We apply a recently developed surface-bulk partitioning model to interpret the effects of individual Hofmeister cations and anions on the surface tension of water. The most surface-excluded salt (Na2SO4) provides a minimum estimate for the number of water molecules per unit area of the surface region of 0.2 H2O A-2. This corresponds to a lower bound thickness of the surface region of approximately 6 A, which we assume is a property of this region and not of the salt investigated. At salt concentrations < or = 1 m, single-ion partition coefficients Kp,i, defined relative to Kp,Na+ = Kp,SO42- = 0, are found to be independent of bulk salt concentration and additive for different salt ions. Semiquantitative agreement with surface-sensitive spectroscopy data and molecular dynamics simulations is attained. In most cases, the rank orders of Kp,i for both anions and cations follow the conventional Hofmeister series, qualitative rankings of ions based on their effects on protein processes (folding, precipitation, assembly). Most anions that favor processes that expose protein surface to water (e.g., SCN-), and hence must interact favorably with (i.e., accumulate at) protein surface, are also accumulated at the air-water interface (Kp >1, e.g., Kp,SCN- =1.6). Most anions that favor processes that remove protein surface from water (e.g., F-), and hence are excluded from protein surface, are also excluded from the air-water interface (Kp,F- = 0.5). The guanidinium cation, a strong protein denaturant and therefore accumulated at the protein surface exposed in unfolding, is somewhat excluded from the air-water surface (Kp,GuH+ = 0.7), but is much less excluded than alkali metal cations (e.g., Kp,Na+ identical with 0, Kp,K+ = 0.1). Hence, cation Kp values for the air-water surface appear shifted (toward exclusion) as compared with values inferred for interactions of these cations with protein surface.

To explain the large, opposite effects of urea and glycine betaine (GB) on stability of folded proteins and protein complexes, we quantify and interpret preferential interactions of urea with 45 model compounds displaying protein functional groups and compare with a previous analysis of GB. This information is needed to use urea as a probe of coupled folding in protein processes and to tune molecular dynamics force fields. Preferential interactions between urea and model compounds relative to their interactions with water are determined by osmometry or solubility and dissected using a unique coarse-grained analysis to obtain interaction potentials quantifying the interaction of urea with each significant type of protein surface (aliphatic, aromatic hydrocarbon (C); polar and charged N and O). Microscopic local-bulk partition coefficients K p for the accumulation or exclusion of urea in the water of hydration of these surfaces relative to bulk water are obtained. K p values reveal that urea accumulates moderately at amide O and weakly at aliphatic C, whereas GB is excluded from both. These results provide both thermodynamic and molecular explanations for the opposite effects of urea and glycine betaine on protein stability, as well as deductions about strengths of amide NH-amide O and amide NH-amide N hydrogen bonds relative to hydrogen bonds to water. Interestingly, urea, like GB, is moderately accumulated at aromatic C surface. Urea m-values for protein folding and other protein processes are quantitatively interpreted and predicted using these urea interaction potentials or K p values. U rea and glycine betaine (GB) rank at opposite ends of a series of small nonelectrolyte solutes in terms of their effects on protein folding and other protein processes. Stabilities (ΔG o obs ) of folded proteins and of site-specific protein-DNA complexes decrease linearly with increasing urea molarity and increase with increasing GB molarity (1-5). This solute series parallels the Hofmeister anion and cation series of non-Coulombic effects of salt ions on protein processes; guanidinium cation and thiosulfate or iodide anions are highly destabilizing but alkali metal cations are not destabilizing and sulfate or fluoride anions are stabilizing (6-8). Similar rank orders but smaller ranges of solute and nonCoulombic salt effects are observed on DNA and RNA duplex formation; urea and salt ions that greatly destabilize proteins when added at molar concentrations also greatly destabilize DNA duplexes, but GB and Hofmeister salt ions that stabilize proteins do not stabilize nucleic acids duplexes (4,5,(8)(9)(10)(11). Another range of solute effects is observed for the series of solutes from ethylene glycol (EG) to PEG, where the monomer EG destabilizes both hairpin and duplex DNA helices, whereas polymeric PEGs greatly stabilize the duplex and eliminate the destabilization of the hairpin helix (12). In our research, we use molecular thermodynamic analyses of model compound data to interpret and predict the effects of these solutes an...

Quantitative interpretation and prediction of Hofmeister ion effects on protein processes, including folding and crystallization, have been elusive goals of a century of research. Here, a quantitative thermodynamic analysis, developed to treat noncoulombic interactions of solutes with biopolymer surface and recently extended to analyze the effects of Hofmeister salts on the surface tension of water, is applied to literature solubility data for small hydrocarbons and model peptides. This analysis allows us to obtain a minimum estimate of the hydration b 1 (H 2 O Å −2 ) of hydrocarbon surface and partition coefficients K p characterizing the distribution of salts and salt ions between this hydration water and bulk water. Assuming that Na + and ions of Na 2 SO 4 (the salt giving the largest reduction in hydrocarbon solubility as well as the largest increase in surface tension) are fully excluded from the hydration water at the hydrocarbon surface, we obtain the same b 1 as for air-water surface (∼0.18 H 2 O Å −2 ). Rank orders of cation and anion partition coefficients for nonpolar surface follow the Hofmeister series for protein processes, but are strongly offset for cations in the direction of exclusion (preferential hydration). Assuming a coarse-grained decomposition of water accessible surface area (ASA) into nonpolar, polar amide, and other polar ASA and the same hydration b 1 to interpret peptide solubility increments, we determine salt partition coefficients for amide surface. These partition coefficients are separated into single-ion contributions based on the observation that both Cl − and Na + (also K + ) occupy neutral positions in the middle of the anion and cation Hofmeister series for protein processes. Independent of this assignment, we find that all cations investigated are strongly accumulated at amide surface while most anions are excluded. Ion effects are independent and additive, allowing successful prediction of Hofmeister salt effects on micelle formation and other processes from structural information (ASA).

The majority (∼70%) of surface buried in protein folding is hydrocarbon, whereas in DNA helix formation, the majority (∼65%) of surface buried is relatively polar nitrogen and oxygen. Our previous quantification of salt exclusion from hydrocarbon (C) accessible surface area (ASA) and accumulation at amide nitrogen (N) and oxygen (O) ASA leads to a prediction of very different Hofmeister effects on processes that bury mostly polar (N, O) surface compared to the range of effects commonly observed for processes that bury mainly nonpolar (C) surface, e.g., micelle formation and protein folding. Here we quantify the effects of salts on folding of the monomeric DNA binding domain (DBD) of lac repressor (lac DBD) and on formation of an oligomeric DNA duplex. In accord with this prediction, no salt investigated has a stabilizing Hofmeister effect on DNA helix formation. Our ASA-based analyses of model compound data and estimates of the surface area buried in protein folding and DNA helix formation allow us to predict Hofmeister effects on these processes. We observe semiquantitative to quantitative agreement between these predictions and the experimental values, obtained from a novel separation of coulombic and Hofmeister effects. Possible explanations of deviations, including salt-dependent unfolded ensembles and interactions with other types of surface, are discussed.Hofmeister salts | m-values | thermodynamics S alts typically exert both specific (Hofmeister) and nonspecific (coulombic) effects on biomolecular processes (1-6). To manipulate and probe biopolymer processes using salts, it is extremely important to develop quantitative methods to interpret and predict these effects in terms of structure. coulombic, valencespecific effects of salt ions (due to screening of surface charges) are most significant at relatively low salt concentrations (<0.1 M). At higher concentrations (>0.1 M), ion-specific effects and relatively nonspecific osmotic effects (due to the lowering of water activity) become increasingly significant. In 1888, Franz Hofmeister discovered that the effectiveness of salts for protein precipitation generally followed a specific order, regardless of the protein being investigated (7). Since then, the so-called Hofmeister series of salt effects has been observed in physical properties of aqueous salt solutions (e.g., surface tension and surface potential) (8, 9), as well as salt effects on a variety of macromolecular processes (e.g., micelle formation, "salting out" nonpolar compounds, and protein folding) (10-13). The general ranking of ions, in decreasing order of effectiveness (best to worst) in driving processes where surface area is buried (e.g., folding and precipitation) or macroscopic surface is lost (transfer of water from the air-water interface to bulk), is as follows (14):Although it is generally accepted that interactions of salts with hydrocarbon surface are unfavorable and salt-specific, following the above order (1,3,11,(14)(15)(16), less is known about the interactions of Hofmeister sa...

Noncovalent self-assembly of biopolymers is driven by molecular interactions between functional groups on complementary biopolymer surfaces, replacing interactions with water. Since individually these interactions are comparable in strength to interactions with water, they have been difficult to quantify. Solutes (osmolytes, denaturants) exert often-large effects on these self-assembly interactions, determined in sign and magnitude by how well the solute competes with water to interact with the relevant biopolymer surfaces. Here, an osmometric method and a water-accessible surface area (ASA) analysis are developed to quantify and interpret the interactions of the remarkable osmolyte glycine betaine (GB) with molecular surfaces in water. We find that GB, lacking hydrogen bond donors, is unable to compete with water to interact with anionic and amide oxygens; this explains its effectiveness as an osmolyte in the E. coli cytoplasm. GB competes effectively with water to interact with amide and cationic nitrogens (hydrogen bonding) and especially with aromatic hydrocarbon (cation-pi). The large stabilizing effect of GB on lac-repressor-lac operator binding is predicted quantitatively from ASA information and shown to result largely from dehydration of anionic DNA phosphate oxygens in the protein-DNA interface. The incorporation of these results into theoretical and computational analyses will likely improve the ability to accurately model intraand inter-protein interactions. Additionally, these results pave the way for development of solutes as kinetic/mechanistic and thermodynamic probes of conformational changes and formation/disruption of molecular interfaces that occur in the steps of biomolecular self-assembly processes.Biopolymer self-assembly (folding, binding) in vivo and in vitro involves the replacement of interactions with water by more favorable interactions between biopolymer functional groups. 1 The ability (or inability) of solutes and Hofmeister salt ions to compete with water to interact Correspondence to: M. Thomas Record, Jr., mtrecord@wisc.edu. † University of Wisconsin-Madison ‡ latex template bug; also affiliation necessary for compilation ¶ Current address: Curriculum in Neurobiology, University of North Carolina, Chapel Hill, North Carolina 27599 § Current address: Department of Physiology and Biophysics, Case Western University, Cleveland, Ohio 44106 * This work was supported by National Institutes of Health Grants GM47022 and GM23467 (to M.T.R.). Tables containing water accessible Supporting Information Available NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2010 November 3. with biopolymer functional groups results in often-large destabilizing (or stabilizing) effects on these assembled states. 2,3 To understand the energetics of self-assembly and how solutes modulate these processes, the strength of interactions of functional groups with water relative to the strength of their interactions with one another must be determined. 4 To accomplish ...