexation of sodium ions by crowders. We also tried to prove it directly by potentiometric measurements using ion-selective electrodes (ISEs) in the crowded solutions as well as with the crowders separated by the dialysis membrane. However, we met experimental difficulties, which are described in detail in the Supporting Information, section S7.

The influence of crowders is related not only to the effect on IS but also to the direct impact on the substrates of the reaction. For instance, depending on the type (e.g., ionic or nonionic) and concentration of crowders, the enzymatic activity can decrease or increase, and protein stability is altered. (44) It was shown that the presence of the crowder molecule near the local neighborhood of the protein substrate may affect its dielectric properties and its hydration structure. (45) Crowders bind water molecules, which can influence the effective amount of available solvent. (46) In section S8 of the Supporting Information, we show the estimated effect of water-binding by crowder molecules and how it may affect concentrations of ions and thus the κ constant. Additionally, the effect of depletion forces that occurs when crowders exclude the effective reactive volume should also be considered and included in the calculation (see Figure 5).

Figure 5

Figure 5. Contribution of interactions in a crowder system. The bar plots (violet) represent experimentally measured K values of 13 bp oligonucleotide hybridization in 100 mM buffer: without crowders, with presence of 40 wt % PEG 400, and in theoretical system where 73 mM of sodium ions were complexed by 40 wt % PEG 400 (κ = 1 M–1) without secondary interactions. The future quantitative analysis forces separation of the contributions of ion complexation and the depletion forces.

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To conclude this study, we applied the brightness analysis method to investigate two factors that affect noncovalent complex formation: crowded environment and ionic strength. We showed that an increase in interactions between substrates is increased by ionic strength. However, after exceeding a certain value, it augmented the reducing effect on the bound fraction, which is unintuitive at first sight. This observation may be especially important for further analysis of biochemical reactions of organisms in a highly saline environment or with less access to water. In further analysis, we showed the applicability of the hybridization of DNA reaction as an indicator of sodium ion concentration. The experiments performed in the crowded environment showed that ion complexation, but not molecular crowding, may be responsible for the most changes in K values of biochemical interactions. In addition, we confirmed the results obtained by measurements using an ion-selective electrode. On the basis of this observation, we determined the complexation of sodium cations (on average, κ ≈ 0.49 M–1) by the most popular crowders differentiated in size and chemical structure. Our results will help to plan precise in vitro experiments to mimic conditions in living cells.

Supporting Information

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The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.jpclett.1c03596.

  • Detailed description of materials and experimental methods, microscope setup, the full description with equations of brightness-based method to determine the equilibrium K constant of biochemical reactions, validation of K constant of DNA hybridization by FRET method, changes of solution properties (e.g., viscosity and pH) after addition of crowders, results of measurements of sodium cation complexation by nonionic crowders with the use of ion-selective electrode, and the discussion and results of water complexation by nonionic crowders (PDF)

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  • Corresponding Author
  • Authors
    • Krzysztof Bielec - Institute of Physical Chemistry, Polish Academy of Sciences, 01-224 Warsaw, Poland;  Institute of Chemical Sciences and Engineering, EPFL CH C2 425, Bâtiment CH, Station 6, Lausanne CH-1015, Switzerland;  Orcidhttps://orcid.org/0000-0002-6023-5499
    • Adam Kowalski - Institute of Physical Chemistry, Polish Academy of Sciences, 01-224 Warsaw, Poland;  Orcidhttps://orcid.org/0000-0003-0172-2622
    • Grzegorz Bubak - Institute of Physical Chemistry, Polish Academy of Sciences, 01-224 Warsaw, Poland;  Orcidhttps://orcid.org/0000-0001-7938-4016
    • Emilia Witkowska Nery - Institute of Physical Chemistry, Polish Academy of Sciences, 01-224 Warsaw, Poland
  • Notes
    The authors declare no competing financial interest.

Acknowledgments

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This work was supported by the National Science Centre, Poland, within the grant Preludium Bis 2020/39/O/ST4/00877.

References

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    The influence of macromolecular crowding and macromolecular confinement on biochemical reactions in physiological media
    Minton, Allen P.
    Journal of Biological Chemistry (2001), 276 (14), 10577-10580CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
    A review, with 48 refs., on the influence of macromol. crowding and macromol. confinement on biochem. reactions in physiol. media.
  9. 9
    Knowles, D.; Shkel, I. A.; Phan, N. M.; Sternke, M.; Lingeman, E.; Cheng, X.; Cheng, L.; O’Connor, K.; Record, M. T. Chemical interactions of polyethylene glycols (PEGs) and glycerol with protein functional groups: applications to effects of PEG and glycerol on protein processes. Biochemistry 2015, 54, 3528– 3542,  DOI: 10.1021/acs.biochem.5b00246
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    9
    Chemical Interactions of Polyethylene Glycols (PEGs) and Glycerol with Protein Functional Groups: Applications to Effects of PEG and Glycerol on Protein Processes
    Knowles, D. B.; Shkel, Irina A.; Phan, Noel M.; Sternke, Matt; Lingeman, Emily; Cheng, Xian; Cheng, Lixue; O'Connor, Kevin; Record, M. Thomas
    Biochemistry (2015), 54 (22), 3528-3542CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
    The authors obtain the data needed to predict chem. interactions of polyethylene glycols (PEGs) and glycerol with proteins and related org. compds. and thereby interpret or predict chem. effects of PEGs on protein processes. To accomplish this, the authors det. interactions of glycerol and tetraEG with >30 model compds. displaying the major C, N, and O functional groups of proteins. Anal. of these data yields coeffs. (α values) that quantify interactions of glycerol, tetraEG, and PEG end (-CH2OH) and interior (-CH2OCH2-) groups with these groups, relative to interactions with water. TetraEG (strongly) and glycerol (weakly) interact favorably with arom. C, amide N, and cationic N, but unfavorably with amide O, carboxylate O, and salt ions. Strongly unfavorable O and salt anion interactions help make both small and large PEGs effective protein precipitants. Interactions of tetraEG and PEG interior groups with aliph. C are quite favorable, while interactions of glycerol and PEG end groups with aliph. C are not. Hence, tetraEG and PEG300 favor unfolding of the DNA-binding domain of lac repressor (lacDBD), while glycerol and di- and monoethylene glycol are stabilizers. Favorable interactions with arom. and aliph. C explain why PEG400 greatly increases the soly. of arom. hydrocarbons and steroids. PEG400-steroid interactions are unusually favorable, presumably because of simultaneous interactions of multiple PEG interior groups with the fused ring system of the steroid. Using α values reported here, chem. contributions to PEG m-values can be predicted or interpreted in terms of changes in water-accessible surface area (ΔASA) and sepd. from excluded vol. effects.
  10. 10
    Knowles, D.; LaCroix, A. S.; Deines, N. F.; Shkel, I.; Record, M. T. Separation of preferential interaction and excluded volume effects on DNA duplex and hairpin stability. Proc. Natl. Acad. Sci. U. S. A. 2011, 108, 12699– 12704,  DOI: 10.1073/pnas.1103382108
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    10
    Separation of preferential interaction and excluded volume effects on DNA duplex and hairpin stability
    Knowles, D. B.; LaCroix, Andrew S.; Deines, Nickolas F.; Shkel, Irina; Record, M. Thomas, Jr.
    Proceedings of the National Academy of Sciences of the United States of America (2011), 108 (31), 12699-12704, S12699/1-S12699/6CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
    Small solutes affect protein and nucleic acid processes because of favorable or unfavorable chem. interactions of the solute with the biopolymer surface exposed or buried in the process. Large solutes also exclude vol. and affect processes where biopolymer molecularity and/or shape changes. Here, we develop an anal. to sep. and interpret or predict excluded vol. and chem. effects of a flexible coil polymer on a process. We report a study of the concn.-dependent effects of the full series from monomeric to polymeric PEG on intramol. hairpin and intermol. duplex formation by 12-nucleotide DNA strands. We find that chem. effects of PEG on these processes increase in proportion to the product of the amt. of DNA surface exposed on melting and the amt. of PEG surface that is accessible to this DNA, and these effects are completely described by two interaction terms that quantify the interactions between this DNA surface and PEG end and interior groups. We find that excluded vol. effects, once sepd. from these chem. effects, are quant. described by the anal. theory of Hermans, which predicts the excluded vol. between a flexible polymer and a rigid mol. From this anal., we show that at const. concn. of PEG monomer, increasing PEG size increases the excluded vol. effect but decreases the chem. interaction effect, because in a large PEG coil a smaller fraction of the monomers are accessible to the DNA. Vol. exclusion by PEG has a much larger effect on intermol. duplex formation than on intramol. hairpin formation.
  11. 11
    Bubak, G.; Kwapiszewska, K.; Kalwarczyk, T.; Bielec, K.; Andryszewski, T.; Iwan, M.; Bubak, S.; Hołyst, R. Quantifying Nanoscale Viscosity and Structures of Living Cells Nucleus from Mobility Measurements. J. Phys. Chem. Lett. 2021, 12, 294– 301,  DOI: 10.1021/acs.jpclett.0c03052
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    11
    Quantifying Nanoscale Viscosity and Structures of Living Cells Nucleus from Mobility Measurements
    Bubak, Grzegorz; Kwapiszewska, Karina; Kalwarczyk, Tomasz; Bielec, Krzysztof; Andryszewski, Tomasz; Iwan, Michalina; Bubak, Szymon; Holyst, Robert
    Journal of Physical Chemistry Letters (2021), 12 (1), 294-301CODEN: JPCLCD; ISSN:1948-7185. (American Chemical Society)
    Understanding the mobility of nano-objects in the eukaryotic cell nucleus, at multiple length-scales, is essential for dissecting nuclear structure-function relationships both in space and in time. Here, we demonstrate, using single-mol. fluorescent correlation spectroscopies, that motion of inert probes (proteins, polymers, or nanoparticles) with diams. ranging from 2.6 to 150 nm is mostly unobstructed in a nucleus. Supported by the anal. of electron tomog. images, these results advocate the ~ 150 nm-wide interchromosomal channels filled with the aq. dild. protein soln. The nucleus is percolated by these channels to allow various cargos to migrate freely at the nanoscale. We detd. the vol. of interchromosomal channels in the HeLa cell nucleus to 237 ± 61 fL, which constitutes 34% of the cell nucleus vol. The vol. fraction of mobile proteins in channels equals 16% ± 4%, and the concn. is 1 mM.
  12. 12
    Sozański, K.; Ruhnow, F.; Wiśniewska, A.; Tabaka, M.; Diez, S.; Hołyst, R. Small crowders slow down kinesin-1 stepping by hindering motor domain diffusion. Physical review letters 2015, 115, 218102,  DOI: 10.1103/PhysRevLett.115.218102
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    12
    Small crowders slow down kinesin-1 stepping by hindering motor domain diffusion
    Sozanski, Krzysztof; Ruhnow, Felix; Wisniewska, Agnieszka; Tabaka, Marcin; Diez, Stefan; Holyst, Robert
    Physical Review Letters (2015), 115 (21), 218102/1-218102/5CODEN: PRLTAO; ISSN:0031-9007. (American Physical Society)
    The dimeric motor protein kinesin-1 moves processively along microtubules against forces of up to 7 pN. However, the mechanism of force generation is still debated. Here, we point to the crucial importance of diffusion of the tethered motor domain for the stepping of kinesin-1: small crowders stop the motor at a viscosity of 5 mPa·s-corresponding to a hydrodynamic load in the sub-fN (∼10-4 pN) range-whereas large crowders have no impact even at viscosities above 100 mPa·s. This indicates that the scale-dependent, effective viscosity experienced by the tethered motor domain is a key factor detg. kinesin's functionality. Our results emphasize the role of diffusion in the kinesin-1 stepping mechanism and the general importance of the viscosity scaling paradigm in nanomechanics.
  13. 13
    Hou, S.; Ziebacz, N.; Kalwarczyk, T.; Kaminski, T. S.; Wieczorek, S. A.; Holyst, R. Influence of nano-viscosity and depletion interactions on cleavage of DNA by enzymes in glycerol and poly (ethylene glycol) solutions: qualitative analysis. Soft Matter 2011, 7, 3092– 3099,  DOI: 10.1039/C0SM00899K
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    13
    Influence of nano-viscosity and depletion interactions on cleavage of DNA by enzymes in glycerol and poly(ethylene glycol) solutions: qualitative analysis
    Hou, Sen; Ziebacz, Natalia; Kalwarczyk, Tomasz; Kaminski, Tomasz S.; Wieczorek, Stefan A.; Holyst, Robert
    Soft Matter (2011), 7 (7), 3092-3099CODEN: SMOABF; ISSN:1744-683X. (Royal Society of Chemistry)
    Biochem. reactions in living systems take place in an environment crowded by various macromols. and ligands. Therefore exptl. data obtained in buffer do not reflect in vivo conditions. We have used glycerol, poly(ethylene glycol) (PEG) 6000 and PEG 8 M solns. to investigate the influence of the crowded environment on cleavage of plasmid DNA by restriction enzyme HindIII. PEG 6000 soln. can effectively slow down the cleavage process. However, neither PEG 8 M soln. of the same viscosity as PEG 6000 soln. nor glycerol soln. of the same concn. as PEG 6000 soln. slows the cleavage of DNA appreciably. The viscosity experienced by the biomols. (here called nano-viscosity) and aggregation induced by the depletion interactions between DNA mols. in polymer soln. (PEG 6000) are two factors responsible for slow cleavage of DNA. We have ruled out the change of pH and denaturation of HindIII as possible sources for the effect.
  14. 14
    Baltierra-Jasso, L. E.; Morten, M. J.; Laflor, L.; Quinn, S. D.; Magennis, S. W. Crowding-induced hybridization of single DNA hairpins. J. Am. Chem. Soc. 2015, 137, 16020– 16023,  DOI: 10.1021/jacs.5b11829
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    14
    Crowding-Induced Hybridization of Single DNA Hairpins
    Baltierra-Jasso, Laura E.; Morten, Michael J.; Laflor, Linda; Quinn, Steven D.; Magennis, Steven W.
    Journal of the American Chemical Society (2015), 137 (51), 16020-16023CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    It is clear that a crowded environment influences the structure, dynamics, and interactions of biol. mols., but the complexity of this phenomenon demands the development of new exptl. and theor. approaches. Here we use two complementary single-mol. FRET techniques to show that the kinetics of DNA base pairing and unpairing, which are fundamental to both the biol. role of DNA and its technol. applications, are strongly modulated by a crowded environment. We directly obsd. single DNA hairpins, which are excellent model systems for studying hybridization, either freely diffusing in soln. or immobilized on a surface under crowding conditions. The hairpins followed two-state folding dynamics with a closing rate increasing by 4-fold and the opening rate decreasing 2-fold, for only modest concns. of crowder [10% (wt./wt.) polyethylene glycol (PEG)]. These expts. serve both to unambiguously highlight the impact of a crowded environment on a fundamental biol. process, DNA base pairing, and to illustrate the benefits of single-mol. approaches to probing the structure and dynamics of complex biomol. systems.
  15. 15
    Mitchison, T. Colloid osmotic parameterization and measurement of subcellular crowding. Molecular biology of the cell 2019, 30, 173– 180,  DOI: 10.1091/mbc.E18-09-0549
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    15
    Colloid osmotic parameterization and measurement of subcellular crowding
    Mitchison, T. J.
    Molecular Biology of the Cell (2019), 30 (2), 173-180CODEN: MBCEEV; ISSN:1939-4586. (American Society for Cell Biology)
    A review. Crowding of the subcellular environment by macromols. is thought to promote protein aggregation and phase sepn. A challenge is how to parameterize the degree of crowding of the cell interior or artificial solns. that is relevant to these reactions. Here I review colloid osmotic pressure as a crowding metric. This pressure is generated by solns. of macromols. in contact with pores that are permeable to water and ions but not macromols. It generates depletion forces that push macromols. together in crowded solns. and thus promotes aggregation and phase sepn. I discuss measurements of colloid osmotic pressure inside cells using the nucleus, the cytoplasmic gel, and fluorescence resonant energy transfer (FRET) biosensors as osmometers, which return a range of values from 1 to 20 kPa. I argue for a low value, 1-2 kPa, in frog eggs and perhaps more generally. This value is close to the linear range on concn.-pressure curves and is thus not crowded from an osmotic perspective. I discuss the implications of a low crowding pressure inside cells for phase sepn. biol., buffer design, and proteome evolution. I also discuss a pressure-tension model for nuclear shape, where colloid osmotic pressure generated by nuclear protein import inflates the nucleus.
  16. 16
    Balcells, C.; Pastor, I.; Pitulice, L.; Hernández, C.; Via, M.; Garcés, J. L.; Madurga, S.; Vilaseca, E.; Isvoran, A.; Cascante, M. Macromolecular crowding upon in-vivo-like enzyme-kinetics: effect of enzyme-obstacle size ratio. New Frontiers in Chemistry 2015, 24, 3
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    Hou, S.; Trochimczyk, P.; Sun, L.; Wisniewska, A.; Kalwarczyk, T.; Zhang, X.; Wielgus-Kutrowska, B.; Bzowska, A.; Holyst, R. How can macromolecular crowding inhibit biological reactions? The enhanced formation of DNA nanoparticles. Sci. Rep. 2016, 6, 22033,  DOI: 10.1038/srep22033
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    17
    How can macromolecular crowding inhibit biological reactions? The enhanced formation of DNA nanoparticles
    Hou, Sen; Trochimczyk, Piotr; Sun, Lili; Wisniewska, Agnieszka; Kalwarczyk, Tomasz; Zhang, Xuzhu; Wielgus-Kutrowska, Beata; Bzowska, Agnieszka; Holyst, Robert
    Scientific Reports (2016), 6 (), 22033CODEN: SRCEC3; ISSN:2045-2322. (Nature Publishing Group)
    In contrast to the already known effect that macromol. crowding usually promotes biol. reactions, solns. of PEG 6k at high concns. stop the cleavage of DNA by HindIII enzyme, due to the formation of DNA nanoparticles. We characterized the DNA nanoparticles and probed the prerequisites for their formation using multiple techniques such as fluorescence correlation spectroscopy, dynamic light scattering, fluorescence anal. ultracentrifugation etc. In >25% PEG 6k soln., macromol. crowding promotes the formation of DNA nanoparticles with dimensions of several hundreds of nanometers. The formation of DNA nanoparticles is a fast and reversible process. Both plasmid DNA (2686 bp) and double-stranded/single-stranded DNA fragment (66bp/nt) can form nanoparticles. We attribute the enhanced nanoparticle formation to the depletion effect of macromol. crowding. This study presents our idea to enhance the formation of DNA nanoparticles by macromol. crowding, providing the first step towards a final soln. to efficient gene therapy.
  18. 18
    Bielec, K.; Bubak, G.; Kalwarczyk, T.; Holyst, R. Analysis of Brightness of a Single Fluorophore for Quantitative Characterization of Biochemical Reactions. J. Phys. Chem. B 2020, 124, 1941– 1948,  DOI: 10.1021/acs.jpcb.0c00770
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    18
    Analysis of Brightness of a Single Fluorophore for Quantitative Characterization of Biochemical Reactions
    Bielec, Krzysztof; Bubak, Grzegorz; Kalwarczyk, Tomasz; Holyst, Robert
    Journal of Physical Chemistry B (2020), 124 (10), 1941-1948CODEN: JPCBFK; ISSN:1520-5207. (American Chemical Society)
    Intrinsic mol. brightness (MB) is a no. of emitted photons per s per mol. When a substrate labeled by a fluorophore and a second unlabeled substrate form a complex in soln., the MB of the fluorophore changes. Here the authors use this change to det. the equil. const. (K) for the formation of the complex at pM concns. To illustrate this method, the authors used a reaction of DNA hybridization, where only one of the strands was fluorescently labeled. The authors detd. K at the substrate concns. from 80 pM to 30 nM. The authors validated this method against FRET. This method is much simpler than FRET as it requires only one fluorophore in the complex with a very small (a few percent) change in MB.
  19. 19
    Zhou, Y.; Bielec, K.; Pasitsuparoad, P.; Hołyst, R. Single-molecule brightness analysis for the determination of anticancer drug interactions with DNA. Analyst 2020, 145, 6600– 6606,  DOI: 10.1039/D0AN01108H
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    19
    Single-molecule brightness analysis for the determination of anticancer drug interactions with DNA
    Zhou, Ying; Bielec, Krzysztof; Pasitsuparoad, Pakorn; Holyst, Robert
    Analyst (Cambridge, United Kingdom) (2020), 145 (20), 6600-6606CODEN: ANALAO; ISSN:0003-2654. (Royal Society of Chemistry)
    Anthracyclines are one of the most studied anticancer drugs approved for medical treatment. The equil. const. (K) of the reaction between these drugs with DNA in both in vitro and in vivo expts. lacks consensus. The K values vary from 104 up to 108 M-1, which suggest a 1000-fold error in detg. the effective concn. needed to form the drug-DNA complex. Until 2014, only one study by Garcia showed that the binding of anthracycline representative doxorubicin occurs in two reactions. We support this result by brightness anal. at a single mol. level for the four most common anthracyclines: doxorubicin, daunorubicin, epirubicin, and idarubicin.
  20. 20
    Belder, D.; Warnke, J. Electrokinetic effects in poly (ethylene glycol)-coated capillaries induced by specific adsorption of cations. Langmuir 2001, 17, 4962– 4966,  DOI: 10.1021/la010115w
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    20
    Electrokinetic Effects in Poly(ethylene glycol)-Coated Capillaries Induced by Specific Adsorption of Cations
    Belder, Detlev; Warnke, Joerg
    Langmuir (2001), 17 (16), 4962-4966CODEN: LANGD5; ISSN:0743-7463. (American Chemical Society)
    Capillaries coated with poly(ethylene glycol) (PEG) exhibit unique electrokinetic properties because of the specific adsorption of cations at the solid/liq. interface. The adsorption of cations present in methanol- or acetonitrile-contg. electrolytes can induce a pos. surface charge on PEG-coated capillaries. This results in an adjustable anodic electroosmotic flow (EOF) in nonaq. electrolytes, whereas a reduced cathodic EOF is obsd. in aq. electrolytes. The EOF is dependent on the electrolyte constitution, namely, on the type of solvent used and esp. on the abilities of the background cations to interact with polyethers such as PEG. The dependency of the EOF on the electrolyte concn. in nonaq. electrolytes can be explained by two counterbalancing mechanisms: (i) the increase in surface charge d. and (ii) the decrease in double-layer thickness. Using different alkali metal and alk. earth metal cations dissolved in methanol the EOF can be varied over a wide range. The magnitude of the induced anodic EOF was found to vary in the order Ba2+ > Ca2+ > Mg2+ for alk. earth metal ions and K+ > Cs+ > Na+ for alkali metal ions, whereas with lithium ions, a cathodic EOF was obsd. The magnitude of the anodic EOF of PEG-coated capillaries in nonaq. electrolytes was found to be dependent on the coating thickness.
  21. 21
    Lüsse, S.; Arnold, K. The interaction of poly (ethylene glycol) with water studied by 1H and 2H NMR relaxation time measurements. Macromolecules 1996, 29, 4251– 4257,  DOI: 10.1021/ma9508616
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    The Interaction of Poly(ethylene glycol) with Water Studied by 1H and 2H NMR Relaxation Time Measurements
    Luesse, S.; Arnold, K.
    Macromolecules (1996), 29 (12), 4251-4257CODEN: MAMOBX; ISSN:0024-9297. (American Chemical Society)
    NMR relaxation time investigations of water protons (1H) and deuterons (2H) in aq. poly(ethylene glycol) (PEG) solns. are presented for the water content range of 3-90 wt.-%. The no. of bound water mols. per PEG repeat unit is estd. to be 1 from the water relaxation times by a model of fast exchange of water mols. between a bound and an unbound water fraction. For water contents <1 water mol. per PEG monomer, large restrictions in polymer mobility occur which also influence water NMR relaxation. For these low water contents, small ranges of oriented PEG chains are present in the solns. resulting in observable quadrupole splittings in the 2H NMR spectra.
  22. 22
    Banka, P.; Selser, J.; Wang, B.; Shenoy, D.; Martin, R. Nonionic Polymer- Salt Interactions in Dilute Solution: The Poly (ethylene oxide)/LiClO4/Methanol System. Macromolecules 1996, 29, 3956– 3959,  DOI: 10.1021/ma9518159
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    Nonionic Polymer-Salt Interactions in Dilute Solution: The Poly(ethylene oxide)/LiClO4/Methanol System
    Banka, P. A.; Selser, J. C.; Wang, B.; Shenoy, D. K.; Martin, R.
    Macromolecules (1996), 29 (11), 3956-9CODEN: MAMOBX; ISSN:0024-9297. (American Chemical Society)
    The interaction of lithium perchlorate with high mol. wt. poly(ethylene oxide) (PEO) in dil. methanol solns. was studied using cloud point measurements, static and dynamic light scattering, and viscometry. For both good and poor solvent conditions, PEO/LiClO4 complexes form in coil outer portions and charge-charge repulsions between these complexes swell the PEO coils and result in repulsive interactions between coils. A noteworthy consequence of this nonionic polymer → polyelectrolyte conversion is the "salting in" of PEO in methanol by LiClO4.
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    Lu, J. E.; Chen, S. Organized assembling of poly (ethylene glycol)-functionalized Janus nanoparticles induced by select alkali metal ions. Inorg. Chem. Commun. 2019, 110, 107586,  DOI: 10.1016/j.inoche.2019.107586
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    Organized assembling of poly(ethylene glycol)-functionalized Janus nanoparticles induced by select alkali metal ions
    Lu, Jia En; Chen, Shaowei
    Inorganic Chemistry Communications (2019), 110 (), 107586CODEN: ICCOFP; ISSN:1387-7003. (Elsevier B.V.)
    Gold Janus nanoparticles were prepd. by interfacial ligand exchange, with hydrophilic poly(ethylene glycol) (PEG) ligands on one hemisphere and hydrophobic hexanethiolates on the other. Due to specific interaction of PEG with alkali metal ions, the Janus nanoparticles exhibited marked conformational changes forming organized ensembles in the presence of Na+ and K+, as manifested in dynamic light scattering, UV-visible absorption and transmission electron microscopic measurements, whereas no apparent variation was obsd. with other alkali metal ions (e.g., Li+, Rb+), bulk-exchange nanoparticles where the two types of capping ligands were homogeneously mixed on the nanoparticle surface, or nanoparticles capped with the PEG ligands alone. The ion complexation was further probed in NMR measurements. Results from this study indicate that select doping of alkali metal ions into PEG-functionalized nanoparticles may be used for controlled assembly of the Janus nanoparticles.
  24. 24
    Ohki, T.; Harada, M.; Okada, T. Structural and thermodynamic aspects of ionic solvation in concentrated aqueous poly (ethylene glycol). J. Phys. Chem. B 2007, 111, 7245– 7252,  DOI: 10.1021/jp071666j
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    24
    Structural and Thermodynamic Aspects of Ionic Solvation in Concentrated Aqueous Poly(ethylene glycol)
    Ohki, Takumi; Harada, Makoto; Okada, Tetsuo
    Journal of Physical Chemistry B (2007), 111 (25), 7245-7252CODEN: JPCBFK; ISSN:1520-6106. (American Chemical Society)
    Solvation of ions in concd. aq. poly(ethylene glycol) (PEG) has been studied from thermodn. and structural viewpoints using ion-transfer voltammetry at the interface between aq. and nitrobenzene phases and X-ray absorption fine structure (XAFS). Systematic changes in the ion-transfer potential from water to aq. PEG have been confirmed for several ions relative to the corresponding potential of tetraethylammonium ion (Et4N+), which is almost independent of PEG concn. The results obtained for alkali cations strongly suggest the involvement of their complexation with PEG even in relatively dild. PEG solns. It has been implied that the solvation circumstances of Br- and ClO4- are drastically altered when the PEG concn. becomes higher than particular crit. values (e.g., 30-50% PEG200), where free water mols. are diminished because of the hydration of PEG. XAFS measurements have also been performed for K+ and Br- to get direct evidence for these findings. Although the spectra at the K K-edge clearly indicate the presence of a PEG complex of K+ in relatively dild. PEG solns. (∼33% PEG200), an obvious increase in its ion-transfer potential has been detected at lower PEG concns., indicating that complexes formed at the interface rather than in bulk soln. are transferred into an org. phase. Br- is fully hydrated in 0-50% PEG solns., whereas some water mols. are replaced by PEG when the PEG concn. increases. Increasing the PEG concn. causes decreases in the coordination no. from 6 in water to 2-3 in neat PEG. Thus, the present approach not only has elucidated the structural and thermodn. aspects of ionic solvation in aq. PEG but also has provided the information of the hydration of PEG.
  25. 25
    Murari, G. F.; Penido, J. A.; da Silva, H. M.; Baêta, B. E. L.; de Aquino, S. F.; de Lemos, L. R.; Rodrigues, G. D.; Mageste, A. B. Use of aqueous two-phase PEG-salt systems for the removal of anionic surfactant from effluents. Journal of environmental management 2017, 198, 43– 49,  DOI: 10.1016/j.jenvman.2017.04.046
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    25
    Use of aqueous two-phase PEG-salt systems for the removal of anionic surfactant from effluents
    Murari, Gabriella Frade; Penido, Jussara Alves; Morais da Silva, Heriveltom; Baeta, Bruno Eduardo Lobo; Francisco de Aquino, Sergio; Rodrigues de Lemos, Leandro; Rodrigues, Guilherme Dias; Mageste, Aparecida Barbosa
    Journal of Environmental Management (2017), 198 (Part_1), 43-49CODEN: JEVMAW; ISSN:0301-4797. (Elsevier Ltd.)
    Linear alkylbenzene sulfonates (LAS) are synthetic anionic surfactants that are extensively used in many industries. As a result, large vols. of effluents contg. high levels of these compds. are discharged into water bodies, causing risks to aquatic flora and fauna. Then, there is a need for environmentally safe and economically viable technologies for the removal of LAS from aq. matrixes. The present work evaluates the use of aq. two-phase systems (ATPS) composed of PEG and sulfate salts for this purpose, considering the effects of tie line length (TLL), molar mass of polymer, and type of cation-forming salt on the partitioning behavior of LAS. All the LAS partition coeff. (KLAS) values were greater than unity, and the LAS extn. efficiencies (%ELAS) were higher than 97%. The system consisting of PEG 1500 + (NH4)2SO4 + H2O provided the highest KLAS (1083.34) and %ELAS (99.9%), indicating that the method provided good extn. of LAS to the top phase. This system was applied using a real effluent sample in lab.-scale expts. as well as in bench-scale batch trials. The results obtained at the lab. scale showed %ELAS values greater than 98%, while the best KLAS value obtained in the batch expts. was 8.50 (±1.75) (%ELAS = 78.17%). These values demonstrated the potential of ATPS for the removal of LAS from industrial effluents.
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    Morini, M. A.; Messina, P. V.; Schulz, P. C. The interaction of electrolytes with non-ionic surfactant micelles. Colloid Polym. Sci. 2005, 283, 1206– 1218,  DOI: 10.1007/s00396-005-1312-7
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    The interaction of electrolytes with non-ionic surfactant micelles
    Morini, Marcela A.; Messina, Paula V.; Schulz, Pablo C.
    Colloid and Polymer Science (2005), 283 (11), 1206-1218CODEN: CPMSB6; ISSN:0303-402X. (Springer)
    The effect of NaCl and HCl on two non-ionic surfactant micelles was studied using several techniques, including cond. and ion-selective electrodes. Both surfactants exhibit opposite behavior. When Tween 20 is titrated with HCl the cond. notably increases in comparison with water, whereas that of Triton X-100 solns. do not change with respect to water until a certain HCl concn. is reached, when it increases. The hydrogen ion activity is lower in Triton X-100 solns. and higher in Tween 20 solns. than in pure water. Chloride ion activity is higher in Tween 20 solns. than in water, whereas in Triton X-100 the activity does not significantly differ from that in water. The activity of sodium ion is lower in Tween 20 solns. than in water, whereas that in Triton X-100 solns. does not differ from the titrn. of water. These phenomena are explained by the changes in conformation of the non-ionic headgroups, which capture water, and in some cases ions, modifying the activity of ions in the intermicellar soln.
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    Breton, M. F.; Discala, F.; Bacri, L.; Foster, D.; Pelta, J.; Oukhaled, A. Exploration of neutral versus polyelectrolyte behavior of poly (ethylene glycol) s in alkali ion solutions using single-nanopore recording. J. Phys. Chem. Lett. 2013, 4, 2202– 2208,  DOI: 10.1021/jz400938q
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    27
    Exploration of Neutral Versus Polyelectrolyte Behavior of Poly(ethylene glycol)s in Alkali Ion Solutions using Single-Nanopore Recording
    Breton, Marie France; Discala, Francoise; Bacri, Laurent; Foster, Damien; Pelta, Juan; Oukhaled, Abdelghani
    Journal of Physical Chemistry Letters (2013), 4 (13), 2202-2208CODEN: JPCLCD; ISSN:1948-7185. (American Chemical Society)
    We examine the effect of alkali ions (Li+, Na+, K+, Rb+, Cs+) on the partitioning of neutral and flexible poly(ethylene glycol) into the alpha-hemolysin (α-HL) nanopore for a large range of applied voltages at high salt concn. The neutral polymer behaves as if charged, i.e., the event frequency increases with applied voltage, and the residence times decrease with the elec. force for all cations except Li+. In contrast, in the presence of LiCl, we find the classical partitioning behavior of neutral polymers, i.e., the event frequency and the residence times are independent of the applied voltage. Assuming that lithium does not assoc. with PEG enabled us to quantify the relative magnitude of the entropic and enthalpic contribution to the free- energy barrier and the no. of complexed cations using two different arguments; the first est. is based on the balance of forces, and the second is found comparing the blockade ratio in the presence of LiCl (no complexed ions) to the blockade ratio of chains in the presence of the other salts (with complexed ions). This est. is in agreement with recent simulations. These findings demonstrate that the nanopore could prove useful for the rapid probing of the capabilities of different neutral mols. to form complexes with different ions.
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    Terashima, T.; Kawabe, M.; Miyabara, Y.; Yoda, H.; Sawamoto, M. Polymeric pseudo-crown ether for cation recognition via cation template-assisted cyclopolymerization. Nat. Commun. 2013, 4, 2321,  DOI: 10.1038/ncomms3321
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    Polymeric pseudo-crown ether for cation recognition via cation template-assisted cyclopolymerization
    Terashima Takaya; Kawabe Minami; Miyabara Yuichiro; Yoda Hiroaki; Sawamoto Mitsuo
    Nature communications (2013), 4 (), 2321 ISSN:.
    Cyclopolymerization is a chain polymerization of bifunctional monomers via alternating processes of intramolecular cyclization and intermolecular addition, to give soluble linear polymers consisting of in-chain cyclic structures. Though cyclopolymers comprising in-chain multiple large rings potentially show unique functionality, they generally require the elaborate design of bifunctional monomers. Here we report cation template-assisted cyclopolymerization of poly(ethylene glycol) dimethacrylates as an efficient strategy directly yielding polymeric pseudo-crown ethers with large in-chain cavities (up to 30-membered rings) for selective molecular recognition. The key is to select a size-fit metal cation for the spacer unit of the divinyl monomers to form a pseudo-cyclic conformation, where the two vinyl groups are suitably positioned for intramolecular cyclization. The marriage of supramolecular chemistry and polymer chemistry affords efficient, one-pot chemical transformation from common chemical reagents with simple templates to functional cyclopolymers.
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    Nakano, S.-i.; Fujimoto, M.; Hara, H.; Sugimoto, N. Nucleic acid duplex stability: influence of base composition on cation effects. Nucleic acids research 1999, 27, 2957– 2965,  DOI: 10.1093/nar/27.14.2957
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    Nucleic acid duplex stability: influence of base composition on cation effects
    Nakano, Shu-Ichi; Fujimoto, Mariko; Hara, Hideyuki; Sugimoto, Naoki
    Nucleic Acids Research (1999), 27 (14), 2957-2965CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)
    The effects of counter ion on a nucleic acid duplex stability were investigated. Since a linear free energy relationship for the thermostability of oligonucleotide duplexes between those in 1 M and in 100 mM NaCl-phosphate buffer were obsd. regardless of whether they are DNA-DNA, RNA-RNA or RNA-DNA duplexes, simple prediction systems for ΔG037 as well as Tm values in 100 mM NaCl-phosphate buffer were established. These predictions were successful with an av. error of only 2.4°C for Tm and 5.7% for G037 values. The no. of Na+ newly bound to a duplex when the duplex forms (-Δn) was significantly influenced by the base compn., and -Δn for d(GCCAGTTAA)/d(TTAACTGGC) was different for MgCl2, CaCl2, BaCl2 and MnCl2 (from 0.70 to 0.76 with the same order of the duplex stability). Almost no additive effects on the duplex stability was obsd. for NaCl and MgCl2, suggesting a competitive binding for these cations. The sequence-dependent manner of Δn suggests the presence of preferential base pairs or nearest-neighbor base pairs for the cation binding, which would affect nearest-neighbor parameters.
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    Miyoshi, D.; Sugimoto, N. Molecular crowding effects on structure and stability of DNA. Biochimie 2008, 90, 1040– 1051,  DOI: 10.1016/j.biochi.2008.02.009
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    Molecular crowding effects on structure and stability of DNA
    Miyoshi, Daisuke; Sugimoto, Naoki
    Biochimie (2008), 90 (7), 1040-1051CODEN: BICMBE; ISSN:0300-9084. (Elsevier B.V.)
    A review. Living cells contain a variety of biomols. including nucleic acids, proteins, polysaccharides, and metabolites as well as other sol. and insol. components. These biomols. occupy a significant fraction (20-40%) of the cellular vol. The total concn. of biomols. reaches 400 g/L, leading to a crowded intracellular environment referred to as mol. crowding. Therefore, an understanding of the effects of mol. crowding conditions on biomols. is important to broad research fields such as biochem., medical, and pharmaceutical sciences. Here, the authors describe mol. conditions in the cytoplasm and nucleus, which are totally different from in vitro conditions, and then show the biochem. and biophys. consequences of mol. crowding. Finally, the authors discuss the effect of mol. crowding on the structure, stability, and function of nucleic acids and the significance of mol. crowding in biotechnol. and nanotechnol.
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    Kulshrestha, N.; Chatterjee, B.; Gupta, P. Structural, thermal, electrical, and dielectric properties of synthesized nanocomposite solid polymer electrolytes. High Perform. Polym. 2014, 26, 677– 688,  DOI: 10.1177/0954008314541820
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    Structural, thermal, electrical, and dielectric properties of synthesized nanocomposite solid polymer electrolytes
    Kulshrestha, N.; Chatterjee, B.; Gupta, P. N.
    High Performance Polymers (2014), 26 (6), 677-688, 12 pp.CODEN: HPPOEX; ISSN:0954-0083. (Sage Publications Ltd.)
    In the present article, polyvinyl alc. (PVA) polymer, sodium iodide (NaI) salt, and fumed silica nanoparticles nanofiller have been used for the prepn. of solid polymer electrolyte films. Fourier transform IR spectroscopy has been performed to study the vibrational change due to the complexation among polymer, salt, and nanofiller. X-ray diffraction has been carried out to study the structural changes in the PVA:NaI (60:40) polymer electrolyte films with fumed silica nanoparticles as dopant. Differential scanning calorimetry studies show decreasing trend in the glass transition temp. for nanocomposite polymer electrolyte films. Thermogravimetric anal. has been performed to study the thermal degrdn. of the sample. Detn. of transference no. using Wagner's polarization technique indicates that the ions are the dominant mobile species. Maximum cond. of approx. 3.8 U+00D7 10-3 S cm-1 at room temp. has been estd. for PVA:NaI (60:40) film contg. 0.5% fumed silica nanoparticles with low value of activation energy. Dielec. relaxation studies with temp. show shifts of the relaxation time toward higher value for samples of nanocomposite polymer electrolyte films.
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    Alekseev, Y. E.; Garnovskii, A. D.; Zhdanov, Y. A. Complexes of natural carbohydrates with metal cations. Russian chemical reviews 1998, 67, 649– 669,  DOI: 10.1070/RC1998v067n08ABEH000343
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    Yang, L.; Su, Y.; Xu, Y.; Wang, Z.; Guo, Z.; Weng, S.; Yan, C.; Zhang, S.; Wu, J. Interactions between metal ions and carbohydrates. Coordination behavior of neutral erythritol to Ca (II) and lanthanide ions. Inorganic chemistry 2003, 42, 5844– 5856,  DOI: 10.1021/ic0300464
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    Interactions between Metal Ions and Carbohydrates. Coordination Behavior of Neutral Erythritol to Ca(II) and Lanthanide Ions
    Yang, Limin; Su, Yunlan; Xu, Yizhuang; Wang, Zheming; Guo, Zonghui; Weng, Shifu; Yan, Chunhua; Zhang, Shiwei; Wu, Jinguang
    Inorganic Chemistry (2003), 42 (19), 5844-5856CODEN: INOCAJ; ISSN:0020-1669. (American Chemical Society)
    The study of the sugar-metal ion interactions remains one of the main objectives of carbohydrate coordination chem. because the interactions between metal ions and carbohydrates are involved in many biochem. processes. This paper presents a comparison of coordination structures of erythritol with alk.-earth-metal and lanthanide chloride and nitrate in the solid state using FTIR and x-ray diffraction. Neutral, nondeprotonated erythritol (E) reacts with CaCl2 to give three CaCl2-erythritol (CaE(I), CaE(II), CaE(III)) complexes, showing that three of the five general features of calcium-carbohydrate complexes deduced in the ref. encounter contrary examples. Different coordination structures were obsd. for calcium and lanthanide chloride and nitrates. The coordination of carbohydrates to metal ions is complicated, and erythritol, chloride ions, nitrates, water mols., and ethanol (crystn. medium and reaction solvents) have the chance to coordinate to metal ions. IR spectral results show that different lanthanide ions, from LaCl3 to TbCl3, have similar coordination structures with erythritol. Erythritol can act as two bidentate neutral ligands (CaE(I), CaE(II), CaE(III), CaEN, PrE, NdE) or as a three-hydroxyl donor (NdEN). The IR results are consistent with the crystal structures.
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    Fang, Y.; Giesecke, M.; Furo, I. Complexing cations by poly (ethylene oxide): Binding site and binding mode. J. Phys. Chem. B 2017, 121, 2179– 2188,  DOI: 10.1021/acs.jpcb.6b12381
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    Complexing Cations by Poly(ethylene oxide): Binding Site and Binding Mode
    Fang, Yuan; Giesecke, Marianne; Furo, Istvan
    Journal of Physical Chemistry B (2017), 121 (9), 2179-2188CODEN: JPCBFK; ISSN:1520-5207. (American Chemical Society)
    The binding in methanol of K+ and Ba2+ cations to short polyethylene oxide (PEO) chains with ca 4-25 monomeric units was studied via detg. the effective charge of the polymer by a combination of electrophoretic NMR (eNMR) and diffusion NMR expts. These cations were previously found to bind on similar strong manner to long PEO chains. In addn., 1H chem. shift and longitudinal spin relaxation rate changes upon binding were quantified. For both systems, binding was stronger for the short chains than that for longer chains that is attributed mainly to interactions between bound ions. For K+ ions, the equil. binding const. of a cation to a binding site was measured. For both cations, the binding site was estd. to consist of ca 6 monomeric units that coordinate the resp. ions. For the systems with barium, a significant fraction of the bound ions are (BaAnion)+ ion pairs. This leads to a strong anion effect in the effective charge of the oligomers acquired upon barium ion binding. For K+, the coordinating oligomer segment remains rather mobile and individual oligomers exchange rapidly (<<s) between their free and ion-complexing states. In contrast, segmental dynamics slows significantly for the oligomer section that coordinates barium species and, for individual oligomers, binding and non-binding sections do not exchange over the time scale of seconds. Hence, oligomers exchange also slowly (>s) between their states free and complexing barium species.
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    Okada, T. Polyethers in inorganic capillary electrophoresis. Journal of Chromatography A 1999, 834, 73– 87,  DOI: 10.1016/S0021-9673(98)00738-9
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    Polyethers in inorganic capillary electrophoresis
    Okada, Tetsuo
    Journal of Chromatography A (1999), 834 (1 + 2), 73-87CODEN: JCRAEY; ISSN:0021-9673. (Elsevier Science B.V.)
    A review with 57 refs. Various additives are employed in running solns. in capillary electrophoresis (CE) to enhance sepn. performance and selectivity. Complexing agents are successfully employed (in most cases indispensable) in inorg. CE, esp. for the sepn. of metal ions. Studies in inorg. anal. chem. were mostly directed to the methodol. developments of the analyses of transition metal ions, and extensive efforts have permitted development of a no. of effective reagents for their detn. Thus, there are numerous reagents forming complexes with transition metal ions. In contrast, ligands effectively complexing main group metal cations are very few. Polyethers are rather unique examples of such ligands capable of effectively interacting with hard cations. Thus, this naturally leads to designs of sepn. where polyethers are incorporated in running soln. of CE to achieve better sepns. of hard cations. Polyethers have another interesting feature which is also potentially useful in CE sepn.; polyether-H2O mixts. provide unique electrophoretic sepn. media, which allow one to modify electrophoretic sepn. selectivity much more effectively than usual org. solvents. In this review, the author discusses roles of polyethers in inorg. CE from two different viewpoints, (1) complex formation and (2) sepn. medium modifiers, and to provide the perspectives of these useful compds. in inorg. CE. Some new data are also presented to show the ability of polyethers as medium modifiers.
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    Czyrko, J.; Sliwiak, J.; Imiolczyk, B.; Gdaniec, Z.; Jaskolski, M.; Brzezinski, K. Metal-cation regulation of enzyme dynamics is a key factor influencing the activity of S-adenosyl-L-homocysteine hydrolase from Pseudomonas aeruginosa. Sci. Rep. 2018, 8, 11334,  DOI: 10.1038/s41598-018-29535-y
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    Metal-cation regulation of enzyme dynamics is a key factor influencing the activity of S-adenosyl-L-homocysteine hydrolase from Pseudomonas aeruginosa
    Czyrko Justyna; Brzezinski Krzysztof; Sliwiak Joanna; Imiolczyk Barbara; Jaskolski Mariusz; Gdaniec Zofia; Jaskolski Mariusz
    Scientific reports (2018), 8 (1), 11334 ISSN:.
    S-adenosyl-L-homocysteine hydrolase from Pseudomonas aeruginosa (PaSAHase) coordinates one K(+) ion and one Zn(2+) ion in the substrate binding area. The cations affect the enzymatic activity and substrate binding but the molecular mechanisms of their action are unknown. Enzymatic and isothermal titration calorimetry studies demonstrated that the K(+) ions stimulate the highest activity and strongest ligand binding in comparison to other alkali cations, while the Zn(2+) ions inhibit the enzyme activity. PaSAHase was crystallized in the presence of adenine nucleosides and K(+) or Rb(+) ions. The crystal structures show that the alkali ion is coordinated in close proximity of the purine ring and a (23)Na NMR study showed that the monovalent cation coordination site is formed upon ligand binding. The cation, bound in the area of a molecular hinge, orders and accurately positions the amide group of Q65 residue to allow its interaction with the ligand. Moreover, binding of potassium is required to enable unique dynamic properties of the enzyme that ensure its maximum catalytic activity. The Zn(2+) ion is bound in the area of a molecular gate that regulates access to the active site. Zn(2+) coordination switches the gate to a shut state and arrests the enzyme in its closed, inactive conformation.
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    Hu, Y.; Komoto, J.; Huang, Y.; Gomi, T.; Ogawa, H.; Takata, Y.; Fujioka, M.; Takusagawa, F. Crystal structure of S-adenosylhomocysteine hydrolase from rat liver. Biochemistry 1999, 38, 8323– 8333,  DOI: 10.1021/bi990332k
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    Crystal structure of S-adenosylhomocysteine hydrolase from rat liver
    Hu, Yongbo; Komoto, Junichi; Huang, Yafei; Gomi, Tomoharu; Ogawa, Hirofumi; Takata, Yoshimi; Fujioka, Motoji; Takusagawa, Fusao
    Biochemistry (1999), 38 (26), 8323-8333CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
    The crystal structure of rat liver S-adenosyl-L-homocysteine hydrolase (EC 3.3.1.1) (I), which catalyzes the reversible hydrolysis of S-adenosylhomocysteine (AdoHcy), was detd. at 2.8 Å resoln. I from rat liver was a tetrameric enzyme with 431 amino acid residues in each identical subunit. The subunit was composed of the catalytic domain, the NAD-binding domain, and the small C-terminal domain. Both catalytic and NAD-binding domains were folded into an ellipsoid with a typical α/β twisted open sheet structure. The C-terminal section was far from the main body of the subunit and extended into the opposite subunit. An NAD mol. bound to the consensus NAD-binding cleft of the NAD-binding domain. The peptide folding pattern of the catalytic domain was quite similar to the patterns obsd. in many methyltransferases. Although the crystal structure did not contain AdoHcy or its analog, there was a well-formed AdoHcy-binding crevice in the catalytic domain. Without introducing any major structural changes, an AdoHcy mol. could be placed in the catalytic domain. In the structure described here, the catalytic and NAD-binding domains were quite far apart from each other. Thus, I appears to have an "open" conformation in the absence of substrate. It is likely that binding of AdoHcy induces a large conformational change so as to place the ribose moiety of AdoHcy in close proximity to the nicotinamide moiety of NAD. A catalytic mechanism for I was proposed on the basis of this crystal structure. Glu-155 acts as a proton acceptor from the O3'-H when the proton of C3'-H is abstracted by NAD. His-54 or Asp-130 acts as a general acid-base catalyst, while Cys-194 modulates the oxidn. state of the bound NAD. The polypeptide folding pattern of the catalytic domain suggests that AdoHcy mols. can travel freely to and from I and methyltransferases to properly regulate methyltransferase activities. The authors believe that the crystal structure described here can provide insight into the mol. architecture of this important regulatory enzyme.
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    Riordan, J. F. The role of metals in enzyme activity. Ann. Clin. Lab. Sci. 1977, 7, 119– 129
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    The role of metals in enzyme activity
    Riordan, James F.
    Annals of Clinical and Laboratory Science (1977), 7 (2), 119-29CODEN: ACLSCP; ISSN:0091-7370.
    A review with 32 refs.
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    Kilpin, K. J.; Dyson, P. J. Enzyme inhibition by metal complexes: concepts, strategies and applications. Chemical Science 2013, 4, 1410– 1419,  DOI: 10.1039/c3sc22349c
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    Enzyme inhibition by metal complexes: concepts, strategies and applications
    Kilpin, Kelly J.; Dyson, Paul J.
    Chemical Science (2013), 4 (4), 1410-1419CODEN: CSHCCN; ISSN:2041-6520. (Royal Society of Chemistry)
    A review. Metal complexes are increasingly being used to inhibit enzymes. The reasons for this increased interest arise from the special features that metal complexes offer, e.g. the facile construction of 3D architectures that tightly fill enzyme active sites increasing selectivity and the possibility of facile coordination to protein residues that enhances enzyme inhibition. In this review we classify the main modes of enzyme inhibition by metal-based complexes and correlate the enzyme inhibition activity to macroscopic properties such as anticancer activity.
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    Fonseca Guerra, C.; Bickelhaupt, F. M.; Snijders, J. G.; Baerends, E. J. Hydrogen bonding in DNA base pairs: reconciliation of theory and experiment. J. Am. Chem. Soc. 2000, 122, 4117– 4128,  DOI: 10.1021/ja993262d
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    Auffinger, P.; Westhof, E. Water and ion binding around RNA and DNA (C, G) oligomers. Journal of molecular biology 2000, 300, 1113– 1131,  DOI: 10.1006/jmbi.2000.3894
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    Water and Ion Binding Around RNA and DNA (C,G) Oligomers
    Auffinger, Pascal; Westhof, Eric
    Journal of Molecular Biology (2000), 300 (5), 1113-1131CODEN: JMOBAK; ISSN:0022-2836. (Academic Press)
    The dynamics, hydration, and ion-binding features of two duplexes, the A(r(CG)12) and the B(d(CG)12), in a neutralizing aq. environment with 0.25 M added KCl have been investigated by mol. dynamics (MD) simulations. The regular repeats of the same C=G base-pair motif have been exploited as a statistical alternative to long MD simulations in order to extend the sampling of the conformational space. The trajectories demonstrate the larger flexibility of DNA compared to RNA helixes. This flexibility results in less well defined hydration patterns around the DNA than around the RNA backbone atoms. Yet, 22 hydration sites are clearly characterized around both nucleic acid structures. With addnl. results from MD simulations, the following hydration scale for C=G pairs can be deduced: A-DNA<RNA (+3 H2O) and B-DNA<RNA (+2 H2O). The calcd. residence times of water mols. in the first hydration shell of the helixes range from 0.5 to 1 ns, in good agreement with available exptl. data. Such water mols. are essentially found in the vicinity of the phosphate groups and in the DNA minor groove. The calcd. no. of ions that break into the first hydration shell of the nucleic acids is close to 0.5 per base-pair for both RNA and DNA. These ions form contacts essentially with the oxygen atoms of the phosphate groups and with the guanine N7 and O6 atoms; they display residence times in the deep/major groove approaching 500 ps. Further, a significant sequence-dependent effect on ion binding has been noted. Despite slight structural differences, K+ binds essentially to GpC and not to CpG steps. These results may be of importance for understanding various sequence-dependent binding affinities. Addnl., the data help to rationalize the exptl. obsd. differences in gel electrophoretic mobility between RNA and DNA as due to the difference in hydration (two water mols. in favor of RNA) rather than to strong ion-binding features, which are largely similar for both nucleic acid structures. (c) 2000 Academic Press.
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    Smith, A. M.; Lee, A. A.; Perkin, S. The electrostatic screening length in concentrated electrolytes increases with concentration. journal of physical chemistry letters 2016, 7, 2157– 2163,  DOI: 10.1021/acs.jpclett.6b00867
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    The Electrostatic Screening Length in Concentrated Electrolytes Increases with Concentration
    Smith, Alexander M.; Lee, Alpha A.; Perkin, Susan
    Journal of Physical Chemistry Letters (2016), 7 (12), 2157-2163CODEN: JPCLCD; ISSN:1948-7185. (American Chemical Society)
    According to classical electrolyte theories interactions in dil. (low ion d.) electrolytes decay exponentially with distance, with the Debye screening length the characteristic length scale. This decay length decreases monotonically with increasing ion concn. due to effective screening of charges over short distances. Thus, within the Debye model no long-range forces are expected in concd. electrolytes. Here the authors reveal, using exptl. detection of the interaction between two planar charged surfaces across a wide range of electrolytes, that beyond the dil. (Debye-Hueckel) regime the screening length increases with increasing concn. The screening lengths for all electrolytes studied-including aq. NaCl solns., ionic liqs. dild. with propylene carbonate, and pure ionic liqs.-collapse onto a single curve when scaled by the dielec. const. This non-monotonic variation of the screening length with concn., and its generality across ionic liqs. and aq. salt solns., demonstrates an important characteristic of concd. electrolytes of substantial relevance from biol. to energy storage.
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    Winkler, K.; Paszewski, M.; Kalwarczyk, T.; Kalwarczyk, E.; Wojciechowski, T.; Gorecka, E.; Pociecha, D.; Holyst, R.; Fialkowski, M. Ionic strength-controlled deposition of charged nanoparticles on a solid substrate. J. Phys. Chem. C 2011, 115, 19096– 19103,  DOI: 10.1021/jp206704s
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    Ionic Strength-Controlled Deposition of Charged Nanoparticles on a Solid Substrate
    Winkler, Katarzyna; Paszewski, Maciej; Kalwarczyk, Tomasz; Kalwarczyk, Ewelina; Wojciechowski, Tomasz; Gorecka, Ewa; Pociecha, Damian; Holyst, Robert; Fialkowski, Marcin
    Journal of Physical Chemistry C (2011), 115 (39), 19096-19103CODEN: JPCCCK; ISSN:1932-7447. (American Chemical Society)
    There is exptl. evidence that in solns. of low ionic strength, charged nanoparticles (NPs) adsorbing onto an oppositely charged substrate cannot form dense monolayers due to the electrostatic repulsion. Here, we investigated the adsorption onto neg. charged substrates occurring in solns. contg. pos. charged NPs and salt for a wide range of ionic strengths. We found that the salt added in high concn., above 2M, stabilizes the soln. and the NPs adsorb on the substrate to form dense coatings characterized by the surface coverage of about 50%. In this regime, the adsorption rate was found to grow with the square of the NP concn. This effect of salt on the deposition process provides a facile method of coating of solid substrates with NP monolayers. The d. of such monolayers depends on and can be easily controlled by the deposition time, as well as by the salt and NP concn. in the plating soln.
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    Zhou, H.-X.; Rivas, G.; Minton, A. P. Macromolecular crowding and confinement: biochemical, biophysical, and potential physiological consequences. Annu. Rev. Biophys. 2008, 37, 375– 397,  DOI: 10.1146/annurev.biophys.37.032807.125817
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    Macromolecular crowding and confinement: Biochemical, biophysical, and potential physiological consequences
    Zhou, Huan-Xiang; Rivas, German; Minton, Allen P.
    Annual Review of Biophysics (2008), 37 (), 375-397CODEN: ARBNCV ISSN:. (Annual Reviews Inc.)
    A review. Expected and obsd. effects of vol. exclusion on the free energy of rigid and flexible macromols. in crowded and confined systems, and consequent effects of crowding and confinement on macromol. reaction rates and equil. are summarized. Findings from relevant theor./simulation and exptl. literature published from 2004 onward are reviewed. Addnl. complexity arising from the heterogeneity of local environments in biol. media, and the presence of nonspecific interactions between macromols. over and above steric repulsion, are discussed. Theor. and exptl. approaches to the characterization of crowding- and confinement-induced effects in systems approaching the complexity of living organisms are suggested.
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    Harada, R.; Sugita, Y.; Feig, M. Protein crowding affects hydration structure and dynamics. J. Am. Chem. Soc. 2012, 134, 4842– 4849,  DOI: 10.1021/ja211115q
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    Protein Crowding Affects Hydration Structure and Dynamics
    Harada, Ryuhei; Sugita, Yuji; Feig, Michael
    Journal of the American Chemical Society (2012), 134 (10), 4842-4849CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    The effect of protein crowding on the structure and dynamics of water was examd. from explicit solvent mol. dynamics simulations of a series of protein G and protein G/villin systems at different protein concns. Hydration structure was analyzed in terms of radial distribution functions, three-dimensional hydration sites, and preservation of tetrahedral coordination. Anal. of hydration dynamics focused on self-diffusion rates and dielec. consts. as a function of crowding. The results show significant changes in both structure and dynamics of water under highly crowded conditions. The structure of water is altered mostly beyond the first solvation shell. Diffusion rates and dielec. consts. are significantly reduced following linear trends as a function of crowding reflecting highly constrained water in crowded environments. The reduced dynamics of diffusion is expected to be strongly related to hydrodynamic properties of crowded cellular environments while the reduced dielec. const. under crowded conditions has implications for the stability of biomols. in crowded environments. The results from this study suggest a prescription for modeling solvation in simulations of cellular environments.
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    Branca, C.; Magazu, S.; Maisano, G.; Migliardo, F.; Migliardo, P.; Romeo, G. Hydration study of PEG/water mixtures by quasi elastic light scattering, acoustic and rheological measurements. J. Phys. Chem. B 2002, 106, 10272– 10276,  DOI: 10.1021/jp014345v
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    Hydration Study of PEG/Water Mixtures by Quasi Elastic Light Scattering, Acoustic and Rheological Measurements
    Branca, C.; Magazu, S.; Maisano, G.; Migliardo, F.; Migliardo, P.; Romeo, G.
    Journal of Physical Chemistry B (2002), 106 (39), 10272-10276CODEN: JPCBFK; ISSN:1520-6106. (American Chemical Society)
    To study the hydration effect of poly(ethylene glycol) (PEG) and its dependence on the mol. wt., we report viscosity, compressibility, and quasi elastic light-scattering measurements on aq. solns. of PEG with different mean mol. wt., Mw, at different concn. and temp. values. In particular, ultrasonic technique allows to evaluate the hydration no. for PEG samples at different polymn. degrees. The values deduced by ultrasonic technique are then compared with those deduced from viscosity data following the Linow and Philipp's model. PCS technique allows to obtain information on the hydrodynamic radius and its dependence on the polymer Mw at different temp. values.

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Cite this: J. Phys. Chem. Lett. 2022, 13, 1, 112–117
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https://doi.org/10.1021/acs.jpclett.1c03596
Published December 28, 2021

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  • Abstract

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    Figure 1

    Figure 1. Hybridization of complementary DNA oligonucleotides was used as a model biochemical reaction. (a) The change of the fluorescent properties as a result of the complex formation makes it possible to use the brightness analysis method. (18,19) We investigated two biophysical factors that affect the formation of the noncovalent complex: ionic strength and crowded environment. (b) Ionic strength. The formation of the complex was observed at different ionic strengths. (c) Crowded environment. We used the most common biorelated crowders of different sizes and chemical structures. The influence of cosolute is negligible even at 109 excess over the concentration of substrates (region I). There is no correlation between sizes and structures of different agents (region II). (d) The comparison of the interactions in a crowded environment with respect to different ionic strengths allowed the determination of the sodium cation complexation by crowder molecules.

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    Figure 2

    Figure 2. Hybridization reaction was measured at constant ionic strength in the presence of various crowder agents. The effect of crowder molecules on reaction components (presented in nanomolar concentration) is negligible below a few wt % of crowder concentration (∼200 mM). However, when the molar concentration of the crowder is approximately a few molar, the reaction is affected.

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    Figure 3

    Figure 3. Influence of ionic strength on the hybridization of complementary DNA oligonucleotide strands (violet points). The data were compared with the observed change of Debye length recorded by Smith et al. (42) The scatters represent data points, and the line (both marked in green) shows dependence of Debye length as a function of ionic strength according to Debye–Hückel theory.

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    Figure 4

    Figure 4. (a) Hybridization constant depending on the concentration of sodium ions without the addition of cosolute (violet dots) and in the presence of 40 wt % PEG 400 (green dots). The arrows in the plot correspond to the difference between the K values and thus the number of complexed sodium cations. (b) The sodium ion complexation by PEG 400 at various concentrations of sodium cations in the solution. The violet line represents the average value of the constant κ determined per one PEG 400 monomer (nmer ≈ 8.5). Considering those results, we infer that the complexation of sodium ions by crowders, but not its direct interactions, is in fact mostly responsible for the changes in equilibrium constants. (c) Comparison of determining sodium complexation equilibrium constant by different crowders, excluding depletion force interaction. (d) (left panel) In an ionic solution, the partially charged compound is surrounded by an electrical double layer. Sodium cations screen negative charge of the DNA backbone, which facilities the complex formation. (right panel) After the addition of crowders, sodium cations are getting complexed by weak interaction with crowder molecules. In high concentration of crowders, repulsion of the DNA strands became more pronounced because of reduction of screening charges on the DNA backbone.

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    Figure 5

    Figure 5. Contribution of interactions in a crowder system. The bar plots (violet) represent experimentally measured K values of 13 bp oligonucleotide hybridization in 100 mM buffer: without crowders, with presence of 40 wt % PEG 400, and in theoretical system where 73 mM of sodium ions were complexed by 40 wt % PEG 400 (κ = 1 M–1) without secondary interactions. The future quantitative analysis forces separation of the contributions of ion complexation and the depletion forces.

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  • References

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      A review. Recent progresses in microscopy and spectroscopy technologies for intracellular measurements, quant. studies using exptl. model systems, and computer simulations have led to an improved understanding of the interactions and reactions of nucleic acids under the macromol. crowding and small-mol. crowding conditions, which are the central theme of this review.
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      Chemical Interactions of Polyethylene Glycols (PEGs) and Glycerol with Protein Functional Groups: Applications to Effects of PEG and Glycerol on Protein Processes
      Knowles, D. B.; Shkel, Irina A.; Phan, Noel M.; Sternke, Matt; Lingeman, Emily; Cheng, Xian; Cheng, Lixue; O'Connor, Kevin; Record, M. Thomas
      Biochemistry (2015), 54 (22), 3528-3542CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
      The authors obtain the data needed to predict chem. interactions of polyethylene glycols (PEGs) and glycerol with proteins and related org. compds. and thereby interpret or predict chem. effects of PEGs on protein processes. To accomplish this, the authors det. interactions of glycerol and tetraEG with >30 model compds. displaying the major C, N, and O functional groups of proteins. Anal. of these data yields coeffs. (α values) that quantify interactions of glycerol, tetraEG, and PEG end (-CH2OH) and interior (-CH2OCH2-) groups with these groups, relative to interactions with water. TetraEG (strongly) and glycerol (weakly) interact favorably with arom. C, amide N, and cationic N, but unfavorably with amide O, carboxylate O, and salt ions. Strongly unfavorable O and salt anion interactions help make both small and large PEGs effective protein precipitants. Interactions of tetraEG and PEG interior groups with aliph. C are quite favorable, while interactions of glycerol and PEG end groups with aliph. C are not. Hence, tetraEG and PEG300 favor unfolding of the DNA-binding domain of lac repressor (lacDBD), while glycerol and di- and monoethylene glycol are stabilizers. Favorable interactions with arom. and aliph. C explain why PEG400 greatly increases the soly. of arom. hydrocarbons and steroids. PEG400-steroid interactions are unusually favorable, presumably because of simultaneous interactions of multiple PEG interior groups with the fused ring system of the steroid. Using α values reported here, chem. contributions to PEG m-values can be predicted or interpreted in terms of changes in water-accessible surface area (ΔASA) and sepd. from excluded vol. effects.
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    11. 11
      Bubak, G.; Kwapiszewska, K.; Kalwarczyk, T.; Bielec, K.; Andryszewski, T.; Iwan, M.; Bubak, S.; Hołyst, R. Quantifying Nanoscale Viscosity and Structures of Living Cells Nucleus from Mobility Measurements. J. Phys. Chem. Lett. 2021, 12, 294– 301,  DOI: 10.1021/acs.jpclett.0c03052
      11
      Quantifying Nanoscale Viscosity and Structures of Living Cells Nucleus from Mobility Measurements
      Bubak, Grzegorz; Kwapiszewska, Karina; Kalwarczyk, Tomasz; Bielec, Krzysztof; Andryszewski, Tomasz; Iwan, Michalina; Bubak, Szymon; Holyst, Robert
      Journal of Physical Chemistry Letters (2021), 12 (1), 294-301CODEN: JPCLCD; ISSN:1948-7185. (American Chemical Society)
      Understanding the mobility of nano-objects in the eukaryotic cell nucleus, at multiple length-scales, is essential for dissecting nuclear structure-function relationships both in space and in time. Here, we demonstrate, using single-mol. fluorescent correlation spectroscopies, that motion of inert probes (proteins, polymers, or nanoparticles) with diams. ranging from 2.6 to 150 nm is mostly unobstructed in a nucleus. Supported by the anal. of electron tomog. images, these results advocate the ~ 150 nm-wide interchromosomal channels filled with the aq. dild. protein soln. The nucleus is percolated by these channels to allow various cargos to migrate freely at the nanoscale. We detd. the vol. of interchromosomal channels in the HeLa cell nucleus to 237 ± 61 fL, which constitutes 34% of the cell nucleus vol. The vol. fraction of mobile proteins in channels equals 16% ± 4%, and the concn. is 1 mM.
    12. 12
      Sozański, K.; Ruhnow, F.; Wiśniewska, A.; Tabaka, M.; Diez, S.; Hołyst, R. Small crowders slow down kinesin-1 stepping by hindering motor domain diffusion. Physical review letters 2015, 115, 218102,  DOI: 10.1103/PhysRevLett.115.218102
      12
      Small crowders slow down kinesin-1 stepping by hindering motor domain diffusion
      Sozanski, Krzysztof; Ruhnow, Felix; Wisniewska, Agnieszka; Tabaka, Marcin; Diez, Stefan; Holyst, Robert
      Physical Review Letters (2015), 115 (21), 218102/1-218102/5CODEN: PRLTAO; ISSN:0031-9007. (American Physical Society)
      The dimeric motor protein kinesin-1 moves processively along microtubules against forces of up to 7 pN. However, the mechanism of force generation is still debated. Here, we point to the crucial importance of diffusion of the tethered motor domain for the stepping of kinesin-1: small crowders stop the motor at a viscosity of 5 mPa·s-corresponding to a hydrodynamic load in the sub-fN (∼10-4 pN) range-whereas large crowders have no impact even at viscosities above 100 mPa·s. This indicates that the scale-dependent, effective viscosity experienced by the tethered motor domain is a key factor detg. kinesin's functionality. Our results emphasize the role of diffusion in the kinesin-1 stepping mechanism and the general importance of the viscosity scaling paradigm in nanomechanics.
    13. 13
      Hou, S.; Ziebacz, N.; Kalwarczyk, T.; Kaminski, T. S.; Wieczorek, S. A.; Holyst, R. Influence of nano-viscosity and depletion interactions on cleavage of DNA by enzymes in glycerol and poly (ethylene glycol) solutions: qualitative analysis. Soft Matter 2011, 7, 3092– 3099,  DOI: 10.1039/C0SM00899K
      13
      Influence of nano-viscosity and depletion interactions on cleavage of DNA by enzymes in glycerol and poly(ethylene glycol) solutions: qualitative analysis
      Hou, Sen; Ziebacz, Natalia; Kalwarczyk, Tomasz; Kaminski, Tomasz S.; Wieczorek, Stefan A.; Holyst, Robert
      Soft Matter (2011), 7 (7), 3092-3099CODEN: SMOABF; ISSN:1744-683X. (Royal Society of Chemistry)
      Biochem. reactions in living systems take place in an environment crowded by various macromols. and ligands. Therefore exptl. data obtained in buffer do not reflect in vivo conditions. We have used glycerol, poly(ethylene glycol) (PEG) 6000 and PEG 8 M solns. to investigate the influence of the crowded environment on cleavage of plasmid DNA by restriction enzyme HindIII. PEG 6000 soln. can effectively slow down the cleavage process. However, neither PEG 8 M soln. of the same viscosity as PEG 6000 soln. nor glycerol soln. of the same concn. as PEG 6000 soln. slows the cleavage of DNA appreciably. The viscosity experienced by the biomols. (here called nano-viscosity) and aggregation induced by the depletion interactions between DNA mols. in polymer soln. (PEG 6000) are two factors responsible for slow cleavage of DNA. We have ruled out the change of pH and denaturation of HindIII as possible sources for the effect.
    14. 14
      Baltierra-Jasso, L. E.; Morten, M. J.; Laflor, L.; Quinn, S. D.; Magennis, S. W. Crowding-induced hybridization of single DNA hairpins. J. Am. Chem. Soc. 2015, 137, 16020– 16023,  DOI: 10.1021/jacs.5b11829
      14
      Crowding-Induced Hybridization of Single DNA Hairpins
      Baltierra-Jasso, Laura E.; Morten, Michael J.; Laflor, Linda; Quinn, Steven D.; Magennis, Steven W.
      Journal of the American Chemical Society (2015), 137 (51), 16020-16023CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      It is clear that a crowded environment influences the structure, dynamics, and interactions of biol. mols., but the complexity of this phenomenon demands the development of new exptl. and theor. approaches. Here we use two complementary single-mol. FRET techniques to show that the kinetics of DNA base pairing and unpairing, which are fundamental to both the biol. role of DNA and its technol. applications, are strongly modulated by a crowded environment. We directly obsd. single DNA hairpins, which are excellent model systems for studying hybridization, either freely diffusing in soln. or immobilized on a surface under crowding conditions. The hairpins followed two-state folding dynamics with a closing rate increasing by 4-fold and the opening rate decreasing 2-fold, for only modest concns. of crowder [10% (wt./wt.) polyethylene glycol (PEG)]. These expts. serve both to unambiguously highlight the impact of a crowded environment on a fundamental biol. process, DNA base pairing, and to illustrate the benefits of single-mol. approaches to probing the structure and dynamics of complex biomol. systems.
    15. 15
      Mitchison, T. Colloid osmotic parameterization and measurement of subcellular crowding. Molecular biology of the cell 2019, 30, 173– 180,  DOI: 10.1091/mbc.E18-09-0549
      15
      Colloid osmotic parameterization and measurement of subcellular crowding
      Mitchison, T. J.
      Molecular Biology of the Cell (2019), 30 (2), 173-180CODEN: MBCEEV; ISSN:1939-4586. (American Society for Cell Biology)
      A review. Crowding of the subcellular environment by macromols. is thought to promote protein aggregation and phase sepn. A challenge is how to parameterize the degree of crowding of the cell interior or artificial solns. that is relevant to these reactions. Here I review colloid osmotic pressure as a crowding metric. This pressure is generated by solns. of macromols. in contact with pores that are permeable to water and ions but not macromols. It generates depletion forces that push macromols. together in crowded solns. and thus promotes aggregation and phase sepn. I discuss measurements of colloid osmotic pressure inside cells using the nucleus, the cytoplasmic gel, and fluorescence resonant energy transfer (FRET) biosensors as osmometers, which return a range of values from 1 to 20 kPa. I argue for a low value, 1-2 kPa, in frog eggs and perhaps more generally. This value is close to the linear range on concn.-pressure curves and is thus not crowded from an osmotic perspective. I discuss the implications of a low crowding pressure inside cells for phase sepn. biol., buffer design, and proteome evolution. I also discuss a pressure-tension model for nuclear shape, where colloid osmotic pressure generated by nuclear protein import inflates the nucleus.
    16. 16
      Balcells, C.; Pastor, I.; Pitulice, L.; Hernández, C.; Via, M.; Garcés, J. L.; Madurga, S.; Vilaseca, E.; Isvoran, A.; Cascante, M. Macromolecular crowding upon in-vivo-like enzyme-kinetics: effect of enzyme-obstacle size ratio. New Frontiers in Chemistry 2015, 24, 3
      There is no corresponding record for this reference.
    17. 17
      Hou, S.; Trochimczyk, P.; Sun, L.; Wisniewska, A.; Kalwarczyk, T.; Zhang, X.; Wielgus-Kutrowska, B.; Bzowska, A.; Holyst, R. How can macromolecular crowding inhibit biological reactions? The enhanced formation of DNA nanoparticles. Sci. Rep. 2016, 6, 22033,  DOI: 10.1038/srep22033
      17
      How can macromolecular crowding inhibit biological reactions? The enhanced formation of DNA nanoparticles
      Hou, Sen; Trochimczyk, Piotr; Sun, Lili; Wisniewska, Agnieszka; Kalwarczyk, Tomasz; Zhang, Xuzhu; Wielgus-Kutrowska, Beata; Bzowska, Agnieszka; Holyst, Robert
      Scientific Reports (2016), 6 (), 22033CODEN: SRCEC3; ISSN:2045-2322. (Nature Publishing Group)
      In contrast to the already known effect that macromol. crowding usually promotes biol. reactions, solns. of PEG 6k at high concns. stop the cleavage of DNA by HindIII enzyme, due to the formation of DNA nanoparticles. We characterized the DNA nanoparticles and probed the prerequisites for their formation using multiple techniques such as fluorescence correlation spectroscopy, dynamic light scattering, fluorescence anal. ultracentrifugation etc. In >25% PEG 6k soln., macromol. crowding promotes the formation of DNA nanoparticles with dimensions of several hundreds of nanometers. The formation of DNA nanoparticles is a fast and reversible process. Both plasmid DNA (2686 bp) and double-stranded/single-stranded DNA fragment (66bp/nt) can form nanoparticles. We attribute the enhanced nanoparticle formation to the depletion effect of macromol. crowding. This study presents our idea to enhance the formation of DNA nanoparticles by macromol. crowding, providing the first step towards a final soln. to efficient gene therapy.
    18. 18
      Bielec, K.; Bubak, G.; Kalwarczyk, T.; Holyst, R. Analysis of Brightness of a Single Fluorophore for Quantitative Characterization of Biochemical Reactions. J. Phys. Chem. B 2020, 124, 1941– 1948,  DOI: 10.1021/acs.jpcb.0c00770
      18
      Analysis of Brightness of a Single Fluorophore for Quantitative Characterization of Biochemical Reactions
      Bielec, Krzysztof; Bubak, Grzegorz; Kalwarczyk, Tomasz; Holyst, Robert
      Journal of Physical Chemistry B (2020), 124 (10), 1941-1948CODEN: JPCBFK; ISSN:1520-5207. (American Chemical Society)
      Intrinsic mol. brightness (MB) is a no. of emitted photons per s per mol. When a substrate labeled by a fluorophore and a second unlabeled substrate form a complex in soln., the MB of the fluorophore changes. Here the authors use this change to det. the equil. const. (K) for the formation of the complex at pM concns. To illustrate this method, the authors used a reaction of DNA hybridization, where only one of the strands was fluorescently labeled. The authors detd. K at the substrate concns. from 80 pM to 30 nM. The authors validated this method against FRET. This method is much simpler than FRET as it requires only one fluorophore in the complex with a very small (a few percent) change in MB.
    19. 19
      Zhou, Y.; Bielec, K.; Pasitsuparoad, P.; Hołyst, R. Single-molecule brightness analysis for the determination of anticancer drug interactions with DNA. Analyst 2020, 145, 6600– 6606,  DOI: 10.1039/D0AN01108H
      19
      Single-molecule brightness analysis for the determination of anticancer drug interactions with DNA
      Zhou, Ying; Bielec, Krzysztof; Pasitsuparoad, Pakorn; Holyst, Robert
      Analyst (Cambridge, United Kingdom) (2020), 145 (20), 6600-6606CODEN: ANALAO; ISSN:0003-2654. (Royal Society of Chemistry)
      Anthracyclines are one of the most studied anticancer drugs approved for medical treatment. The equil. const. (K) of the reaction between these drugs with DNA in both in vitro and in vivo expts. lacks consensus. The K values vary from 104 up to 108 M-1, which suggest a 1000-fold error in detg. the effective concn. needed to form the drug-DNA complex. Until 2014, only one study by Garcia showed that the binding of anthracycline representative doxorubicin occurs in two reactions. We support this result by brightness anal. at a single mol. level for the four most common anthracyclines: doxorubicin, daunorubicin, epirubicin, and idarubicin.
    20. 20
      Belder, D.; Warnke, J. Electrokinetic effects in poly (ethylene glycol)-coated capillaries induced by specific adsorption of cations. Langmuir 2001, 17, 4962– 4966,  DOI: 10.1021/la010115w
      20
      Electrokinetic Effects in Poly(ethylene glycol)-Coated Capillaries Induced by Specific Adsorption of Cations
      Belder, Detlev; Warnke, Joerg
      Langmuir (2001), 17 (16), 4962-4966CODEN: LANGD5; ISSN:0743-7463. (American Chemical Society)
      Capillaries coated with poly(ethylene glycol) (PEG) exhibit unique electrokinetic properties because of the specific adsorption of cations at the solid/liq. interface. The adsorption of cations present in methanol- or acetonitrile-contg. electrolytes can induce a pos. surface charge on PEG-coated capillaries. This results in an adjustable anodic electroosmotic flow (EOF) in nonaq. electrolytes, whereas a reduced cathodic EOF is obsd. in aq. electrolytes. The EOF is dependent on the electrolyte constitution, namely, on the type of solvent used and esp. on the abilities of the background cations to interact with polyethers such as PEG. The dependency of the EOF on the electrolyte concn. in nonaq. electrolytes can be explained by two counterbalancing mechanisms: (i) the increase in surface charge d. and (ii) the decrease in double-layer thickness. Using different alkali metal and alk. earth metal cations dissolved in methanol the EOF can be varied over a wide range. The magnitude of the induced anodic EOF was found to vary in the order Ba2+ > Ca2+ > Mg2+ for alk. earth metal ions and K+ > Cs+ > Na+ for alkali metal ions, whereas with lithium ions, a cathodic EOF was obsd. The magnitude of the anodic EOF of PEG-coated capillaries in nonaq. electrolytes was found to be dependent on the coating thickness.
    21. 21
      Lüsse, S.; Arnold, K. The interaction of poly (ethylene glycol) with water studied by 1H and 2H NMR relaxation time measurements. Macromolecules 1996, 29, 4251– 4257,  DOI: 10.1021/ma9508616
      21
      The Interaction of Poly(ethylene glycol) with Water Studied by 1H and 2H NMR Relaxation Time Measurements
      Luesse, S.; Arnold, K.
      Macromolecules (1996), 29 (12), 4251-4257CODEN: MAMOBX; ISSN:0024-9297. (American Chemical Society)
      NMR relaxation time investigations of water protons (1H) and deuterons (2H) in aq. poly(ethylene glycol) (PEG) solns. are presented for the water content range of 3-90 wt.-%. The no. of bound water mols. per PEG repeat unit is estd. to be 1 from the water relaxation times by a model of fast exchange of water mols. between a bound and an unbound water fraction. For water contents <1 water mol. per PEG monomer, large restrictions in polymer mobility occur which also influence water NMR relaxation. For these low water contents, small ranges of oriented PEG chains are present in the solns. resulting in observable quadrupole splittings in the 2H NMR spectra.
    22. 22
      Banka, P.; Selser, J.; Wang, B.; Shenoy, D.; Martin, R. Nonionic Polymer- Salt Interactions in Dilute Solution: The Poly (ethylene oxide)/LiClO4/Methanol System. Macromolecules 1996, 29, 3956– 3959,  DOI: 10.1021/ma9518159
      22
      Nonionic Polymer-Salt Interactions in Dilute Solution: The Poly(ethylene oxide)/LiClO4/Methanol System
      Banka, P. A.; Selser, J. C.; Wang, B.; Shenoy, D. K.; Martin, R.
      Macromolecules (1996), 29 (11), 3956-9CODEN: MAMOBX; ISSN:0024-9297. (American Chemical Society)
      The interaction of lithium perchlorate with high mol. wt. poly(ethylene oxide) (PEO) in dil. methanol solns. was studied using cloud point measurements, static and dynamic light scattering, and viscometry. For both good and poor solvent conditions, PEO/LiClO4 complexes form in coil outer portions and charge-charge repulsions between these complexes swell the PEO coils and result in repulsive interactions between coils. A noteworthy consequence of this nonionic polymer → polyelectrolyte conversion is the "salting in" of PEO in methanol by LiClO4.
    23. 23
      Lu, J. E.; Chen, S. Organized assembling of poly (ethylene glycol)-functionalized Janus nanoparticles induced by select alkali metal ions. Inorg. Chem. Commun. 2019, 110, 107586,  DOI: 10.1016/j.inoche.2019.107586
      23
      Organized assembling of poly(ethylene glycol)-functionalized Janus nanoparticles induced by select alkali metal ions
      Lu, Jia En; Chen, Shaowei
      Inorganic Chemistry Communications (2019), 110 (), 107586CODEN: ICCOFP; ISSN:1387-7003. (Elsevier B.V.)
      Gold Janus nanoparticles were prepd. by interfacial ligand exchange, with hydrophilic poly(ethylene glycol) (PEG) ligands on one hemisphere and hydrophobic hexanethiolates on the other. Due to specific interaction of PEG with alkali metal ions, the Janus nanoparticles exhibited marked conformational changes forming organized ensembles in the presence of Na+ and K+, as manifested in dynamic light scattering, UV-visible absorption and transmission electron microscopic measurements, whereas no apparent variation was obsd. with other alkali metal ions (e.g., Li+, Rb+), bulk-exchange nanoparticles where the two types of capping ligands were homogeneously mixed on the nanoparticle surface, or nanoparticles capped with the PEG ligands alone. The ion complexation was further probed in NMR measurements. Results from this study indicate that select doping of alkali metal ions into PEG-functionalized nanoparticles may be used for controlled assembly of the Janus nanoparticles.
    24. 24
      Ohki, T.; Harada, M.; Okada, T. Structural and thermodynamic aspects of ionic solvation in concentrated aqueous poly (ethylene glycol). J. Phys. Chem. B 2007, 111, 7245– 7252,  DOI: 10.1021/jp071666j
      24
      Structural and Thermodynamic Aspects of Ionic Solvation in Concentrated Aqueous Poly(ethylene glycol)
      Ohki, Takumi; Harada, Makoto; Okada, Tetsuo
      Journal of Physical Chemistry B (2007), 111 (25), 7245-7252CODEN: JPCBFK; ISSN:1520-6106. (American Chemical Society)
      Solvation of ions in concd. aq. poly(ethylene glycol) (PEG) has been studied from thermodn. and structural viewpoints using ion-transfer voltammetry at the interface between aq. and nitrobenzene phases and X-ray absorption fine structure (XAFS). Systematic changes in the ion-transfer potential from water to aq. PEG have been confirmed for several ions relative to the corresponding potential of tetraethylammonium ion (Et4N+), which is almost independent of PEG concn. The results obtained for alkali cations strongly suggest the involvement of their complexation with PEG even in relatively dild. PEG solns. It has been implied that the solvation circumstances of Br- and ClO4- are drastically altered when the PEG concn. becomes higher than particular crit. values (e.g., 30-50% PEG200), where free water mols. are diminished because of the hydration of PEG. XAFS measurements have also been performed for K+ and Br- to get direct evidence for these findings. Although the spectra at the K K-edge clearly indicate the presence of a PEG complex of K+ in relatively dild. PEG solns. (∼33% PEG200), an obvious increase in its ion-transfer potential has been detected at lower PEG concns., indicating that complexes formed at the interface rather than in bulk soln. are transferred into an org. phase. Br- is fully hydrated in 0-50% PEG solns., whereas some water mols. are replaced by PEG when the PEG concn. increases. Increasing the PEG concn. causes decreases in the coordination no. from 6 in water to 2-3 in neat PEG. Thus, the present approach not only has elucidated the structural and thermodn. aspects of ionic solvation in aq. PEG but also has provided the information of the hydration of PEG.
    25. 25
      Murari, G. F.; Penido, J. A.; da Silva, H. M.; Baêta, B. E. L.; de Aquino, S. F.; de Lemos, L. R.; Rodrigues, G. D.; Mageste, A. B. Use of aqueous two-phase PEG-salt systems for the removal of anionic surfactant from effluents. Journal of environmental management 2017, 198, 43– 49,  DOI: 10.1016/j.jenvman.2017.04.046
      25
      Use of aqueous two-phase PEG-salt systems for the removal of anionic surfactant from effluents
      Murari, Gabriella Frade; Penido, Jussara Alves; Morais da Silva, Heriveltom; Baeta, Bruno Eduardo Lobo; Francisco de Aquino, Sergio; Rodrigues de Lemos, Leandro; Rodrigues, Guilherme Dias; Mageste, Aparecida Barbosa
      Journal of Environmental Management (2017), 198 (Part_1), 43-49CODEN: JEVMAW; ISSN:0301-4797. (Elsevier Ltd.)
      Linear alkylbenzene sulfonates (LAS) are synthetic anionic surfactants that are extensively used in many industries. As a result, large vols. of effluents contg. high levels of these compds. are discharged into water bodies, causing risks to aquatic flora and fauna. Then, there is a need for environmentally safe and economically viable technologies for the removal of LAS from aq. matrixes. The present work evaluates the use of aq. two-phase systems (ATPS) composed of PEG and sulfate salts for this purpose, considering the effects of tie line length (TLL), molar mass of polymer, and type of cation-forming salt on the partitioning behavior of LAS. All the LAS partition coeff. (KLAS) values were greater than unity, and the LAS extn. efficiencies (%ELAS) were higher than 97%. The system consisting of PEG 1500 + (NH4)2SO4 + H2O provided the highest KLAS (1083.34) and %ELAS (99.9%), indicating that the method provided good extn. of LAS to the top phase. This system was applied using a real effluent sample in lab.-scale expts. as well as in bench-scale batch trials. The results obtained at the lab. scale showed %ELAS values greater than 98%, while the best KLAS value obtained in the batch expts. was 8.50 (±1.75) (%ELAS = 78.17%). These values demonstrated the potential of ATPS for the removal of LAS from industrial effluents.
    26. 26
      Morini, M. A.; Messina, P. V.; Schulz, P. C. The interaction of electrolytes with non-ionic surfactant micelles. Colloid Polym. Sci. 2005, 283, 1206– 1218,  DOI: 10.1007/s00396-005-1312-7
      26
      The interaction of electrolytes with non-ionic surfactant micelles
      Morini, Marcela A.; Messina, Paula V.; Schulz, Pablo C.
      Colloid and Polymer Science (2005), 283 (11), 1206-1218CODEN: CPMSB6; ISSN:0303-402X. (Springer)
      The effect of NaCl and HCl on two non-ionic surfactant micelles was studied using several techniques, including cond. and ion-selective electrodes. Both surfactants exhibit opposite behavior. When Tween 20 is titrated with HCl the cond. notably increases in comparison with water, whereas that of Triton X-100 solns. do not change with respect to water until a certain HCl concn. is reached, when it increases. The hydrogen ion activity is lower in Triton X-100 solns. and higher in Tween 20 solns. than in pure water. Chloride ion activity is higher in Tween 20 solns. than in water, whereas in Triton X-100 the activity does not significantly differ from that in water. The activity of sodium ion is lower in Tween 20 solns. than in water, whereas that in Triton X-100 solns. does not differ from the titrn. of water. These phenomena are explained by the changes in conformation of the non-ionic headgroups, which capture water, and in some cases ions, modifying the activity of ions in the intermicellar soln.
    27. 27
      Breton, M. F.; Discala, F.; Bacri, L.; Foster, D.; Pelta, J.; Oukhaled, A. Exploration of neutral versus polyelectrolyte behavior of poly (ethylene glycol) s in alkali ion solutions using single-nanopore recording. J. Phys. Chem. Lett. 2013, 4, 2202– 2208,  DOI: 10.1021/jz400938q
      27
      Exploration of Neutral Versus Polyelectrolyte Behavior of Poly(ethylene glycol)s in Alkali Ion Solutions using Single-Nanopore Recording
      Breton, Marie France; Discala, Francoise; Bacri, Laurent; Foster, Damien; Pelta, Juan; Oukhaled, Abdelghani
      Journal of Physical Chemistry Letters (2013), 4 (13), 2202-2208CODEN: JPCLCD; ISSN:1948-7185. (American Chemical Society)
      We examine the effect of alkali ions (Li+, Na+, K+, Rb+, Cs+) on the partitioning of neutral and flexible poly(ethylene glycol) into the alpha-hemolysin (α-HL) nanopore for a large range of applied voltages at high salt concn. The neutral polymer behaves as if charged, i.e., the event frequency increases with applied voltage, and the residence times decrease with the elec. force for all cations except Li+. In contrast, in the presence of LiCl, we find the classical partitioning behavior of neutral polymers, i.e., the event frequency and the residence times are independent of the applied voltage. Assuming that lithium does not assoc. with PEG enabled us to quantify the relative magnitude of the entropic and enthalpic contribution to the free- energy barrier and the no. of complexed cations using two different arguments; the first est. is based on the balance of forces, and the second is found comparing the blockade ratio in the presence of LiCl (no complexed ions) to the blockade ratio of chains in the presence of the other salts (with complexed ions). This est. is in agreement with recent simulations. These findings demonstrate that the nanopore could prove useful for the rapid probing of the capabilities of different neutral mols. to form complexes with different ions.
    28. 28
      Terashima, T.; Kawabe, M.; Miyabara, Y.; Yoda, H.; Sawamoto, M. Polymeric pseudo-crown ether for cation recognition via cation template-assisted cyclopolymerization. Nat. Commun. 2013, 4, 2321,  DOI: 10.1038/ncomms3321
      28
      Polymeric pseudo-crown ether for cation recognition via cation template-assisted cyclopolymerization
      Terashima Takaya; Kawabe Minami; Miyabara Yuichiro; Yoda Hiroaki; Sawamoto Mitsuo
      Nature communications (2013), 4 (), 2321 ISSN:.
      Cyclopolymerization is a chain polymerization of bifunctional monomers via alternating processes of intramolecular cyclization and intermolecular addition, to give soluble linear polymers consisting of in-chain cyclic structures. Though cyclopolymers comprising in-chain multiple large rings potentially show unique functionality, they generally require the elaborate design of bifunctional monomers. Here we report cation template-assisted cyclopolymerization of poly(ethylene glycol) dimethacrylates as an efficient strategy directly yielding polymeric pseudo-crown ethers with large in-chain cavities (up to 30-membered rings) for selective molecular recognition. The key is to select a size-fit metal cation for the spacer unit of the divinyl monomers to form a pseudo-cyclic conformation, where the two vinyl groups are suitably positioned for intramolecular cyclization. The marriage of supramolecular chemistry and polymer chemistry affords efficient, one-pot chemical transformation from common chemical reagents with simple templates to functional cyclopolymers.
    29. 29
      Nakano, S.-i.; Fujimoto, M.; Hara, H.; Sugimoto, N. Nucleic acid duplex stability: influence of base composition on cation effects. Nucleic acids research 1999, 27, 2957– 2965,  DOI: 10.1093/nar/27.14.2957
      29
      Nucleic acid duplex stability: influence of base composition on cation effects
      Nakano, Shu-Ichi; Fujimoto, Mariko; Hara, Hideyuki; Sugimoto, Naoki
      Nucleic Acids Research (1999), 27 (14), 2957-2965CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)
      The effects of counter ion on a nucleic acid duplex stability were investigated. Since a linear free energy relationship for the thermostability of oligonucleotide duplexes between those in 1 M and in 100 mM NaCl-phosphate buffer were obsd. regardless of whether they are DNA-DNA, RNA-RNA or RNA-DNA duplexes, simple prediction systems for ΔG037 as well as Tm values in 100 mM NaCl-phosphate buffer were established. These predictions were successful with an av. error of only 2.4°C for Tm and 5.7% for G037 values. The no. of Na+ newly bound to a duplex when the duplex forms (-Δn) was significantly influenced by the base compn., and -Δn for d(GCCAGTTAA)/d(TTAACTGGC) was different for MgCl2, CaCl2, BaCl2 and MnCl2 (from 0.70 to 0.76 with the same order of the duplex stability). Almost no additive effects on the duplex stability was obsd. for NaCl and MgCl2, suggesting a competitive binding for these cations. The sequence-dependent manner of Δn suggests the presence of preferential base pairs or nearest-neighbor base pairs for the cation binding, which would affect nearest-neighbor parameters.
    30. 30
      Miyoshi, D.; Sugimoto, N. Molecular crowding effects on structure and stability of DNA. Biochimie 2008, 90, 1040– 1051,  DOI: 10.1016/j.biochi.2008.02.009
      30
      Molecular crowding effects on structure and stability of DNA
      Miyoshi, Daisuke; Sugimoto, Naoki
      Biochimie (2008), 90 (7), 1040-1051CODEN: BICMBE; ISSN:0300-9084. (Elsevier B.V.)
      A review. Living cells contain a variety of biomols. including nucleic acids, proteins, polysaccharides, and metabolites as well as other sol. and insol. components. These biomols. occupy a significant fraction (20-40%) of the cellular vol. The total concn. of biomols. reaches 400 g/L, leading to a crowded intracellular environment referred to as mol. crowding. Therefore, an understanding of the effects of mol. crowding conditions on biomols. is important to broad research fields such as biochem., medical, and pharmaceutical sciences. Here, the authors describe mol. conditions in the cytoplasm and nucleus, which are totally different from in vitro conditions, and then show the biochem. and biophys. consequences of mol. crowding. Finally, the authors discuss the effect of mol. crowding on the structure, stability, and function of nucleic acids and the significance of mol. crowding in biotechnol. and nanotechnol.
    31. 31
      Kulshrestha, N.; Chatterjee, B.; Gupta, P. Structural, thermal, electrical, and dielectric properties of synthesized nanocomposite solid polymer electrolytes. High Perform. Polym. 2014, 26, 677– 688,  DOI: 10.1177/0954008314541820
      31
      Structural, thermal, electrical, and dielectric properties of synthesized nanocomposite solid polymer electrolytes
      Kulshrestha, N.; Chatterjee, B.; Gupta, P. N.
      High Performance Polymers (2014), 26 (6), 677-688, 12 pp.CODEN: HPPOEX; ISSN:0954-0083. (Sage Publications Ltd.)
      In the present article, polyvinyl alc. (PVA) polymer, sodium iodide (NaI) salt, and fumed silica nanoparticles nanofiller have been used for the prepn. of solid polymer electrolyte films. Fourier transform IR spectroscopy has been performed to study the vibrational change due to the complexation among polymer, salt, and nanofiller. X-ray diffraction has been carried out to study the structural changes in the PVA:NaI (60:40) polymer electrolyte films with fumed silica nanoparticles as dopant. Differential scanning calorimetry studies show decreasing trend in the glass transition temp. for nanocomposite polymer electrolyte films. Thermogravimetric anal. has been performed to study the thermal degrdn. of the sample. Detn. of transference no. using Wagner's polarization technique indicates that the ions are the dominant mobile species. Maximum cond. of approx. 3.8 U+00D7 10-3 S cm-1 at room temp. has been estd. for PVA:NaI (60:40) film contg. 0.5% fumed silica nanoparticles with low value of activation energy. Dielec. relaxation studies with temp. show shifts of the relaxation time toward higher value for samples of nanocomposite polymer electrolyte films.
    32. 32
      Alekseev, Y. E.; Garnovskii, A. D.; Zhdanov, Y. A. Complexes of natural carbohydrates with metal cations. Russian chemical reviews 1998, 67, 649– 669,  DOI: 10.1070/RC1998v067n08ABEH000343
      There is no corresponding record for this reference.
    33. 33
      Yang, L.; Su, Y.; Xu, Y.; Wang, Z.; Guo, Z.; Weng, S.; Yan, C.; Zhang, S.; Wu, J. Interactions between metal ions and carbohydrates. Coordination behavior of neutral erythritol to Ca (II) and lanthanide ions. Inorganic chemistry 2003, 42, 5844– 5856,  DOI: 10.1021/ic0300464
      33
      Interactions between Metal Ions and Carbohydrates. Coordination Behavior of Neutral Erythritol to Ca(II) and Lanthanide Ions
      Yang, Limin; Su, Yunlan; Xu, Yizhuang; Wang, Zheming; Guo, Zonghui; Weng, Shifu; Yan, Chunhua; Zhang, Shiwei; Wu, Jinguang
      Inorganic Chemistry (2003), 42 (19), 5844-5856CODEN: INOCAJ; ISSN:0020-1669. (American Chemical Society)
      The study of the sugar-metal ion interactions remains one of the main objectives of carbohydrate coordination chem. because the interactions between metal ions and carbohydrates are involved in many biochem. processes. This paper presents a comparison of coordination structures of erythritol with alk.-earth-metal and lanthanide chloride and nitrate in the solid state using FTIR and x-ray diffraction. Neutral, nondeprotonated erythritol (E) reacts with CaCl2 to give three CaCl2-erythritol (CaE(I), CaE(II), CaE(III)) complexes, showing that three of the five general features of calcium-carbohydrate complexes deduced in the ref. encounter contrary examples. Different coordination structures were obsd. for calcium and lanthanide chloride and nitrates. The coordination of carbohydrates to metal ions is complicated, and erythritol, chloride ions, nitrates, water mols., and ethanol (crystn. medium and reaction solvents) have the chance to coordinate to metal ions. IR spectral results show that different lanthanide ions, from LaCl3 to TbCl3, have similar coordination structures with erythritol. Erythritol can act as two bidentate neutral ligands (CaE(I), CaE(II), CaE(III), CaEN, PrE, NdE) or as a three-hydroxyl donor (NdEN). The IR results are consistent with the crystal structures.
    34. 34
      Fang, Y.; Giesecke, M.; Furo, I. Complexing cations by poly (ethylene oxide): Binding site and binding mode. J. Phys. Chem. B 2017, 121, 2179– 2188,  DOI: 10.1021/acs.jpcb.6b12381
      34
      Complexing Cations by Poly(ethylene oxide): Binding Site and Binding Mode
      Fang, Yuan; Giesecke, Marianne; Furo, Istvan
      Journal of Physical Chemistry B (2017), 121 (9), 2179-2188CODEN: JPCBFK; ISSN:1520-5207. (American Chemical Society)
      The binding in methanol of K+ and Ba2+ cations to short polyethylene oxide (PEO) chains with ca 4-25 monomeric units was studied via detg. the effective charge of the polymer by a combination of electrophoretic NMR (eNMR) and diffusion NMR expts. These cations were previously found to bind on similar strong manner to long PEO chains. In addn., 1H chem. shift and longitudinal spin relaxation rate changes upon binding were quantified. For both systems, binding was stronger for the short chains than that for longer chains that is attributed mainly to interactions between bound ions. For K+ ions, the equil. binding const. of a cation to a binding site was measured. For both cations, the binding site was estd. to consist of ca 6 monomeric units that coordinate the resp. ions. For the systems with barium, a significant fraction of the bound ions are (BaAnion)+ ion pairs. This leads to a strong anion effect in the effective charge of the oligomers acquired upon barium ion binding. For K+, the coordinating oligomer segment remains rather mobile and individual oligomers exchange rapidly (<<s) between their free and ion-complexing states. In contrast, segmental dynamics slows significantly for the oligomer section that coordinates barium species and, for individual oligomers, binding and non-binding sections do not exchange over the time scale of seconds. Hence, oligomers exchange also slowly (>s) between their states free and complexing barium species.
    35. 35
      Okada, T. Polyethers in inorganic capillary electrophoresis. Journal of Chromatography A 1999, 834, 73– 87,  DOI: 10.1016/S0021-9673(98)00738-9
      35
      Polyethers in inorganic capillary electrophoresis
      Okada, Tetsuo
      Journal of Chromatography A (1999), 834 (1 + 2), 73-87CODEN: JCRAEY; ISSN:0021-9673. (Elsevier Science B.V.)
      A review with 57 refs. Various additives are employed in running solns. in capillary electrophoresis (CE) to enhance sepn. performance and selectivity. Complexing agents are successfully employed (in most cases indispensable) in inorg. CE, esp. for the sepn. of metal ions. Studies in inorg. anal. chem. were mostly directed to the methodol. developments of the analyses of transition metal ions, and extensive efforts have permitted development of a no. of effective reagents for their detn. Thus, there are numerous reagents forming complexes with transition metal ions. In contrast, ligands effectively complexing main group metal cations are very few. Polyethers are rather unique examples of such ligands capable of effectively interacting with hard cations. Thus, this naturally leads to designs of sepn. where polyethers are incorporated in running soln. of CE to achieve better sepns. of hard cations. Polyethers have another interesting feature which is also potentially useful in CE sepn.; polyether-H2O mixts. provide unique electrophoretic sepn. media, which allow one to modify electrophoretic sepn. selectivity much more effectively than usual org. solvents. In this review, the author discusses roles of polyethers in inorg. CE from two different viewpoints, (1) complex formation and (2) sepn. medium modifiers, and to provide the perspectives of these useful compds. in inorg. CE. Some new data are also presented to show the ability of polyethers as medium modifiers.
    36. 36
      Czyrko, J.; Sliwiak, J.; Imiolczyk, B.; Gdaniec, Z.; Jaskolski, M.; Brzezinski, K. Metal-cation regulation of enzyme dynamics is a key factor influencing the activity of S-adenosyl-L-homocysteine hydrolase from Pseudomonas aeruginosa. Sci. Rep. 2018, 8, 11334,  DOI: 10.1038/s41598-018-29535-y
      36
      Metal-cation regulation of enzyme dynamics is a key factor influencing the activity of S-adenosyl-L-homocysteine hydrolase from Pseudomonas aeruginosa
      Czyrko Justyna; Brzezinski Krzysztof; Sliwiak Joanna; Imiolczyk Barbara; Jaskolski Mariusz; Gdaniec Zofia; Jaskolski Mariusz
      Scientific reports (2018), 8 (1), 11334 ISSN:.
      S-adenosyl-L-homocysteine hydrolase from Pseudomonas aeruginosa (PaSAHase) coordinates one K(+) ion and one Zn(2+) ion in the substrate binding area. The cations affect the enzymatic activity and substrate binding but the molecular mechanisms of their action are unknown. Enzymatic and isothermal titration calorimetry studies demonstrated that the K(+) ions stimulate the highest activity and strongest ligand binding in comparison to other alkali cations, while the Zn(2+) ions inhibit the enzyme activity. PaSAHase was crystallized in the presence of adenine nucleosides and K(+) or Rb(+) ions. The crystal structures show that the alkali ion is coordinated in close proximity of the purine ring and a (23)Na NMR study showed that the monovalent cation coordination site is formed upon ligand binding. The cation, bound in the area of a molecular hinge, orders and accurately positions the amide group of Q65 residue to allow its interaction with the ligand. Moreover, binding of potassium is required to enable unique dynamic properties of the enzyme that ensure its maximum catalytic activity. The Zn(2+) ion is bound in the area of a molecular gate that regulates access to the active site. Zn(2+) coordination switches the gate to a shut state and arrests the enzyme in its closed, inactive conformation.
    37. 37
      Hu, Y.; Komoto, J.; Huang, Y.; Gomi, T.; Ogawa, H.; Takata, Y.; Fujioka, M.; Takusagawa, F. Crystal structure of S-adenosylhomocysteine hydrolase from rat liver. Biochemistry 1999, 38, 8323– 8333,  DOI: 10.1021/bi990332k
      37
      Crystal structure of S-adenosylhomocysteine hydrolase from rat liver
      Hu, Yongbo; Komoto, Junichi; Huang, Yafei; Gomi, Tomoharu; Ogawa, Hirofumi; Takata, Yoshimi; Fujioka, Motoji; Takusagawa, Fusao
      Biochemistry (1999), 38 (26), 8323-8333CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
      The crystal structure of rat liver S-adenosyl-L-homocysteine hydrolase (EC 3.3.1.1) (I), which catalyzes the reversible hydrolysis of S-adenosylhomocysteine (AdoHcy), was detd. at 2.8 Å resoln. I from rat liver was a tetrameric enzyme with 431 amino acid residues in each identical subunit. The subunit was composed of the catalytic domain, the NAD-binding domain, and the small C-terminal domain. Both catalytic and NAD-binding domains were folded into an ellipsoid with a typical α/β twisted open sheet structure. The C-terminal section was far from the main body of the subunit and extended into the opposite subunit. An NAD mol. bound to the consensus NAD-binding cleft of the NAD-binding domain. The peptide folding pattern of the catalytic domain was quite similar to the patterns obsd. in many methyltransferases. Although the crystal structure did not contain AdoHcy or its analog, there was a well-formed AdoHcy-binding crevice in the catalytic domain. Without introducing any major structural changes, an AdoHcy mol. could be placed in the catalytic domain. In the structure described here, the catalytic and NAD-binding domains were quite far apart from each other. Thus, I appears to have an "open" conformation in the absence of substrate. It is likely that binding of AdoHcy induces a large conformational change so as to place the ribose moiety of AdoHcy in close proximity to the nicotinamide moiety of NAD. A catalytic mechanism for I was proposed on the basis of this crystal structure. Glu-155 acts as a proton acceptor from the O3'-H when the proton of C3'-H is abstracted by NAD. His-54 or Asp-130 acts as a general acid-base catalyst, while Cys-194 modulates the oxidn. state of the bound NAD. The polypeptide folding pattern of the catalytic domain suggests that AdoHcy mols. can travel freely to and from I and methyltransferases to properly regulate methyltransferase activities. The authors believe that the crystal structure described here can provide insight into the mol. architecture of this important regulatory enzyme.
    38. 38
      Riordan, J. F. The role of metals in enzyme activity. Ann. Clin. Lab. Sci. 1977, 7, 119– 129
      38
      The role of metals in enzyme activity
      Riordan, James F.
      Annals of Clinical and Laboratory Science (1977), 7 (2), 119-29CODEN: ACLSCP; ISSN:0091-7370.
      A review with 32 refs.
    39. 39
      Kilpin, K. J.; Dyson, P. J. Enzyme inhibition by metal complexes: concepts, strategies and applications. Chemical Science 2013, 4, 1410– 1419,  DOI: 10.1039/c3sc22349c
      39
      Enzyme inhibition by metal complexes: concepts, strategies and applications
      Kilpin, Kelly J.; Dyson, Paul J.
      Chemical Science (2013), 4 (4), 1410-1419CODEN: CSHCCN; ISSN:2041-6520. (Royal Society of Chemistry)
      A review. Metal complexes are increasingly being used to inhibit enzymes. The reasons for this increased interest arise from the special features that metal complexes offer, e.g. the facile construction of 3D architectures that tightly fill enzyme active sites increasing selectivity and the possibility of facile coordination to protein residues that enhances enzyme inhibition. In this review we classify the main modes of enzyme inhibition by metal-based complexes and correlate the enzyme inhibition activity to macroscopic properties such as anticancer activity.
    40. 40
      Fonseca Guerra, C.; Bickelhaupt, F. M.; Snijders, J. G.; Baerends, E. J. Hydrogen bonding in DNA base pairs: reconciliation of theory and experiment. J. Am. Chem. Soc. 2000, 122, 4117– 4128,  DOI: 10.1021/ja993262d
      There is no corresponding record for this reference.
    41. 41
      Auffinger, P.; Westhof, E. Water and ion binding around RNA and DNA (C, G) oligomers. Journal of molecular biology 2000, 300, 1113– 1131,  DOI: 10.1006/jmbi.2000.3894
      41
      Water and Ion Binding Around RNA and DNA (C,G) Oligomers
      Auffinger, Pascal; Westhof, Eric
      Journal of Molecular Biology (2000), 300 (5), 1113-1131CODEN: JMOBAK; ISSN:0022-2836. (Academic Press)
      The dynamics, hydration, and ion-binding features of two duplexes, the A(r(CG)12) and the B(d(CG)12), in a neutralizing aq. environment with 0.25 M added KCl have been investigated by mol. dynamics (MD) simulations. The regular repeats of the same C=G base-pair motif have been exploited as a statistical alternative to long MD simulations in order to extend the sampling of the conformational space. The trajectories demonstrate the larger flexibility of DNA compared to RNA helixes. This flexibility results in less well defined hydration patterns around the DNA than around the RNA backbone atoms. Yet, 22 hydration sites are clearly characterized around both nucleic acid structures. With addnl. results from MD simulations, the following hydration scale for C=G pairs can be deduced: A-DNA<RNA (+3 H2O) and B-DNA<RNA (+2 H2O). The calcd. residence times of water mols. in the first hydration shell of the helixes range from 0.5 to 1 ns, in good agreement with available exptl. data. Such water mols. are essentially found in the vicinity of the phosphate groups and in the DNA minor groove. The calcd. no. of ions that break into the first hydration shell of the nucleic acids is close to 0.5 per base-pair for both RNA and DNA. These ions form contacts essentially with the oxygen atoms of the phosphate groups and with the guanine N7 and O6 atoms; they display residence times in the deep/major groove approaching 500 ps. Further, a significant sequence-dependent effect on ion binding has been noted. Despite slight structural differences, K+ binds essentially to GpC and not to CpG steps. These results may be of importance for understanding various sequence-dependent binding affinities. Addnl., the data help to rationalize the exptl. obsd. differences in gel electrophoretic mobility between RNA and DNA as due to the difference in hydration (two water mols. in favor of RNA) rather than to strong ion-binding features, which are largely similar for both nucleic acid structures. (c) 2000 Academic Press.
    42. 42
      Smith, A. M.; Lee, A. A.; Perkin, S. The electrostatic screening length in concentrated electrolytes increases with concentration. journal of physical chemistry letters 2016, 7, 2157– 2163,  DOI: 10.1021/acs.jpclett.6b00867
      42
      The Electrostatic Screening Length in Concentrated Electrolytes Increases with Concentration
      Smith, Alexander M.; Lee, Alpha A.; Perkin, Susan
      Journal of Physical Chemistry Letters (2016), 7 (12), 2157-2163CODEN: JPCLCD; ISSN:1948-7185. (American Chemical Society)
      According to classical electrolyte theories interactions in dil. (low ion d.) electrolytes decay exponentially with distance, with the Debye screening length the characteristic length scale. This decay length decreases monotonically with increasing ion concn. due to effective screening of charges over short distances. Thus, within the Debye model no long-range forces are expected in concd. electrolytes. Here the authors reveal, using exptl. detection of the interaction between two planar charged surfaces across a wide range of electrolytes, that beyond the dil. (Debye-Hueckel) regime the screening length increases with increasing concn. The screening lengths for all electrolytes studied-including aq. NaCl solns., ionic liqs. dild. with propylene carbonate, and pure ionic liqs.-collapse onto a single curve when scaled by the dielec. const. This non-monotonic variation of the screening length with concn., and its generality across ionic liqs. and aq. salt solns., demonstrates an important characteristic of concd. electrolytes of substantial relevance from biol. to energy storage.
    43. 43
      Winkler, K.; Paszewski, M.; Kalwarczyk, T.; Kalwarczyk, E.; Wojciechowski, T.; Gorecka, E.; Pociecha, D.; Holyst, R.; Fialkowski, M. Ionic strength-controlled deposition of charged nanoparticles on a solid substrate. J. Phys. Chem. C 2011, 115, 19096– 19103,  DOI: 10.1021/jp206704s
      43
      Ionic Strength-Controlled Deposition of Charged Nanoparticles on a Solid Substrate
      Winkler, Katarzyna; Paszewski, Maciej; Kalwarczyk, Tomasz; Kalwarczyk, Ewelina; Wojciechowski, Tomasz; Gorecka, Ewa; Pociecha, Damian; Holyst, Robert; Fialkowski, Marcin
      Journal of Physical Chemistry C (2011), 115 (39), 19096-19103CODEN: JPCCCK; ISSN:1932-7447. (American Chemical Society)
      There is exptl. evidence that in solns. of low ionic strength, charged nanoparticles (NPs) adsorbing onto an oppositely charged substrate cannot form dense monolayers due to the electrostatic repulsion. Here, we investigated the adsorption onto neg. charged substrates occurring in solns. contg. pos. charged NPs and salt for a wide range of ionic strengths. We found that the salt added in high concn., above 2M, stabilizes the soln. and the NPs adsorb on the substrate to form dense coatings characterized by the surface coverage of about 50%. In this regime, the adsorption rate was found to grow with the square of the NP concn. This effect of salt on the deposition process provides a facile method of coating of solid substrates with NP monolayers. The d. of such monolayers depends on and can be easily controlled by the deposition time, as well as by the salt and NP concn. in the plating soln.
    44. 44
      Zhou, H.-X.; Rivas, G.; Minton, A. P. Macromolecular crowding and confinement: biochemical, biophysical, and potential physiological consequences. Annu. Rev. Biophys. 2008, 37, 375– 397,  DOI: 10.1146/annurev.biophys.37.032807.125817
      44
      Macromolecular crowding and confinement: Biochemical, biophysical, and potential physiological consequences
      Zhou, Huan-Xiang; Rivas, German; Minton, Allen P.
      Annual Review of Biophysics (2008), 37 (), 375-397CODEN: ARBNCV ISSN:. (Annual Reviews Inc.)
      A review. Expected and obsd. effects of vol. exclusion on the free energy of rigid and flexible macromols. in crowded and confined systems, and consequent effects of crowding and confinement on macromol. reaction rates and equil. are summarized. Findings from relevant theor./simulation and exptl. literature published from 2004 onward are reviewed. Addnl. complexity arising from the heterogeneity of local environments in biol. media, and the presence of nonspecific interactions between macromols. over and above steric repulsion, are discussed. Theor. and exptl. approaches to the characterization of crowding- and confinement-induced effects in systems approaching the complexity of living organisms are suggested.
    45. 45
      Harada, R.; Sugita, Y.; Feig, M. Protein crowding affects hydration structure and dynamics. J. Am. Chem. Soc. 2012, 134, 4842– 4849,  DOI: 10.1021/ja211115q
      45
      Protein Crowding Affects Hydration Structure and Dynamics
      Harada, Ryuhei; Sugita, Yuji; Feig, Michael
      Journal of the American Chemical Society (2012), 134 (10), 4842-4849CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      The effect of protein crowding on the structure and dynamics of water was examd. from explicit solvent mol. dynamics simulations of a series of protein G and protein G/villin systems at different protein concns. Hydration structure was analyzed in terms of radial distribution functions, three-dimensional hydration sites, and preservation of tetrahedral coordination. Anal. of hydration dynamics focused on self-diffusion rates and dielec. consts. as a function of crowding. The results show significant changes in both structure and dynamics of water under highly crowded conditions. The structure of water is altered mostly beyond the first solvation shell. Diffusion rates and dielec. consts. are significantly reduced following linear trends as a function of crowding reflecting highly constrained water in crowded environments. The reduced dynamics of diffusion is expected to be strongly related to hydrodynamic properties of crowded cellular environments while the reduced dielec. const. under crowded conditions has implications for the stability of biomols. in crowded environments. The results from this study suggest a prescription for modeling solvation in simulations of cellular environments.
    46. 46
      Branca, C.; Magazu, S.; Maisano, G.; Migliardo, F.; Migliardo, P.; Romeo, G. Hydration study of PEG/water mixtures by quasi elastic light scattering, acoustic and rheological measurements. J. Phys. Chem. B 2002, 106, 10272– 10276,  DOI: 10.1021/jp014345v
      46
      Hydration Study of PEG/Water Mixtures by Quasi Elastic Light Scattering, Acoustic and Rheological Measurements
      Branca, C.; Magazu, S.; Maisano, G.; Migliardo, F.; Migliardo, P.; Romeo, G.
      Journal of Physical Chemistry B (2002), 106 (39), 10272-10276CODEN: JPCBFK; ISSN:1520-6106. (American Chemical Society)
      To study the hydration effect of poly(ethylene glycol) (PEG) and its dependence on the mol. wt., we report viscosity, compressibility, and quasi elastic light-scattering measurements on aq. solns. of PEG with different mean mol. wt., Mw, at different concn. and temp. values. In particular, ultrasonic technique allows to evaluate the hydration no. for PEG samples at different polymn. degrees. The values deduced by ultrasonic technique are then compared with those deduced from viscosity data following the Linow and Philipp's model. PCS technique allows to obtain information on the hydrodynamic radius and its dependence on the polymer Mw at different temp. values.

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    • Detailed description of materials and experimental methods, microscope setup, the full description with equations of brightness-based method to determine the equilibrium K constant of biochemical reactions, validation of K constant of DNA hybridization by FRET method, changes of solution properties (e.g., viscosity and pH) after addition of crowders, results of measurements of sodium cation complexation by nonionic crowders with the use of ion-selective electrode, and the discussion and results of water complexation by nonionic crowders (PDF)


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