Molecular-dynamics simulations of alkaline-earth metal cations in water by atom-bond electronegativity equalization method fused into molecular mechanics

Intermolecular potential for alkaline-earth metal (Be(2+), Mg(2+), and Ca(2+)) cations in water has been derived using the atom-bond electronegativity equalization method fused into molecular mechanics (ABEEM/MM), and it is consistent with what was previously applied to the hydration study of the monovalent cations. Parameters for the effective interaction between a cation and a water molecule were determined, reproducing the ab initio results. The static, dynamic, and thermodynamic properties of Be(2+)(aq), Mg(2+)(aq), and Ca(2+)(aq) were studied using these potential parameters. Be(2+) requires a more complicated form of the potential function than Mg(2+) and Ca(2+) in order to obtain better fits. Strong influences of the twofold charged cations on the structures of the hydration shells and some other properties of aqueous ionic solutions are discussed and compared with the results of a previous study of monovalent cations in water. At the same time, comparative study of the hydration properties of each cation is also discussed. This work demonstrates that ABEEM/MM provides a useful tool in the exploration of the hydration of double-charged cations in water.     

Effects of cations on the hydrogen bond network of liquid water: new results from X-ray absorption spectroscopy of liquid microjets

Oxygen K-edge X-ray absorption spectra (XAS) of aqueous chloride solutions have been measured for Li(+), Na(+), K(+), NH(4)(+), C(NH(2))(3)(+), Mg(2+), and Ca(2+) at 2 and 4 M cation concentrations. Marked changes in the liquid water XAS are observed upon addition of the various monovalent cation chlorides that are nearly independent of the identity of the cation. This indicates that interactions with the dissolved monovalent cations do not significantly perturb the unoccupied molecular orbitals of water molecules in the vicinity of the cations and that water-chloride interactions are primarily responsible for the observed spectral changes. In contrast, the addition of the divalent cations engenders changes unique from the case of the monovalent cations, as well as from each other. Density functional theory calculations suggest that the ion-specific spectral variations arise primarily from direct electronic perturbation of the unoccupied orbitals due to the presence of the ions, probably as a result of differences in charge transfer from the water molecules onto the divalent cations.     

Folding of the thrombin aptamer into a G-quadruplex with Sr(2+): stability, heat, and hydration.

It has been shown that the DNA aptamer d(G(2)T(2)G(2)TGTG(2)T(2)G(2)) adopts an intramolecular G-quadruplex structure in the presence of K+. Its affinity for trombin has been associated with the inhibition of thrombin-catalyzed fibrin clot formation. In this work, we used a combination of spectroscopy, calorimetry, density, and ultrasound techniques to determine the spectral characteristics, thermodynamics, and hydration effects for the formation of G-quadruplexes with a variety of monovalent and divalent metal ions. The formation of cation-aptamer complexes is relatively fast and highly reproducible. The comparison of their CD spectra and melting profiles as a function of strand concentration shows that K+, Rb+, NH(4)+, Sr(2+), and Ba(2+) form intramolecular cation-aptamer complexes with transition temperatures above 25 degrees C. However, the cations Li+, Na+, Cs+, Mg(2+), and Ca(2+) form weaker complexes at very low temperatures. This is consistent with the observation that metal ions with ionic radii in the range 1.3-1.5 A fit well within the two G-quartets of the complex, while the other cations cannot. The comparison of thermodynamic unfolding profiles of the Sr(2+)-aptamer and K+ -aptamer complexes shows that the Sr(2+)-aptamer complex is more stable, by approximately 18 degrees C, and unfolds with a lower endothermic heat of 8.3 kcal/mol. This is in excellent agreement with the exothermic heats of -16.8 kcal/mol and -25.7 kcal/mol for the binding of Sr(2+) and K+ to the aptamer, respectively. Furthermore, volume and compressibility parameters of cation binding show hydration effects resulting mainly from two contributions: the dehydration of both cation and guanine atomic groups and water uptake upon the folding of a single-strand into a G- quadruplex structure.     

Folding and unfolding kinetics of DNA hairpins in flowing solution by multiparameter fluorescence correlation spectroscopy

Dynamic equilibrium between the folded and unfolded conformations of single stranded DNA hairpin molecules containing polythymine hairpin loops was investigated using simultaneous two-beam fluorescence cross-correlation spectroscopy and single beam autocorrelation spectroscopy. The hairpins were end-labeled with a fluorescent dye and a quencher, such that folding and unfolding of the DNA hairpin primary structure caused the dye fluorescence to fluctuate on the same characteristic time scale as the folding and unfolding reaction. These fluctuations were observed as the molecules flowed sequentially between two spatially offset, microscopic detection volumes. Cross-correlation analysis of fluorescence from the two detection volumes revealed the translational diffusion and flow properties of the hairpins, as well as the average molecular occupancy of the two volumes. Autocorrelation analysis of the fluorescence from the individual detection volumes revealed the kinetics of hairpin folding and unfolding, with the parameters relating to diffusion, flow, and molecular occupancy constrained to the values determined from the cross-correlation analysis. This allowed unambiguous characterization of the folding and unfolding kinetics, without the need to determine the hydrodynamic properties by analyzing a separate control sample. The analysis revealed nonexponential relaxation kinetics and DNA size-dependent folding times characteristic of dynamic heterogeneity in the DNA hairpin-forming mechanism.     

Cold denaturation of the hammerhead ribozyme

Cold denaturation is a thermodynamic phenomenon resulting from a difference in the heat capacities, DeltaCp, of the folded and unfolded states of a macromolecule. Whereas this phenomenon has been extensively studied in proteins, it has been thought not to occur in nucleic acids due to a negligible DeltaCp of folding. Questioning the validity of this assumption, the low-temperature structure of the hammerhead ribozyme, a small catalytic RNA, was investigated by circular dichroism spectroscopy. In the presence of 10 mM Mg2+ at pH 5.0 and 40% methanol, a cold unfolding event likely corresponding to tertiary structure loss was observed with a Tm of -20 degrees C. In 500 mM NaCl at pH 6.6, and 40% methanol, large-scale unfolding of the ribozyme at both hot (Tm = 53 degrees C) and cold (Tm = -1 degrees C) temperatures occurred. Fitting of these data to a two-state model allowed determination of DeltaCp = 3.4 kJ mol-1 K-1, corresponding to >/=0.18 kJ K-1 (mol base pair)-1, in good agreement with recently published calorimetric values for DNA duplexes. These results constitute the first direct observation of cold denaturation of a nucleic acid, and point to the importance of DeltaCp terms in the thermodynamics of nucleic acid folding.     

Aqueous divalent metal-nitrate interactions: hydration versus ion pairing

Nitrate aqueous solutions, Mg(NO(3))(2), Ca(NO(3))(2), Sr(NO(3))(2), and Pb(NO(3))(2), are investigated using Raman spectroscopy and free energy profiles from molecular dynamics (MD) simulations. Analysis of the in-plane deformation, symmetric stretch, and asymmetric stretch vibrational modes of the nitrate ions reveal perturbation caused by the metal cations and hydrating water molecules. Results show that Pb(2+) has a strong tendency to form contact ion pairs with nitrate relative to Sr(2+), Ca(2+), and Mg(2+), and contact ion pair formation decreases with decreasing cation size and increasing cation charge density: Pb(2+) > Sr(2+) > Ca(2+) > Mg(2+). In the case of Mg(2+), the Mg(2+)-OH(2) intermolecular modes indicate strong hydration by water molecules and no contact ion pairing with nitrate. Free energy profiles provide evidence for the experimentally observed trend and clarification between solvent-separated, solvent-shared, and contact ion pairs, particularly for Mg(2+) relative to other cations.     

Effect of exchangeable cation on the swelling property of 2:1 dioctahedral smectite--a periodic first principle study

We used both localized and periodic calculations on a series of monovalent (Li+, Na+, K+, Rb+, Cs+) and divalent (Mg2+, Ca2+, Sr2+, Ba2+) cations to monitor their effect on the swelling of clays. The activity order obtained for the exchangeable cations among all the monovalent and divalent series studied: Ca2+ > Sr2+ > Mg2+ > Rb+ > Ba2+ > Na+ > Li+ > Cs+ > K+. We have shown that, in case of dioctahedral smectite, the hydroxyl groups play a major role in their interaction with water and other polar molecules in the presence of an interlayer cation. We studied both type of clays, with a different surface structure and with/without water using a periodic calculation. Interlayer cations and charged 2:1 clay surfaces interact strongly with polar solvents; when it is in an aqueous medium, clay expands and the phenomenon is known as crystalline swelling. The extent of swelling is controlled by a balance between relatively strong swelling forces and electrostatic forces of attraction between the negatively charged phyllosilicate layer and the positively charged interlayer cation. We have calculated the solvation energy at the first hydration shell of an exchangeable cation, but the results do not correspond directly to the experimental d-spacing values. A novel quantitative scale is proposed with the numbers generated by the relative nucleophilicity of the active cation sites in their hydrated state through Fukui functions within the helm of the hard soft acid base principle. The solvation effect thus measured show a perfect match with experiment, which proposes that the reactivity index calculation with a first hydration shell could rationalize the swelling mechanism for exchangeable cations. The conformers after electron donation or acceptance propose the swelling mechanism for monovalent and divalent cations.     

Is Poisson-Boltzmann theory insufficient for protein folding simulations?

The Poisson-Boltzmann theory has been widely used in the studies of energetics and conformations of biological macromolecules. Recently, introduction of the efficient generalized Born approximation has greatly extended its applicability to areas such as protein folding simulations where highly efficient computation is crucial. However, limitations have been found in the folding simulations of a well-studied beta hairpin with several generalized Born implementations and different force fields. These studies have raised the question whether the underlining Poisson-Boltzmann theory, on which the generalized Born model is calibrated, is adequate in the treatment of polar interactions for the challenging protein folding simulations. To address the question whether the Poisson-Boltzmann theory in the current formalism might be insufficient, we directly tested our efficient numerical Poisson-Boltzmann implementation in the beta-hairpin folding simulation. Good agreement between simulation and experiment was found for the beta-hairpin equilibrium structures when the numerical Poisson-Boltzmann solvent and a recently improved generalized Born solvent were used. In addition simulated thermodynamic properties also agree well with experiment in both solvents. Finally, an overall agreement on the beta-hairpin folding mechanism was found between the current and previous studies. Thus, our simulations indicate that previously observed limitations are most likely due to imperfect calibration in previous generalized Born models but not due to the limitation of the Poisson-Boltzmann theory.     

Evidence for a thermodynamically distinct Mg2+ ion associated with formation of an RNA tertiary structure

A folding strategy adopted by some RNAs is to chelate cations in pockets or cavities, where the ions neutralize charge from solvent-inaccessible phosphate. Although such buried Mg(2+)-RNA chelates could be responsible for a significant fraction of the Mg(2+)-dependent stabilization free energy of some RNA tertiary structures, direct measurements have not been feasible because of the difficulty of finding conditions under which the free energy of Mg(2+) chelation is uncoupled from RNA folding and from unfavorable interactions with Mg(2+) ions in other environments. In a 58mer rRNA fragment, we have used a high-affinity thermophilic ribosomal protein to trap the RNA in a structure nearly identical to native; Mg(2+)- and protein-stabilized structures differ in the solvent exposure of a single nucleotide located at the chelation site. Under these conditions, titration of a high affinity chelation site takes place in a micromolar range of Mg(2+) concentration, and is partially resolved from the accumulation of Mg(2+) in the ion atmosphere. From these experiments, we estimate the total and site-specific Mg(2+)-RNA interaction free energies over the range of accessed Mg(2+) concentrations. At 0.1 mM Mg(2+) and 60 mM K(+), specific site binding contributes ～-3 kcal/mol of the total Mg(2+) interaction free energy of ～-13 kcal/mol from all sources; at higher Mg(2+) concentrations the site-binding contribution becomes a smaller proportion of the total (-4.5 vs -33 kcal/mol). Under approximately physiological ionic conditions, the specific binding site will be saturated but will provide only a fraction of the total free energy of Mg(2+)-RNA interactions.     

Fluorescence anisotropy and FRET studies of G-quadruplex formation in presence of different cations

Results of the steady-state fluorescence, anisotropy and FRET measurements of G-quadruplex formation in the presence of selected cations (Li(+), Na(+), K(+), NEt(4)(+) and Mg(2+)) are reported. Three different fluorescent oligonucleotides with human telomeric sequence labeled with fluorescein (FAM) and tetramethylrhodamine (TAMRA) were investigated: a dual-labeled 21-mer denoted as PSO (Potassium Sensing Oligonucleotide) and two 5'- and 3'- single-labeled probes, FAM-21 and 21-TAMRA, respectively. The fluorescence signal of FAM-21 increased significantly for all systems and the fluorescence enhancement was comparable in magnitude for monovalent cations but it was more pronounced for Mg(2+) cation. This phenomenon was attributed to the protolytic equilibria of FAM affected by the variation in ionic strength. On the other hand, fluorescence of TAMRA was enhanced selectively by Na(I) cation that was explained by the dequenching of TAMRA emission originated from the peculiarity of the basket-type structure of Na(I)-quadruplex. Anisotropy of FAM-21 (but not 21-TAMRA) appeared to be sensitive to the G-quadruplex formation, showing significant increase with an increase in cation concentration and indicating some restrictions in rotational depolarization of FAM. FRET experiments revealed that all tested cations caused quenching of FAM fluorescence in PSO, but only Na(+) and K(+) ions produced sensitized emission of TAMRA acceptor. Higher FRET efficiency observed in the presence of sodium ion was attributed to the specific spectral factor and steric interactions in the basket-type Na(I)-quadruplex.     

Folding/unfolding/refolding of proteins: present methodologies in comparison with capillary zone electrophoresis

A series of techniques for monitoring protein folding/unfolding/misfolding equilibria are here assessed and compared with capillary zone electrophoresis (CZE). They include spectroscopic techniques, such as circular dichroism, intrinsic fluorescence, nuclear magnetic resonance, Fourier transform infrared and Raman spectroscopy, small-angle X-ray scattering, as well as techniques based on biological assays, such as limited proteolysis and immunochemical analysis of different conformational states. Some unusual probes, such as mass spectrometry for probing unfolding transitions, are also discussed. Size-exclusion chromatography is also evaluated in view of the fact that this technique, like all electrophoretic techniques, and unlike spectroscopic probes, which can only see an average signal in mixed populations, can indeed physically separate folded vs. unfolded macromolecules, especially in the case of slow equilibria. Particular emphasis is devoted to electrophoretic techniques, such as gel-slab electrophoresis in transverse urea or thermal gradients, and CZE. In the latter case, a number of applications are shown, demonstrating the excellent correlation of CZE with more traditional probes, such as intrinsic fluorescence monitoring. It is additionally shown that CZE can be used for measuring the deltaG degrees of unfolding over the pH scale, in good agreement with theoretical calculations on the electrostatic free energy of folding vs. pH, as calculated with a linearized Poisson-Boltzmann equation. Finally, it is demonstrated that CZE can probe also aggregate formation in the presence of helix-inducing agents, such as trifluorethanol.     

Determining the Mg2+ stoichiometry for folding an RNA metal ion core

The folding and catalytic function of RNA molecules depend on their interactions with divalent metal ions, such as magnesium. As with every molecular process, the most basic knowledge required for understanding the close relationship of an RNA with its metal ions is the stoichiometry of the interaction. Unfortunately, inventories of the numbers of divalent ions associated with unfolded and folded RNA states have been unattainable. A common approach has been to interpret Hill coefficients fit to folding equilibria as the number of metal ions bound upon folding. However, this approach is vitiated by the presence of diffusely associated divalent ions in a dynamic ion atmosphere and by the likelihood of multiple transitions along a folding pathway. We demonstrate that the use of molar concentrations of background monovalent salt can alleviate these complications. These simplifying solution conditions allow a precise determination of the stoichiometry of the magnesium ions involved in folding the metal ion core of the P4-P6 domain of the Tetrahymena group I ribozyme. Hill analysis of hydroxyl radical footprinting data suggests that the P4-P6 RNA core folds cooperatively upon the association of two metal ions. This unexpectedly small stoichiometry is strongly supported by counting magnesium ions associated with the P4-P6 RNA via fluorescence titration and atomic emission spectroscopy. By pinpointing the metal ion stoichiometry, these measurements provide a critical but previously missing step in the thermodynamic dissection of the coupling between metal ion binding and RNA folding.     

RNA catalysis in model protocell vesicles

We are engaged in a long-term effort to synthesize chemical systems capable of Darwinian evolution, based on the encapsulation of self-replicating nucleic acids in self-replicating membrane vesicles. Here, we address the issue of the compatibility of these two replicating systems. Fatty acids form vesicles that are able to grow and divide, but vesicles composed solely of fatty acids are incompatible with the folding and activity of most ribozymes, because low concentrations of divalent cations (e.g., Mg(2+)) cause fatty acids to precipitate. Furthermore, vesicles that grow and divide must be permeable to the cations and substrates required for internal metabolism. We used a mixture of myristoleic acid and its glycerol monoester to construct vesicles that were Mg(2+)-tolerant and found that Mg(2+) cations can permeate the membrane and equilibrate within a few minutes. In vesicles encapsulating a hammerhead ribozyme, the addition of external Mg(2+) led to the activation and self-cleavage of the ribozyme molecules. Vesicles composed of these amphiphiles grew spontaneously through osmotically driven competition between vesicles, and further modification of the membrane composition allowed growth following mixed micelle addition. Our results show that membranes made from simple amphiphiles can form vesicles that are stable enough to retain encapsulated RNAs in the presence of divalent cations, yet dynamic enough to grow spontaneously and allow the passage of Mg(2+) and mononucleotides without specific macromolecular transporters. This combination of stability and dynamics is critical for building model protocells in the laboratory and may have been important for early cellular evolution.     

Ion-specific and charge effects in counterion binding to poly(styrenesulfonate) anions

In order to obtain a deeper insight into effects occurring when an electrolyte solution is added to a solution of a strong polyelectrolyte, the microcalorimetric and potentiometric titrations of poly(sodium 4-styrenesulfonate) (Na(+)PSS(-)) solution with different alkali, earth-alkali and tetraalkylammonium nitrate, perchlorate and chloride solutions were performed. From the calorimetric titrations the differences in sign and magnitude of enthalpy change upon addition of various electrolytes were observed depending on the salt used. Potentiometric titrations using a sodium ion selective electrode have revealed that addition of an electrolyte is accompanied by the increase in sodium activity until a certain critical value is reached, which seems to be the consequence of counterion substitution on the polyelectrolyte chain. In the case of addition of lithium and sodium salts the experimental results for ΔH of mixing can be qualitatively correctly explained by the Poisson-Boltzmann and Monte Carlo calculations based on the continuum solvent models. This is not the case for the mixtures with KNO(3), RbNO(3) and CsNO(3) salts. The results suggest that the ion-specific effects, associated with the changes in the water structure, have to be taken into account when thermodynamic properties of polyelectrolytes in solution are concerned. The calorimetric results imply that the enthalpically observed cation specificity for binding to a poly(styrenesulfonate) group could be correlated with corresponding cation hydration enthalpies. The counterion substitution of sodium with divalent cations was found to be endothermic, which is in qualitative agreement with the electrostatic theory.     

SAXS study on ionic aggregates of styrene‐isoprene‐sulfonated isoprene terpolymer ionomer

The small‐angle X‐ray scattering profile of styrene‐isoprene‐sulfonated isoprene terpolymer ionomers was studied to clarify the structure of ionic aggregates at ambient temperature as a function of the degree of neutralization of Na or Mg cations. An ionic cluster peak was observed for ionomers with a degree of neutralization greater than 25%. The ionic cluster peak was analyzed by the modified hard sphere model proposed by Yarusso and Cooper [Macromolecules, 1983, 16, 1871], and the size of the ion cluster, the closest approach distance between the clusters, and the average system volume per ion cluster were evaluated by a curve‐fitting method. The size of the ion clusters of the ionomer with monovalent Na cation increased with the degree of neutralization, but for divalent Mg cation slightly changed. The number of ion clusters of the styrene‐isoprene‐sulfonated isoprene ionomer with Na and Mg cations characteristically increased with the neutralization. © 2000 John Wiley & Sons, Inc. J Polym Sci B: Polym Phys 38: 1307–1311, 2000     

The importance of cerium substituted phosphates as cation exchanger-some unique properties and related application potentials

Seven different samples of an inorganic ion exchanger, cerium phosphate, suitable for column use have been prepared under varying conditions. The property of these exchangers has been characterized by Inductively Coupled Plasma Spectroscopy. These exchangers are stable in water, dilute mineral acids, ethanol, methanol, acetone and ether. However, in concentrated HCl and HNO(3) they decompose. They retain about 50% of their exchange value after drying at 80 degrees C, and can be regenerated twice without any decrease in exchange capacity. The distribution coefficient measurements for alkaline earth metals, tellurium, iodine and molybdenum using these seven ion exchangers were studied. This revealed the relative affinity for each exchanger, where the sorption in general was most effective at pH 6-8. The titration curves of cerium phosphate (disodium) with alkaline earth metals showed that the selectivity sequence Ba(2+)>Sr(2+)>Ca(2+)>Mg(2+) is observed. Furthermore, it could be deduced that the adsorption of alkaline earth metal cations greatly depends on the cation. These studies have also shown that cerium phosphates with divalent ions are strongly preferred to monovalent ones. Therefore, as for the cerium phosphates with large monovalent ions, the lack of exchange for Ba(2+), Mg(2+) or other alkali earth metal ions should be essentially due to steric hindrance and this could include any one of the following: the large crystalline radius of metal ions or large hydrated ionic radius and high energy of hydration for other divalent ions. Three binary separations of Te(IV)-Mo(VI), Te(IV)-I(I) and Mo(VI)-I(I) has been developed and the recovery ranging from 90 to 100% has been achieved on cerium phosphate (disodium) columns.     

Parameters of monovalent ions in the AMBER-99 forcefield: assessment of inaccuracies and proposed improvements

The monovalent ion parameters used by the AMBER-99 forcefield are shown to exhibit physically inaccurate behavior in molecular dynamics simulations of strong 1:1 electrolytes. These errors arise from an ad hoc adaptation of Aqvist's cation parameters. The result is the rapid formation of large, unphysical clusters at concentrations that are well below solubility limits. The observed unphysical behavior poses a serious challenge for simulating ions around highly charged polymers such as nucleic acids. In this communication, we explain the source of this unphysical behavior. To facilitate the continued use of the popular AMBER parameters, we prescribe a simple fix whereby Aqvist's cations and anions are used in conjunction with the AMBER forcefield for nucleic acids. A preliminary test of this strategy suggests that the proposed fix is reasonable and is likely to be generalizable for simulating diffuse and specific ion binding to nucleic acids.     

Effect of metal ion exchange on the photocurrent response from bacteriorhodopsin on tin oxide electrodes

The transient photocurrent response from bacteriorhodopsin (bR) on tin oxide electrodes was strongly influenced by metal ions bound to bR molecules. The photocurrent polarity reversal pH, which corresponded to the pH value for the reversal of the proton release/uptake sequence in the bR photocycle, of cation-substituted purple membrane (PM) was shifted to lower pH with the increase in the cation affinities to carboxyl groups and a close correlation was noted between the two values. This suggests that the metal ion present in the extracellular region of a bR molecule modulates the pK(a) of proton release groups of bR by stabilizing the ionized state of the proton-releasing glutamic acids. The behavior of photocurrents at light-off in alkaline media, reflecting the proton uptake by bR, was unchanged by binding monovalent (Na(+) and K(+)) or divalent cations (Mg(2+) and Ca(2+)), but was drastically changed by binding La(3+) ions. This can be explained by invoking a substantial slowing of the proton uptake process in the presence of La(3+).