Ionophore A23187: the effect of H+ concentration on complex formation with divalent and monovalent cations and the demonstration of K+ transport in mitochondria mediated by A23187.

The two-phase extraction technique has been used to study the equilibrium between A23187, metal cations, and H+. Under these conditions the ionophore forms charge neutral isostoichiometric complexes with divalent cations in which both carboxylate groups of the 2:1 A23187:M2+ complexes are deprotonated. In ethanol, however, the methyl ester of A23187 also binds divalent cations indicating that protonated complexes between A23187 and cations should also exist. With monovalent cations, A23187 forms two charge-neutral complexes of stoichiometries and relative stabilities: A2HM greater than AM. Examination of energy utilization K+ and H+ movements, and light scattering capacity of mitochondria in the presence of divalent cation chelators, A23187, and valinomycin demonstrates that A23187 can act as a nigericin type K+ ionophore under appropriate conditions. Formation constants for the A2HM complexes with monovalent cations indicate that with appropriate conditions transport of Li+ and Na+ mediated by A23187 would also be expected. The binding constant data and associated free energies of complex formation are compared as a function of ionic radius and of cation charge. The data indicate that lack of conformational mobility in A23187 is responsible for the high cation size selectivity of this compound. To explain the transport selectivity of A23187 for divalent cations, it is proposed that this ionophore forms a family of five complexes, isostoichiometric between cations of different valence but of which only charge-neutral species are permeant to membranes. The charge of a given complex is in turn determined by that of the cation. The concept is consistent with the divalent cation transport specificity of A23187, explains the observed monovalent cation transport, and is useful in rationalizing the differences in charge selectivity between A23187 and X-537A.     

Pyruvate kinase: Activation by and catalytic role of the monovalent and divalent cations

Summary This mini review is primarily concerned with the monovalent and divalent cation activation of pyruvate kinase. All preparations of pyruvate kinase from vertebrate tissue which have been examined require monovalent cations such as K⁺ for catalysis. However, several microbial preparations are not activated by monovalent cations. In fact,E. coli synthesizes depending on growth conditions, 2 different forms of the enzyme; one form is not activated while the other is activated by monovalent cations. The monovalent cation was shown by NMR techniques to bind within 4–8 ? of the divalent cation activat or and apparently plays a direct role in the catalytic process. As with all kinases, pyruvate kinase requires a divalent cation for catalysis. Mg⁺² is optimal for the physiological reaction, however, Co⁺², Mn⁺², and Ni⁺² also activate. The divalent cation activation of several non-physiological reactions catalyzed by pyruvate kinase are reviewed. Several lines of evidence suggest that 2 moles of the divalent cation are required in the catalytic event. However, the specific role of both atoms in the catalytic event have not been thoroughly elucidated.     

Channel permeant cations compete selectively with noncompetitive inhibitors of the nicotinic acetylcholine receptor.

Previous work suggests that noncompetitive inhibitor (NCI) ligands and channel permeant cations bind to sites within the nicotinic acetylcholine receptor ion channel. We have used ethidium as a fluorescent probe of the NCI site to investigate interactions between NCI ligands and channel permeant cations. We found that ethidium can be completely displaced from the receptor by a variety of inorganic monovalent and divalent cations. The rank order of monovalent cation affinities was found to be Tl+ greater than Rb+ greater than or equal to K+ greater than Cs+ greater than Na+ greater than Li+. The monovalent cation Kd values vary markedly over a 40-fold range, from 3 to 121 mM. The Kd values and rank order correspond to values determined previously from electrophysiological data. Hill plots of the back titrations yield slopes of 1.0 for all monovalent cations, indicating a single class of independent sites, as shown previously for NCI ligands. Scatchard analysis of ethidium binding in the presence of Tl+ reveals a reduction in affinity and no changes in the maximal number of sites. In the presence of agonist the kinetics of ethidium dissociation induced by the addition of phencyclidine or cations alone or the simultaneous addition of both are nearly identical. The ethidium dissociation rate induced by either phencyclidine or cations is regulated by the occupation of the agonist sites in a similar manner. These results indicate that the effect of cations on NCI ligand binding occurs by mutually exclusive competition. We suggest that NCIs can regulate cation binding at a physiological cation recognition site that is likely part of the cation permeation path through the receptor channel.     

Comparison of the hammerhead cleavage reactions stimulated by monovalent and divalent cations

Although the hammerhead reaction proceeds most efficiently in divalent cations, cleavage in 4 M LiCl is only approximately 10-fold slower than under standard conditions of 10 mM MgCl2 (Murray et al., Chem Biol, 1998, 5:587-595; Curtis & Bartel, RNA, 2001, this issue, pp. 546-552). To determine if the catalytic mechanism with high concentrations of monovalent cations is similar to that with divalent cations, we compared the activities of a series of modified hammerhead ribozymes in the two ionic conditions. Nearly all of the modifications have similar deleterious effects under both reaction conditions, suggesting that the hammerhead adopts the same general catalytic structure with both monovalent and divalent cations. However, modification of three ligands previously implicated in the binding of a functional divalent metal ion have substantially smaller effects on the cleavage rate in Li+ than in Mg2+. This result suggests that an interaction analogous to the interaction made by this divalent metal ion is absent in the monovalent reaction. Although the contribution of this divalent metal ion to the overall reaction rate is relatively modest, its presence is needed to achieve the full catalytic rate. The role of this ion appears to be in facilitating formation of the active structure, and any direct chemical role of metal ions in hammerhead catalysis is small.     

Kinetic and thermodynamic analysis of the interaction of cations with dialkylglycine decarboxylase

Dialkylglycine decarboxylase (DGD) is a tetrameric pyridoxal phosphate (PLP)-dependent enzyme that catalyzes both decarboxylation and transamination in its normal catalytic cycle. Its activity is dependent on cations. Metal-free DGD and DGD complexes with seven monovalent cations (Li(+), Na(+), K(+), Rb(+), Cs(+), NH(4)(+), and Tl(+)) and three divalent cations (Mg(2+), Ca(2+), and Ba(2+)) have been studied. The catalytic rate constants for cation-bound enzyme (ck(cat) and ck(cat)/bK(AIB)) are cation-size-dependent, K(+) being the monovalent cation with the optimal size for catalytic activity. The divalent alkaline earth cations (Mg(2+), Ca(2+), and Ba(2+)) all give approximately 10-fold lower activity compared to monovalent alkali cations of similar ionic radius. The Michaelis constant for aminoisobutyrate (AIB) binding to DGD-PLP complexes with cations (bK(AIB)) varies with ionic radius. The larger cations (K(+), Rb(+), Cs(+), NH(4)(+), and Tl(+)) give smaller bK(AIB) ( approximately 4 mM), while smaller cations (Li(+), Na(+)) give larger values (approximately 10 mM). Cation size and charge dependence is also found with the dissociation constant for PLP binding to DGD-cation complexes (aK(PLP)). K(+) and Rb(+) possess the optimal ionic radius, giving the lowest values of aK(PLP). The divalent alkaline earth cations give aK(PLP) values approximately 10-fold higher than alkali cations of similar ionic radius. The cation dissociation constant for DGD-PLP-AIB-cation complexes (betaK(M)z+) was determined and also shown to be cation-size-dependent, K(+) and Rb(+) yielding the lowest values. The kinetics of PLP association and dissociation from metal-free DGD and its complexes with cations (Na(+), K(+), and Ba(2+)) were analyzed. All three cations tested increase PLP association and decrease PLP dissociation rate constants. Kinetic studies of cation binding show saturation kinetics for the association reaction. The half-life for association with saturating Rb(+) is approximately 24 s, while the half-life for dissociation of Rb(+) from the DGD-PLP-AIB-Rb(+) complex is approximately 12 min.     

The dependence of the conductance of the hemocyanin channel on applied potential and ionic concentration with mono- and divalent cations

Incorporation of Megatura crenulata hemocyanin into phosphatidylcholine black lipid membranes results in the formation of ion channels. Channel properties depend on many factors, three of which are examined in this work: type and concentration of electrolyte and applied voltage. Eight cations at different concentrations have been used. Instantaneous conductance of the channel is a saturating function of both applied voltage and ionic strength of the bathing solution with monovalent cations, but only of ionic strength with divalent cations. Steady-state voltage-conductance relations are nonlinear for both signs but show slight saturation with ionic strength. Relaxation towards the steady state can be fitted by two exponentials with different time constants. All experimental data are fitted postulating the existence of a mechanism of voltage gating of the channel, and of discrete negative charge near its mouth. Specific and nonspecific binding of cations is required.     

Aggregation of nucleosomes by divalent cations.

Conditions of precipitation of nucleosome core particles (NCP) by divalent cations (Ca(2+) and Mg(2+)) have been explored over a large range of nucleosome and cation concentrations. Precipitation of NCP occurs for a threshold of divalent cation concentration, and redissolution is observed for further addition of salt. The phase diagram looks similar to those obtained with DNA and synthetic polyelectrolytes in the presence of multivalent cations, which supports the idea that NCP/NCP interactions are driven by cation condensation. In the phase separation domain the effective charge of the aggregates was determined by measurements of their electrophoretic mobility. Aggregates formed in the presence of divalent cations (Mg(2+)) remain negatively charged over the whole concentration range. They turn positively charged when aggregation is induced by trivalent (spermidine) or tetravalent (spermine) cations. The higher the valency of the counterions, the more significant is the reversal of the effective charge of the aggregates. The sign of the effective charge has no influence on the aspect of the phase diagram. We discuss the possible reasons for this charge reversal in the light of actual theoretical approaches.     

The effect of monovalent and divalent cations on the activity of Streptococcus lactis C10 pyruvate kinase.

The pyruvate kinase (ATP: pyruvate 2-O-phosphotransferase, EC 2.7.1.40) from Streptococcus lactis C10 had an obligatory requirement for both a monovalent cation and divalent cation. NH+4 and K+ activated the enzyme in a sigmoidal manner (nH =1.55) at similar concentrations, whereas Na+ and Li+ could only weakly activate the enzyme. Of eight divalent cations studied, only three (Co2+, Mg2+ and Mn2+) activated the enzyme. The remaining five divalent cations (Cu2+, Zn2+, Ca2+, Ni2+ and Ba2+) inhibited the Mg2+ activated enzyme to varying degrees. (Cu2+ completely inhibited activity at 0.1 mM while Ba2+, the least potent inhibitor, caused 50% inhibition at 3.2 mM). In the presence of 1 mM fructose 1,6-diphosphate (Fru-1,6-P2) the enzyme showed a different kinetic response to each of the three activating divalent cations. For Co2+, Mn2+ and Mg2+ the Hill interaction coefficients (nH) were 1.6, 1.7 and 2.3 respectively and the respective divalent cation concentrations required for 50% maximum activity were 0.9, 0.46 and 0.9 mM. Only with Mn2+ as the divalent cation was there significatn activity in the absence of Fru-1,6-P2. When Mn2+ replaced Mg2+, the Fru-1,6-P2 activation changed from sigmoidal (nH = 2.0) to hyperbolic (nH = 1.0) kinetics and the Fru-1,6-P2 concentration required for 50% maximum activity decreased from 0.35 to 0.015 mM. The cooperativity of phosphoenolpyruvate binding increased (nH 1.2 to 1.8) and the value of the phosphoenolpyruvate concentration giving half maximal velocity decreased (0.18 to 0.015 mM phosphoenolyruvate) when Mg2+ was replaced by Mn2+ in the presence of 1 mM Fru-1,6-P2. The kinetic response to ADP was not altered significantly when Mn2+ was substituted for Mg2+. The effects of pH on the binding of phosphoenolpyruvate and Fru-1,6-P2 were different depending on whether Mg2+ or Mn2+ was the divalent cation.     

Adsorption of divalent cations to bilayer membranes containing phosphatidylserine

The Stern equation, a combination of the Langmuir adsorption isotherm, the Boltzmann relation, and the Grahame equation from the theory of the diffuse double layer, provides a simple theoretical framework for describing the adsorption of charged molecules to surfaces. The ability of this equation to describe the adsorption of divalent cations to membranes containing brain phosphatidylserine (PS) was tested in the following manner. Charge reversal measurements were first made to determine the intrinsic 1:1 association constants of the divalent cations with the anionic PS molecules: when the net charge of a PS vesicle is zero one-half of the available sites are occupied by divalent cations. The intrinsic association constant, therefore, is equal to the reciprocal of the divalent cation concentration at which the mobility of a PS vesicle reverses sign. The Stern equation with this association constant is capable of accurately describing both the zeta potential data obtained with PS vesicles at other concentrations of the divalent cations and the data obtained with with vesicles formed from mixtures of PS and zwitterionic phospholipids. Independent measurements of the number of ions adsorbed to sonicated PS vesicles were made with a calcium-sensitive electrode. The results agreed with the zeta potential results obtained with multilamellar vesicles. When membranes are formed at 20 degrees C in 0.1 M NaCl, the intrinsic 1:1 association constants of Ni, Co, Mn, Ba, Sr, Ca, and Mg with PS are 40, 28, 25, 20, 14, 12, and 8 M-1, respectively.     

Ionic currents of channels that are permeable to monovalent and divalent cations.

The cation-selective channel from Tetrahymena cilia is permeable to both monovalent and divalent cations. The single channel conductance in mixed solutions of K+ and Ca2+ was determined by the Gibbs-Donnan ratio of K+ and Ca2+, and the binding sites of this channel were considered to be always occupied by two potassium ions or by one calcium ion under the experimental conditions: 5-90 mM K+ and 0.5-35 mM Ca2+ (Oosawa and Kasai, 1988). A two-barrier model for the channel was introduced and the values of Michaelis-Menten constants and maximum currents carried by K+ and Ca2+ were calculated using this model. Single channel current amplitudes and reversal potentials were calculated from these values. The calculated single-channel currents were compared with those obtained experimentally. The calculated reversal potentials were compared with the resting potentials of Tetrahymena measured in various concentrations of extracellular K+ and Ca2+. The method of calculation of ionic currents and reversal potentials presented here is helpful for understanding the properties of the channels permeable to both monovalent and divalent cations.     

The effect of cations on the amidase activity of human tissue kallikrein: 1-linear competitive inhibition by sodium, potassium, calcium and magnesium. 2-linear mixed inhibition by aluminium

Hydrolysis of D-valyl-L-leucyl-L-arginine p-nitroanilide by human tissue kallikrein (hK1) was studied in the absence and in the presence of increasing concentrations of the following chloride salts: sodium, potassium, calcium, magnesium and aluminium. The data indicate that the inhibition of hK1 by sodium, potassium, calcium and magnesium is linear competitive and that divalent cations are more potent inhibitors of hK1 than univalent cations. However the inhibition of hK1 by aluminium cation is linear mixed, with the cation being able to bind to both the free enzyme and the ES complex. This cation was the best hK1 inhibitor. Aluminium is not a physiological cation, but is a known neurotoxicant for animals and humans. The neurotoxic actions of aluminium may relate to neuro-degenerative diseases.     

Circular dichroism (CD) studies on yeast enolase: activation by divalent cations

The effect of divalent cations on the near ultraviolet circular dichroism (CD) spectrum of yeast enolase showed that calcium, magnesium, and nickel ions produced identical changes. This was interpreted as indicating that the cations bound to the same sites on the enzyme and produced identical changes in tertiary structure. There was no effect of magnesium ion on the far ultraviolet spectrum. Evidently magnesium ion has no effect on the secondary structure. Substrate bound to the enzyme when the above cations were present although calcium permits no enzymatic activity. The CD spectral difference produced by the substrate was nearly the reverse of that produced by the metal ions. Glycolic acid phosphate, a competitive inhibitor lacking carbon-3, produced no effect, indicating carbon-3 was necessary for the CD spectral changes. The CD and visible absorption spectra of nickel and cobalt bound to various sites on the enzyme showed that the binding sites were octahedral or distorted octahedral in coordination and that the ligands appeared to be oxyligands: water molecules, hydroxyl or carboxyl groups. Examination of the effects of substrate and two compounds thought to be "transition state analogues" showed that these perturbed the "conformational" sites of the enzyme. The "catalytic" and "inhibitory" sites did not appear to be very CD active.     

Large divalent cations and electrostatic potentials adjacent to membranes. A theoretical calculation

We have extended the Gouy-Chapman theory of the electrostatic diffuse double layer by considering the finite size of divalent cations in the aqueous phase adjacent to a charged surface. The divalent cations are modeled as either two point charges connected by an infinitely thin, rigid "rod" or two noninteracting point charges connected by an infinitely thin, flexible "string." We use the extended theory to predict the effects of a cation of length 10 A (1 nm) on the zeta and surface potentials of phospholipid bilayer membranes. The predictions of the rod and string models are similar to one another but differ markedly from the predictions of the Gouy-Chapman theory. Specifically, the extended model predicts that a large divalent cation will have a smaller effect on the potential adjacent to a negatively charged bilayer membrane than a point divalent cation, that the magnitude of this discrepancy will decrease as the Debye length increases, and that a large divalent cation will produce a negative zeta potential on a membrane formed from zwitterionic lipids. These predictions agree qualitatively with the experimental results obtained with the large divalent cation hexamethonium. We discuss the biological relevance of our calculations in the context of the interaction of cationic drugs with receptor sites on cell membranes.     

An aspartate ring at the TolC tunnel entrance determines ion selectivity and presents a target for blocking by large cations

The TolC protein of Escherichia coli comprises an outer membrane beta-barrel channel and a contiguous alpha-helical tunnel spanning the periplasm, providing an exit duct for protein export and multidrug efflux. It forms a single transmembrane pore that is open to the outside of the cell but constricted at the peri-plasmic tunnel entrance. This sole constriction is lined by a ring of six aspartate residues, two in each of the three identical monomers. When these were replaced by alanines, the resulting TolC(DADA) protein reconstituted normally in black lipid membranes but showed altered electrophysiological characteristics. In particular, it had lost the strong pH dependence of the wild type and had switched ion selectivity from cations to anions. The function of wild-type TolC as a membrane pore was severely inhibited by divalent and trivalent cations entering the channel tunnel from the channel ("extracurricular") side. Divalent cations bound reversibly to effect complete blocking of the transmembrane ion flux. Trivalent cations were more potent. Hexamminecobalt bound at nanomolar concentrations allowed visualization of single blocking events, whereas the smaller Cr(3+) cation bound irreversibly and could also access the cation binding site via the tunnel entrance. The inhibitory cations had no effect on the mutant TolC(DADA), supporting the view that the aspartate ring is the cation binding site. The electronegative entrance is widely conserved throughout the TolC family, which is essential for efflux and export my Gram-negative bacteria, suggesting that it could present a general target for drugs.     

Fusogenic capacities of divalent cations and effect of liposome size

The initial kinetics of divalent cation (Ca2+, Ba2+, Sr2+) induced fusion of phosphatidylserine (PS) liposomes, LUV, is examined to obtain the fusion rate constant, f11, for two apposed liposomes as a function of bound divalent cation. The aggregation of dimers is rendered very rapid by having Mg2+ in the electrolyte, so that their subsequent fusion is rate limiting to the overall reaction. In this way the fusion kinetics are observed directly. The bound Mg2+, which by itself is unable to induce the PS LUV to fuse, is shown to affect only the aggregation kinetics when the other divalent cations are present. There is a threshold amount of bound divalent cation below which the fusion rate constant f11 is small and above which it rapidly increases with bound divalent cation. These threshold amounts increase in the sequence Ca2+ less than Ba2+ less than Sr2+, which is the same as found previously for sonicated PS liposomes, SUV. While Mg2+ cannot induce fusion of the LUV and much more bound Sr2+ is required to reach the fusion threshold, for Ca2+ and Ba2+ the threshold is the same for PS SUV and LUV. The fusion rate constant for PS liposomes clearly depends upon the amount and identity of bound divalent cation and the size of the liposomes. However, for Ca2+ and Ba2+, this size dependence manifests itself only in the rate of increase of f11 with bound divalent cation, rather than in any greater intrinsic instability of the PS SUV. The destabilization of PS LUV by Mn2+ and Ni2+ is shown to be qualitatively distinct from that induced by the alkaline earth metals.     

Ion regulation of homotypic vacuole fusion in Saccharomyces cerevisiae

Biological membrane fusion employs divalent cations as protein cofactors or as signaling ligands. For example, Mg2+ is a cofactor for the N-ethylmaleimide-sensitive factor (NSF) ATPase, and the Ca2+ signal from neuronal membrane depolarization is required for synaptotagmin activation. Divalent cations also regulate liposome fusion, but the role of such ion interactions with lipid bilayers in Rab- and soluble NSF attachment protein receptor (SNARE)-dependent biological membrane fusion is less clear. Yeast vacuole fusion requires Mg2+ for Sec18p ATPase activity, and vacuole docking triggers an efflux of luminal Ca2+. We now report distinct reaction conditions where divalent or monovalent ions interchangeably regulate Rab- and SNARE-dependent vacuole fusion. In reactions with 5 mm Mg2+, other free divalent ions are not needed. Reactions containing low Mg2+ concentrations are strongly inhibited by the rapid Ca2+ chelator BAPTA. However, addition of the soluble SNARE Vam7p relieves BAPTA inhibition as effectively as Ca2+ or Mg2+, suggesting that Ca2+ does not perform a unique signaling function. When the need for Mg2+, ATP, and Sec18p for fusion is bypassed through the addition of Vam7p, vacuole fusion does not require any appreciable free divalent cations and can even be stimulated by their chelators. The similarity of these findings to those with liposomes, and the higher ion specificity of the regulation of proteins, suggests a working model in which ion interactions with bilayer lipids permit Rab- and SNARE-dependent membrane fusion.     

Structural roles of monovalent cations in the HDV ribozyme

The hepatitis delta virus (HDV) ribozyme catalyzes viral RNA self-cleavage through general acid-base chemistry in which an active-site cytidine and at least one metal ion are involved. Monovalent metal ions support slow catalysis and were proposed to substitute for structural, but not catalytic, divalent metal ions in the RNA. To investigate the role of monovalent cations in ribozyme structure and function, we determined the crystal structure of the precursor HDV ribozyme in the presence of thallium ions (Tl(+)). Two Tl(+) ions can occupy a previously observed divalent metal ion hexahydrate-binding site located near the scissile phosphate, but are easily competed away by cobalt hexammine, a magnesium hexahydrate mimic and potent reaction inhibitor. Intriguingly, a third Tl(+) ion forms direct inner-sphere contacts with the ribose 2'-OH nucleophile and the pro-S(p) scissile phosphate oxygen. We discuss possible structural and catalytic implications of monovalent cation binding for the HDV ribozyme mechanism.     

DNA denaturation in situ. Effect of divalent cations and alcohols

Heat denaturation profiles of rat thymus DNA, in intact cells, reveal the presence of two main DNA fractions differing in sensitivities to heat. The thermosensitive DNA fraction shows certain properties similar to those of free DNA: its stability to heat is decreased by alcohols and is increased in the presence of the divalent cations Ca2+, Mn2+, or Mg2+ at concentrations of 0.1-1.0 mM. Unlike free DNA, however, this fraction denatures over a wide range of temperature, and is heterogeneous, consisting of at least two subfractions with different melting points. The thermoresistant DNA fraction shows lowered stability to heat in the presence of Ca2+, Mn2+, or Mg2+ and increased stability in the presence of alcohols. It denatures within a relatively narrow range of temperature, consists of at least three subfractions, and, most likely, represents DNA masked by histones. The effect of Ca2+, Mn2+, or Mg2+ in lowering the melting point of the thermoresistant DNA fraction is seen at cation concentrations comparable to those required to maintain gross chromatin structure in cell nuclei or to support superhelical DNA conformation in isolated chromatin (0.5-1.0 mM). It is probable that factors involved in the maintenance of gross chromatin organization in situ and/or related to DNA superhelicity also have a role in modulating DNA-histone interactions, and that DNA-protein interactions as revealed by conventional methods using isolated chromatin may be different from those revealed when gross chromatin morphology remains intact.     

Probing non-selective cation binding in the hairpin ribozyme with Tb(III)

Catalysis by the hairpin ribozyme is stimulated by a wide range of both simple and complex metallic and organic cations. This independence from divalent metal ion binding unequivocally excludes inner-sphere coordination to RNA as an obligatory role for metal ions in catalysis. Hence, the hairpin ribozyme is a unique model to study the role of outer-sphere coordinated cations in folding of a catalytically functional RNA structure. Here, we demonstrate that micromolar concentrations of a deprotonated aqueous complex of the lanthanide metal ion terbium(III), Tb(OH)(aq)(2+), reversibly inhibit the ribozyme by competing for a crucial, yet non-selective cation binding site. Tb(OH)(aq)(2+) also reports a likely location of this binding site through backbone hydrolysis, and permits the analysis of metal binding through sensitized luminescence. We propose that the critical cation-binding site is located at a position within the catalytic core that displays an appropriately-sized pocket and a high negative charge density. We show that cationic occupancy of this site is required for tertiary folding and catalysis, yet the site can be productively occupied by a wide variety of cations. It is striking that micromolar Tb(OH)(aq)(2+) concentrations are compatible with tertiary folding, yet interfere with catalysis. The motif implicated here in cation-binding has also been found to organize the structure of multi-helix loops in evolutionary ancient ribosomal RNAs. Our findings, therefore, illuminate general principles of non-selective outer-sphere cation binding in RNA structure and function that may have prevailed in primitive ribozymes of an early "RNA world".     

Electrostatic effects on the stability of condensed DNA in the presence of divalent cations.

Cylindrical cell model Poisson-Boltzmann (P-B) calculations are used to evaluate the electrostatic contributions to the relative stability of various DNA conformations (A, B, C, Z, and single-stranded (ss) with charge spacings of 3.38 and 4.2 A) as a function of interhelix distance in a concentrated solution of divalent cations. The divalent ion concentration was set at 100 mM, to compare with our earlier reports of spectroscopic and calorimetric experiments, which demonstrate substantial disruption of B-DNA geometry. Monovalent cations neutralize the DNA phosphates in two ways, corresponding to different experimental situations: 1) There is no significant contribution to the ionic strength from the neutralizing cations, corresponding to DNA condensation from dilute solution and to osmotic stress experiments in which DNA segments are brought into close proximity to each other in the presence of a large excess of buffer. 2) The solution is uniformly concentrated in DNA, so that the neutralizing cations add significantly to those in the buffer at close DNA packing. In case 1), conformations with lower charge density (Z and ssDNA) have markedly lower electrostatic free energies than B-DNA as the DNA molecules approach closely, due largely to ionic entropy. If the divalent cations bind preferentially to single-stranded DNA or a distorted form of B-DNA, as is the case with transition metals, the base pairing and stacking free energies that stabilize the double helix against electrostatic denaturation may be overcome. Strong binding to the bases is favored by the high concentration of divalent cations at the DNA surface arising from the large negative surface potential; the surface concentration increases sharply as the interhelical distance decreases. In case 2), the concentration of neutralizing monovalent cations becomes very large and the electrostatic free energy difference between secondary structures becomes small as the interhelical spacing decreases. Such high ionic concentrations will be expected to modify the stability of DNA by changing water activity as well as by screening electrostatic interactions. This may be the root of the decreased thermal stability of DNA in the presence of high concentrations of magnesium ions.