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Zwitterionic Osmolytes Resurrect Electrostatic Interactions Screened by Salt Roy Govrin, Shani Tcherner, Tal Obstbaum, and Uri Sivan J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.8b07771 • Publication Date (Web): 09 Oct 2018 Downloaded from http://pubs.acs.org on October 9, 2018

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Journal of the American Chemical Society

Zwitterionic Osmolytes Resurrect Electrostatic Interactions Screened by Salt Roy Govrin,* Shani Tcherner, Tal Obstbaum, and Uri Sivan* Department of Physics and the Russell Berrie Nanotechnology Institute, Technion – Israel Institute of Technology, Technion City, Haifa , Israel ABSTRACT: Many cells synthesize significant quantities of zwitterionic osmolytes to cope with the osmotic stress induced by excess salt. In addition to their primary role in balancing osmotic pressure, these osmolytes also help stabilize protein structure and restore enzymatic activity compromised by high ionic strength. This osmoprotective effect has been studied extensively but its electrostatic aspects have somehow escaped the main stream. Here, we report that despite their overall neutrality, zwitterions may dramatically affect electrostatic interactions in saline solutions of biological relevance. Using atomic force microscopy, we study the combined effect of osmolytes and salts on electrostatic interactions between two negatively charged silica surfaces in mixtures of  salts (NaCl, KCl, CsCl, MgCl, CaCl) and  zwitterionic osmolytes (betaine, proline, trimethylamine N-oxide, glycine, sarcosine) as a function of solutes concentration and pH. All osmolytes are found to counteract the screening effect of salt on electrostatic repulsion, albeit to a different extent. They do so by both increasing the screening length shortened by added salts, and by desorbing bound protons and cations, hence enhancing the negative surface charge. Both effects are traced to the osmolytes' higher molecular polarizability compared with water. In addition to this direct effect on the solution's dielectric constant, we identify an osmolytic Hofmeister effect with the more hydrophobic osmolytes desorbing more efficiently weakly hydrated cations from weakly hydrated silica, and less hydrophobic osmolytes desorbing better strongly hydrated cations from strongly hydrated silica. The combined results shed light on Coulomb interactions in a more realistic model of the cytosol, a relatively unexplored territory.

INTRODUCTION The cytosol of most cells contains a large number of negatively charged surface groups, mostly nucleic acid phosphates and amino acid carboxylates, balanced by positively charged residues and inorganic cations, mainly K and Mg. While low concentration of ions is crucial for the formation of nucleic acid duplexes,1 protein folding, and enzyme activity,2 ion concentrations higher than  M perturb biological function.2,3 Organisms thus invest resources in limiting ionic concentrations by pumping ions out of cells.4 The total osmolarity of charged species and macromolecules in cells then reaches typically M, too low to balance the osmotic stress exerted by typical surroundings such as seawater, saline soil, or renal fluids. To cope with this stress, cells produce small neutral solutes, termed osmolytes, in concentrations that together with the charged species approximately match the external osmolarity. The amount and composition of these osmolytes varies across organisms and between cells within organism, with characteristic concentrations in the  M range.5 Contrary to ions, many of the latter molecules are compatible at these concentrations with macromolecular function.3 It has been known for a while that besides their role in maintaining osmotic balance, osmolytes protect organisms against the adverse effect of excess salt. Specifically, zwitterionic osmolytes such as betaine, proline (Pro) and tri-

methylamine N-oxide (TMAO) have been termed osmoprotectans due to their induction of salt-tolerance in plants and saltwater fish,5 protection of membranes and protein structure against high salt,6 and restoration of enzymatic activity inhibited by salt.7–10 This salt counteracting activity is traditionally attributed to the osmolytes' stabilizing/destabilizing effect on macromolecules by non-Coulombic interactions, as reflected in their depletion or accumulation near macromolecular surfaces and overall effect on water activity.11–15 Despite the importance of electrostatic interactions for salt-dependent biological functions, the direct effect of osmolytes on surface charge and Coulomb interaction received relatively little attention. Interestingly, a very recent publication suggested to include the effect of osmolytes on electrostatics in their general description.16 The results presented below strongly support this view. Clues to the direct effect of osmolytes on electrostatic interaction are found in the dependence of their activity on pH13,14,16,17 and salt concentration.7–10 Often, when protein charge is sensitive to pH, osmolytes dramatically affect protein stability.17 Conversely, at pH and salt concentrations that minimize electrostatic interactions osmolytic effects are minimal.7–9,16 At the microscopic level, the protonation state of surface groups indeed modulates osmolytes activity. For instance, the efficiency of the protein stabi-

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lizer betaine deteriorates at low pH values,18 and its interaction with DNA19 and protein20 decreases with increasing oxygen deprotonation. To study the direct effect of osmoprotectants on Coulomb interaction and charge screening, we have used atomic force microscopy21 to measure the force acting between a flat silica surface and a silica colloid immersed in ternary solutions of salt (NaCl, KCl, CsCl, MgCl, CaCl) and zwitterionic osmolytes (betaine, proline, TMAO, glycine and sarcosine). Silica, an archetype inorganic protic surface was chosen since it mimics many of the properties displayed by negatively charged proteins, without the inherent complications associated with protein structure. In both cases the surface acquires its negative charge by deprotonation of weak acids – carboxyl ones in the case of proteins and silanols in the case of silica. In the absence of osmolytes, the repulsive interaction between the two negatively charged silica surfaces was found to be increasingly screened out by added salt. Despite their charge neutrality, added zwitterions were found to counteract this effect, revealing a remarkable new facet of osmolyte activity – resurrection of Coulomb interactions screened out by excess salt. The detailed force vs distance data provided by AFM unraveled the mechanisms underlying this resurrection. The molecular dipole moment of zwitterionic osmolytes is generally larger than that of water molecules. Their addition to saline solution therefore increases the solution's dielectric constant with two main effects: () Increasing the Debye-Hückel (DH) screening length, and () Suppressing cation and proton binding to the negatively charged silanols, hence increasing the surface charge. Both effects therefore act in concert to resurrect the electrostatic interaction screened by salt. Furthermore, we found that in addition to these Coulomb effects, full account of the force data required elements of surface and molecular hydration physics, similar to those leading to the famous Hofmeister series.

Results and Discussion Electrostatic Interaction Between Two Charged Surfaces, Surface Charge Density and Screening Length. In contact with aqueous solution, surface silanols partially deprotonate to give a pH dependent negative surface charge. This charge is partially compensated by adsorbed cations, which together with the deprotonated silanols form the so called Stern layer22 characterized by a total negative surface charge density . This charge is screened by net accumulation of excess mobile cations, which together with the Stern layer form an electrostatic double layer. When two charged surfaces approach each other, their double layers overlap and the increased ion concentration between them exerts a repulsive osmotic pressure on the two surfaces. This double layer repulsion (first term on the right-hand side (RHS) of eq ) is partially compensated by van der Waals (vdW) attraction (second term) to give the total force

F  h R



4 lD

 0 r

 2e

h

lD



H 6h 2

()

lD 

 0 r kBT

2000 N A Ie 2

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.

()

Here, R is the colloid radius and kB ,T , N A , e,  r ,  0 , I are the Boltzmann constant, the absolute temperature, the Avogadro number, the elementary charge, the relative permittivity, the vacuum permittivity, and the molar ionic strength, respectively. H= pN∙nm21 is the Hamaker constant and lD is the DH screening length. Increased salt concentration (increased I) shortens the screening length and the overall repulsion. This effect on the long-range Coulomb interaction can clearly be offset by an increased r, which is one of the effects of added zwitterions (eq S).23–26 The same change in dielectric constant is also expected to weaken the short-range association of protons and cations to the negatively charged silanols,27 hence releasing them from the surface and enhancing the surface negative charge. Salt Screens the Electrostatic Interaction Between Two Charged Surfaces. Figure A depicts normalized force (force divided by colloid radius) vs surface separation in the presence of increasing concentrations of KCl (pH . As expected, added KCl dramatically weakens the intersurface repulsion. The measured force curves are adequately fitted by eq (solid black lines) and as seen in the inset, the extracted lD indeed shortens with added salt The two terms on the RHS of eq  are drawn separately to show the effect of screening by KCl on double layer repulsion. Betaine Compensates for the Screening of Electrostatic Interaction by Salt. Figure B depicts normalized force curves measured in solutions of  mM KCl, pH , and increasing concentrations of betaine. Addition of betaine recovers the intersurface repulsion seen with lower KCl concentrations and no betaine. At  mM KCl and  M betaine, for example, repulsion is nearly as strong as for  mM KCl without betaine. Opposite to the attenuation of lD with added KCl, at a constant salt the measured lD increases with betaine concentration (inset to Figure B), thus counteracting the effect of salt on screening length. This increase is expected according to eq  and the relatively large r of betaine solutions (eq S). Adsorption of Cations to Silica Depends on Surface and Cation Hydration (Cation-Specific Adsorption). Figure  depicts force curves for three different salts with and without betaine. At pH  silica is weakly hydrated and weakly hydrated cations tend to accumulate next to it by essentially hydrophobic expulsion from solution. The order of surface repulsion strength seen in Figure  without betaine, Na+ > K+ > Cs+, discloses an opposite ordering of cation propensity to the surface28,29 (Na+ < K+ < Cs+). This ordering conforms with the normal Hofmeister series for cation adsorption to weakly hydrated surfaces. Betaine Desorbs Salt Cations from Surface. Comparison between solid ( M betaine) and dotted ( M betaine) lines in Figure , and the extracted  in its inset, show that addition of betaine at pH  significantly increases surface charge density and enhances electrostatic repulsion. Added betaine leads at  M to %, % and % increase


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Journal of the American Chemical Society of surface charge in CsCl, KCl and NaCl, respectively. Charge enhancement by betaine results from liberation of bound cations from the negative surface – the higher the adsorption in the absence of betaine, the larger its effect. AT  M betaine the Hofmeister effect (cation specificity) nearly vanishes.

increase at intermediate concentrations of osmolytes in the order betaine > Pro > sarcosine > Gly, namely, from the least to the most hydrophilic zwitterion. At  M osmolyte, the difference in surface charge between betaine, sarcosine and Pro, seen at intermediate concentrations, diminishes. Even though betaine and TMAO have both three methyl groups, the former counteracts salt more efficiently than the latter. We attribute this difference to the markedly higher dielectric constant of betaine (eq S).23,26

Figure . Betaine resurrects intersurface repulsion and enhances surface charge density in NaCl, KCl and CsCl solutions. Main figure - Normalized intersurface force vs surface separation in different salt solutions without betaine or with  M betaine. Colored lines – experimental data. Solid black lines – best fit by eq . Inset: Surface charge density vs betaine concentration for the three salts.  was extracted by fitting eq  to measured force curves.

Figure . The effect of KCl and betaine on screening length and normalized force between two silica surfaces spaced a distance h apart (pH ). (A) Effect of KCl concentration. Solid colored lines – experimental results, solid black lines – best fit by eq . Dashed blue and black lines depict the corresponding double layer (electrostatic) and vdW terms on the RHS of eq . Inset: Green disks - screening length extracted by fitting eq  to data in Figure A. Solid black line – theoretical expectation based on eq . (B) Effect of added betaine. Solid colored lines – experimental results. Solid black lines - best fit by eq . Inset: Green disks - screening length extracted by fitting eq  to data in Figure B. Solid line - lD predicted by eqs  and S. When error bar is absent, error is smaller than marker size.

Osmolyte Hydrophobicity Affects its Capacity to Desorb Bound Cations (Osmolyte-Specific Desorption). Figure  depicts the increase in negative surface charge due to desorption of bound Cs+ upon addition of  different osmolytes at pH . Force curves for Pro and TMAO are presented in Figure S. Focusing on amino acids and their derivatives, the surface charge density is found to

Osmolytes' capacity to counteract suppression of enzyme activity by salt is known to increase with glycine methylation.9,10 The same trend is seen here with the recovery of Coulomb interaction by glycine and its methylated derivatives, where salt-counteraction increases with the number of methyl groups. Surface Hydration Affects the Cation Desorption Capacity of Osmolytes. The nature of silica surface changes from mildly hydrophobic at pH 5.6 to mildly hydrophilic at pH , with a crossover at ≈ pH . As a result, the Hofmeister ordering of cation adsorption seen at pH  (Figure ) is reversed at pH  (see figure  in ref ) with the strongly hydrated Na+ adsorbing more than the weakly hydrated cations, Cs+ and K+. In analogy with cations, we expected a similar ordering of osmolyte propensity to the surface and its reversal on going from pH  to . To test this conjecture, we have studied the cation desorption power of glycine and two of its methylated derivatives at three pH values and  M concentration (Figure , inset). As expected, at pH  where the silica surface is mildly hydrophobic, the order of activity follows the hydrophobicity level, betaine > sarcosine > Gly. This order is reversed at pH , when the silica turns mildly hydrophilic, the same way the order of cation



propensity to the surface is reversed. This analogy, already highlighted by Yancey et al.,3 suggests that similarly to Cs+, betaine also accumulates near the surface at pH . The decreasing cation desorption capacity of betaine with increased surface deprotonation concurs with the known unfavorable interaction of betaine with deprotonated oxygen in biomolecules.12,30 At pH , on the other hand, when the silica surface is strongly hydrated, the hydrophilic Gly desorbs bound cations more efficiently than betaine.

The analysis of force vs distance curves identifies two distinct mechanisms, one associated with the zwitterions' large dielectric constants compared with water and the other having to do with surface, cation, and osmolyte hydration levels. The first mechanism is not limited to monovalent cations. As seen in Figure S, added betaine also increases the screening length in ternary solutions containing MgCl or CaCl, and enhances surface charge by liberating adsorbed Mg and Ca cations from the silica surface. The release of these ions from negatively charged groups by zwitterionic osmolytes might be important for the function of many enzymes that require them for their activity. The present results complement our earlier investigation31 where we found that urea, with its high dipole moment, acts similarly to the zwitterionic osmolytes, namely, enhances surface charging and double layer repulsion. Glycerol, on the other hand, was found to suppress surface charging and diminish Coulomb repulsion, in agreement with its lower dipole moment compared with water. Taken together, the combined data disclose that osmolytes characterized by dipole moments larger than water enhance surface charging and double layer repulsion while osmolytes of lower dipole moment act in the opposite direction.

Figure . Surface charge density increases as different osmolytes desorb cations from the negatively charged surface. The main figure presents surface charge density vs osmolyte concentration for  zwitterionic osmolytes in  mM CsCl solutions of pH  (see legend). Surface charge density was extracted from force curves (see Figure S for proline and TMAO force curves) fitted by eq . Inset: Surface charge density vs pH in M osmolyte (see legend in main figure) and  mM CsCl.

Accumulation of K+ and Cs+ near the surface at pH  is evident from the shorter screening length depicted in Figure S, compared with Na+ or theory (zero betaine points). For all three salts, the screening length at pH  grows steeply with betaine concentration to values larger than expected based on osmolyte bulk concentration, further supporting the conjecture about betaine accumulation. At pH , on the other hand, the screening length agrees with the predicted values, indicating that betaine indeed no longer accumulates near the surface. Remarkably, the osmolyte-specific effect vanishes at pH , where the strength of silica hydrogen bonds with water matches the strength of water-water ones and silica is neither hydrophilic nor hydrophobic. This is indeed the point where cation-specific propensity to the surface also vanishes.28,29

Conclusion We find that electrostatic interactions between protic surfaces in the presence of ternary solutions of salt and zwitterionic osmolytes can differ significantly from those studied with salt alone. Specifically, we find that added osmolytes oppose the screening effect of salt and resurrect Coulomb interactions to their full strength. This is a new facet of osmolyte activity, largely ignored until now.

It has recently been found that contrary to the traditional view, osmolyte accumulation/depletion near protein surfaces is not necessarily correlated with their stabilizing capacity.15 Both glycerol and zwitterionic osmolytes stabilize proteins but affect oppositely electrostatic interactions. On the same footing, urea and zwitterionic osmolytes enhance electrostatic interactions but operate oppositely to each other on protein stability. We see then that the effect of osmolytes on protein stability is not necessarily correlated with their effect on electrostatic interactions. The osmolytic effect on Coulomb interactions is not expected to affect the osmolytes' primary role in balancing osmotic pressure in organisms living with salt stress. It should have, though, profound impact on Coulomb interactions that govern enzyme-substrate binding and proteinprotein interactions. As most proteins are negatively charged, the adsorption of intracellular cations weakens protein-protein repulsion and may lead to decreased overall solubility of the proteome under salt stress. The discovered compensation mechanism explains how zwitterionic osmolytes may restore protein-protein repulsion and counteract the adverse effect of salt. A recent publication16 reported the effect of different osmolytes and other cosolutes on the dimerization of charged protein units, mimicked here by the two silica surfaces. Beyond similarities in the effect of pH and salt concentration to the present data, they find that addition of side methyl residues to glycine destabilizes the dimer as expected from the enhancement of surface charge observed here.


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Journal of the American Chemical Society

ASSOCIATED CONTENT Supporting Information. The supporting information (pdf) contains Materials and Method section, AFM data for proline and TMAO solutions, extracted screening lengths, and effect of betaine in  salt solutions. The supporting information is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION

(10) (11)

(12) (13) (14) (15)

Corresponding Authors (16)

*E-mail [email protected] *E-mail [email protected]

(17)

Funding Sources This research was supported by the Israeli Science Foundation through grant number 10/1051 and the single molecule ICore center of excellence, grant number 1902/12.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT

(18)

(19)

(20)

(21)

We are grateful to Daniel Harries for useful discussions. (22)

REFERENCES (1) (2) (3) (4) (5) (6) (7) (8) (9)

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