Monovalent Cation Binding Research Articles

Mechanism of monovalent and divalent ion mobility in Nafion membrane: An atomistic simulation study.

Polymer cation exchange membranes (CEMs) are widely used in water treatment processes. The fundamental factors that control the separation and selectivity of cations with different valences in CEMs are not fully understood. In this study, we use atomistic molecular dynamics simulations to investigate the underlying molecular mechanisms that control the mobility of cations with different valences in Nafion membranes. Our results indicate structural differences in binding of monovalent and divalent cations, which gives rise to differences in ion diffusion in Nafion. Monovalent cations are shown to be "territorially" bound, i.e., bound while partially hydrated, to the fixed charge groups whereas divalent cations are shown to be "site" bound, i.e., bound while fully dehydrated, to the charge groups on the polymer. This difference in binding structure gives rise to differences in transport characteristics of cations in Nafion.

D-Alanine–d-alanine ligase as a model for the activation of ATP-grasp enzymes by monovalent cations

The ATP-grasp superfamily of enzymes shares an atypical nucleotide-binding site known as the ATP-grasp fold. These enzymes are involved in many biological pathways in all domains of life. One ATP-grasp enzyme, d-alanine-d-alanine ligase (Ddl), catalyzes ATP-dependent formation of the d-alanyl-d-alanine dipeptide essential for bacterial cell wall biosynthesis and is therefore an important antibiotic drug target. Ddl is activated by the monovalent cation (MVC) K+, but despite its clinical relevance and decades of research, how this activation occurs has not been elucidated. We demonstrate here that activating MVCs bind adjacent to the active site of Ddl from Thermus thermophilus and used a combined biochemical and structural approach to characterize MVC activation. We found that TtDdl is a type II MVC-activated enzyme, retaining activity in the absence of MVCs. However, the efficiency of TtDdl increased ∼20-fold in the presence of activating MVCs, and it was maximally activated by K+ and Rb+ ions. A strict dependence on ionic radius of the MVC was observed, with Li+ and Na+ providing little to no TtDdl activation. To understand the mechanism of MVC activation, we solved crystal structures of TtDdl representing distinct catalytic stages in complex with K+, Rb+, or Cs+ Comparison of these structures with apo TtDdl revealed no evident conformational change on MVC binding. Of note, the identified MVC binding site is structurally conserved within the ATP-grasp superfamily. We propose that MVCs activate Ddl by altering the charge distribution of its active site. These findings provide insight into the catalytic mechanism of ATP-grasp enzymes.

An Anionic Surfactant on an Anionic Substrate: Monovalent Cation Binding.

Neutron reflectometry has been used to study the adsorption of the anionic surfactant bis(2-ethylhexyl) sulfosuccinate cesium salt on the anionic surface of mica. Evidence of significant adsorption is reported. The adsorption is reversible and changes little with pH. This unexpected adsorption behavior of an anionic molecule on an anionic surface is discussed in terms of recent models for surfactant adsorption such as cation bridging, where adsorption has been reported with the divalent ion calcium but not previously observed with monovalent ions.

Cloning and molecular characterization of the betaine aldehyde dehydrogenase involved in the biosynthesis of glycine betaine in white shrimp (Litopenaeus vannamei)

The enzyme betaine aldehyde dehydrogenase (BADH) catalyzes the irreversible oxidation of betaine aldehyde to glycine betaine (GB), a very efficient osmolyte accumulated during osmotic stress. In this study, we determined the nucleotide sequence of the cDNA for the BADH from the white shrimp Litopenaeus vannamei (LvBADH). The cDNA was 1882 bp long, with a complete open reading frame of 1524 bp, encoding 507 amino acids with a predicted molecular mass of 54.15 kDa and a pI of 5.4. The predicted LvBADH amino acid sequence shares a high degree of identity with marine invertebrate BADHs. Catalytic residues (C-298, E-264 and N-167) and the decapeptide VTLELGGKSP involved in nucleotide binding and highly conserved in BADHs were identified in the amino acid sequence. Phylogenetic analyses classified LvBADH in a clade that includes ALDH9 sequences from marine invertebrates. Molecular modeling of LvBADH revealed that the protein has amino acid residues and sequence motifs essential for the function of the ALDH9 family of enzymes. LvBADH modeling showed three potential monovalent cation binding sites, one site is located in an intra-subunit cavity; other in an inter-subunit cavity and a third in a central-cavity of the protein. The results show that LvBADH shares a high degree of identity with BADH sequences from marine invertebrates and enzymes that belong to the ALDH9 family. Our findings suggest that the LvBADH has molecular mechanisms of regulation similar to those of other BADHs belonging to the ALDH9 family, and that BADH might be playing a role in the osmoregulation capacity of L. vannamei.

Differential binding of monovalent cations to KcsA: Deciphering the mechanisms of potassium channel selectivity

This work explores whether the ion selectivity and permeation properties of a model potassium channel, KcsA, could be explained based on ion binding features. Non-permeant Na+ or Li+ bind with low affinity (millimolar KD's) to a single set of sites contributed by the S1 and S4 sites seen at the selectivity filter in the KcsA crystal structure. Conversely, permeant K+, Rb+, Tl+ and even Cs+ bind to two different sets of sites as their concentration increases, consistent with crystallographic evidence on the ability of permeant species to induce concentration-dependent transitions between conformational states (non-conductive and conductive) of the channel's selectivity filter. The first set of such sites, assigned also to the crystallographic S1 and S4 sites, shows similarly high affinities for all permeant species (micromolar KD's), thus, securing displacement of potentially competing non-permeant cations. The second set of sites, available only to permeant cations upon the transition to the conductive filter conformation, shows low affinity (millimolar KD's), thus, favoring cation dissociation and permeation and results from the contribution of all S1 through S4 crystallographic sites.The differences in affinities between permeant and non-permeant cations and the similarities in binding behavior within each of these two groups, correlate fully with their permeabilities relative to K+, suggesting that binding is an important determinant of the channel's ion selectivity. Conversely, the complexity observed in permeation features cannot be explained just in terms of binding and likely relates to reported differences in the occupancy of the S2 and S3 sites by the permeant cations.

Binding of Monovalent and Multivalent Metal Cations to Polyethylene Oxide in Methanol Probed by Electrophoretic and Diffusion NMR.

Complex formation in methanol between monodisperse polyethylene oxide (PEO) and a large set of cations was studied by measuring the effective charge acquired by PEO upon complexation. Quantitative data were obtained at a low ionic strength of 2 mM (for some salts, also between 0.5 and 6 mM) by a combination of diffusion nuclear magnetic resonance (NMR) and electrophoretic NMR experiments. For strongly complexing cations, the magnitude of the acquired effective charge was on the order of 1 cation per 100 monomer units. For monovalent cations, the relative strength of binding varies as Na+ < K+ ≈ Rb+ ≈ Cs+, whereas Li+ exhibited no significant binding. All polyvalent cations bind very weakly, except for Ba2+ that exhibited strong binding. Anions do not bind, as is shown by the lack of response to the chemical nature of anionic species (perchlorate, iodide, or acetate). Diffusion experiments directly show that the acetate anion with monovalent cations does not associate with PEO. Considering all cations, we find that the observed binding does not follow any Hofmeister order. Instead, binding occurs below a critical surface charge density, which indicates that the degree of complexation is defined by the solvation shell. A large solvation shell prevents the binding of most multivalent ions.

Circular Dichroism is Sensitive to Monovalent Cation Binding in Monensin Complexes.

Monensin is a natural antibiotic that exhibits high affinity to certain metal ions. In order to explore its potential in coordination chemistry, circular dichroism (CD) spectra of monensic acid A (MonH) and its derivatives containing monovalent cations (Li(+) , Na(+) , K(+) , Rb(+) , Ag(+) , and Et4 N(+) ) in methanolic solutions were measured and compared to computational models. Whereas the conventional CD spectroscopy allowed recording of the transitions down to 192 nm, synchrotron radiation circular dichroism (SRCD) revealed other bands in the 178-192 nm wavelength range. CD signs and intensities significantly varied in the studied compounds, in spite of their similar crystal structure. Computational modeling based on the Density Functional Theory (DFT) and continuum solvent model suggests that the solid state monensin structure is largely conserved in the solutions as well. Time-dependent Density Functional Theory (TDDFT) simulations did not allow band-to-band comparison with experimental spectra due to their limited precision, but indicated that the spectral changes were caused by a combination of minor conformational changes upon the monovalent cation binding and a direct involvement of the metal electrons in monensin electronic transitions. Both the experiment and simulations thus show that the CD spectra of monensin complexes are very sensitive to the captured ions and can be used for their discrimination. Chirality 28:420-428, 2016. © 2016 Wiley Periodicals, Inc.

Probing the kinetic and thermodynamic consequences of the tetraloop/tetraloop receptor monovalent ion-binding site in P4-P6 RNA by smFRET.

Structured RNA molecules play roles in central biological processes and understanding the basic forces and features that govern RNA folding kinetics and thermodynamics can help elucidate principles that underlie biological function. Here we investigate one such feature, the specific interaction of monovalent cations with a structured RNA, the P4-P6 domain of the Tetrahymena ribozyme. We employ single molecule FRET (smFRET) approaches as these allow determination of folding equilibrium and rate constants over a wide range of stabilities and thus allow direct comparisons without the need for extrapolation. These experiments provide additional evidence for specific binding of monovalent cations, Na+ and K+, to the RNA tetraloop-tetraloop receptor (TL-TLR) tertiary motif. These ions facilitate both folding and unfolding, consistent with an ability to help order the TLR for binding and further stabilize the tertiary contact subsequent to attainment of the folding transition state.

A Ribokinase Family Conserved Monovalent Cation Binding Site Enhances the MgATP-induced Inhibition in E. coli Phosphofructokinase-2

The presence of a regulatory site for monovalent cations that affects the conformation of the MgATP-binding pocket leading to enzyme activation has been demonstrated for ribokinases. This site is selective toward the ionic radius of the monovalent cation, accepting those larger than Na+. Phosphofructokinase-2 (Pfk-2) from Escherichia coli is homologous to ribokinase, but unlike other ribokinase family members, presents an additional site for the nucleotide that negatively regulates its enzymatic activity. In this work, we show the effect of monovalent cations on the kinetic parameters of Pfk-2 together with its three-dimensional structure determined by x-ray diffraction in the presence of K+ or Cs+. Kinetic characterization of the enzyme shows that K+ and Na+ alter neither the kcat nor the KM values for fructose-6-P or MgATP. However, the presence of K+ (but not Na+) enhances the allosteric inhibition induced by MgATP. Moreover, binding experiments show that K+ (but not Na+) increases the affinity of MgATP in a saturable fashion. In agreement with the biochemical data, the crystal structure of Pfk-2 obtained in the presence of MgATP shows a cation-binding site at the conserved position predicted for the ribokinase family of proteins. This site is adjacent to the MgATP allosteric binding site and is only observed in the presence of Cs+ or K+. These results indicate that binding of the monovalent metal ions indirectly influences the allosteric site of Pfk-2 by increasing its affinity for MgATP with no alteration in the conformation of residues present at the catalytic site.

Effect of Monovalent Ion Concentration in Molecular Simulation of Electroporation

Monovalent ion concentration gradients, regulated by ion pumps and leak channels, are critical components of many cellular functions. This dynamic balance is disturbed by the electroporative permeabilization of the cell membrane, which bypasses the normal membrane barriers to transmembrane ion flux. A better understanding of ion transport during and after electroporation will enable more efficient and more effective utilization of this method in biomedicine and biotechnology.Molecular dynamics (MD) simulations provide a view of the behavior of ions and biomolecular structures at the molecular level. Previous MD studies have demonstrated some of the effects of monovalent ions on phospholipid bilayers, including decreased area per lipid, higher ordering in head group vertical orientations, and decreased lateral lipid mobility [1-4]. MD simulations of porated membranes have also shown that the binding of monovalent cations to phospholipids can increase pore line tension, which leads to a decrease in pore lifetime [5]. In this work we employ MD simulations to systematically study the effects of varying the concentration of Na+, K+, and Cl− in POPC lipid bilayer systems during different stages of electropore formation.1.Gurtovenko, A.A. and I. Vattulainen, J Phys Chem B, 2008. 112(7): p. 1953-62.2.Vacha, R., et al., Journal of Physical Chemistry A, 2009. 113(26): p. 7235-7243.3.Vacha, R., et al., J Phys Chem B, 2010. 114(29): p. 9504-9.4.Jurkiewicz, P., et al., Biochim Biophys Acta, 2012. 1818(3): p. 609-16.5.Leontiadou, H., A.E. Mark, and S.J. Marrink, Biophys J, 2007. 92(12): p. 4209-15.

Linkage of cation binding and folding in human telomeric quadruplex DNA

Formation of DNA quadruplexes requires monovalent cation binding. To characterize the cation binding stoichiometry and linkage between binding and folding, we carried out KCl titrations of Tel22 (d[A(GGGTTA) 3]), a model of the human telomere sequence, using a fluorescent indicator to determine [K +] free and circular dichroism to assess the extent of folding. At [K +] free = 5 mM (sufficient for > 95% folding), the apparent binding stoichiometry is 3K +/Tel22; at [K +] free = 20 mM, it increased to 8–10K +/Tel22. Thermodynamic analysis shows that at [K +] free = 5 mM, K + binding contributes approximately − 4.9 kcal/mol for folding Tel22. The overall folding free energy is − 2.4 kcal/mol, indicating that there are energetically unfavorable contributions to folding. Thus, quadruplex folding is driven almost entirely by the energy of cation binding with little or no contribution from other weak molecular interactions.

Preferential Site Binding of Monovalent Cations With the Random Coil Form of DNA

Collins and Rogers (Chem. Biol. Interactions 19 197 1977) have suggested that monovalent tetraalkylammonium (TXA+) ions diminish the melting temperature of duplex DNA by preferential binding to the random coil form. We have investigated this suggestion using a known 16 base hairpin as the test oligomer and electrophoretic mobility in free solution as the analytical parameter. Measurements were made in the range 15-60° using background electrolyte solutions (BGE) containing 0.01-0.9 M monovalent cation (M+) and diethylmalonate as the buffering cation at pH 7.3. A 16 base all-T oligonucleotide served as a reference random coil to correct for changes in hairpin mobility contributed by changes in the physical properties of the BGE. Thermal transitions were observed as a decrease in the mobility ratio of the hairpin/coil to 1.00 as the temperature was increased at a fixed [M+]. The Tm of the hairpin increased hyperbolically from 37° as the [M+] increased linearly. The increase in Tm observed with Na+ is exactly that predicted by the DINAMelt algorithm. The span of the increase in Tm has the order Na=Li=K>NH4>TMA>Tris>TEA>TPA>TBA. Cation site binding was analyzed as a change in the mobility ratio with increasing [M+] at 20° where the hairpin conformation predominates. The mobility ratio increases hyperbolically with increasing [M+], consistent with the view that M+ preferentially site binds with the coil form. The magnitude of the increase in mobility ratio from the binding measurements at 20° correlates with the dTm/d(log M+) from the melting measurements, suggesting that the two phenomena are related. We propose that the effect of M+ on DNA melting is the net result of two opposing contributions, preferential stabilization of the counterion cloud about the structured form and preferential site binding with the unstructured random coil form.

Na+ and K+ Allosterically Regulate Cooperative DNA Binding by the Human Progesterone Receptor

Cooperativity is a common mechanism used by transcription factors to generate highly responsive yet stable gene regulation. For the two isoforms of human progesterone receptor (PR-A and PR-B), differences in cooperative DNA binding energetics may account for their differing transcriptional activation properties. Here we report on the molecular origins responsible for cooperativity, finding that it can be activated or repressed with Na(+) and K(+), respectively. We demonstrate that PR self-association and DNA-dependent cooperativity are linked to a monovalent cation binding event and that this binding is coupled to modulation of receptor structure. K(+) and Na(+) are therefore allosteric effectors of PR function. Noting that the apparent binding affinities of Na(+) and K(+) are comparable to their intracellular concentrations and that PR isoforms directly regulate the genes of a number of ion pumps and channels, these results suggest that Na(+) and K(+) may additionally function as physiological regulators of PR action.

Tryptophan Synthase: Structure and Function of the Monovalent Cation Site

The monovalent cation (MVC) site of the tryptophan synthase from Salmonella typhimurium plays essential roles in catalysis and in the regulation of substrate channeling. In vitro, MVCs affect the equilibrium distribution of intermediates formed in the reaction of l-Ser with the alpha(2)beta(2) complex; the MVC-free, Cs(+)-bound, and NH(4)(+)-bound enzymes stabilize the alpha-aminoacrylate species, E(A-A), while Na(+) binding stabilizes the l-Ser external aldimine species, E(Aex(1)). Two probes of beta-site reactivity and conformation were used herein, the reactive indole analogue, indoline, and the l-Trp analogue, l-His. MVC-bound E(A-A) reacts rapidly with indoline to give the indoline quinonoid species, E(Q)(indoline), which slowly converts to dihydroiso-l-tryptophan. MVC-free E(A-A) gives very little E(Q)(indoline), and turnover is strongly impaired; the fraction of E(Q)(indoline) formed is <3.5% of that given by the Na(+)-bound form. The reaction of l-Ser with the MVC-free internal aldimine species, E(Ain), initially gives small amounts of an active E(A-A) which converts to an inactive species on a slower, conformational, time scale. This inactivation is abolished by the binding of MVCs. The inactive E(A-A) appears to have a closed beta-subunit conformation with an altered substrate binding site that is different from the known conformations of tryptophan synthase. Reaction of l-His with E(Ain) gives an equilibrating mixture of external aldimine and quinonoid species, E(Aex)(his) and E(Q)(his). The MVC-free and Na(+) forms of the enzyme gave trace amounts of E(Q)(his) ( approximately 1% of the beta-sites). The Cs(+) and NH(4)(+) forms gave approximately 17 and approximately 14%, respectively. The reactivity of MVC-free E(Ain) was restored by the binding of an alpha-site ligand. These studies show MVCs and alpha-site ligands act synergistically to modulate the switching of the beta-subunit from the open to the closed conformation, and this switching is crucial to the regulation of beta-site catalytic activity. Comparison of the structures of Na(+) and Cs(+) forms of the enzyme shows Cs(+) favors complexes with open indole binding sites poised for the conformational transition to the closed state, whereas the Na(+) form does not. The beta-subunits of Cs(+) complexes exhibit preformed indole subsites; the indole subsites of the open Na(+) complexes are collapsed, distorted, and too small to accommodate indole.

Use of thallium to identify monovalent cation binding sites in GroEL.

GroEL is a bacterial chaperone protein that assembles into a homotetradecameric complex exhibiting D(7) symmetry and utilizes the co-chaperone protein GroES and ATP hydrolysis to assist in the proper folding of a variety of cytosolic proteins. GroEL utilizes two metal cofactors, Mg(2+) and K(+), to bind and hydrolyze ATP. A K(+)-binding site has been proposed to be located next to the nucleotide-binding site, but the available structural data do not firmly support this conclusion. Moreover, more than one functionally significant K(+)-binding site may exist within GroEL. Because K(+) has important and complex effects on GroEL activity and is involved in both positive (intra-ring) and negative (inter-ring) cooperativity for ATP hydrolysis, it is important to determine the exact location of these cation-binding site(s) within GroEL. In this study, the K(+) mimetic Tl(+) was incorporated into GroEL crystals, a moderately redundant 3.94 A resolution X-ray diffraction data set was collected from a single crystal and the strong anomalous scattering signal from the thallium ion was used to identify monovalent cation-binding sites. The results confirmed the previously proposed placement of K(+) next to the nucleotide-binding site and also identified additional binding sites that may be important for GroEL function and cooperativity. These findings also demonstrate the general usefulness of Tl(+) for the identification of monovalent cation-binding sites in protein crystal structures, even when the quality and resolution of the diffraction data are relatively low.

Selectivity and Cooperativity of Modulatory Ions in a Neurotransmitter Receptor

Ions play a modulatory role in many proteins. Kainate receptors, members of the ionotropic glutamate receptor family, require both monovalent anions and cations in the extracellular milieu for normal channel activity. Molecular dynamics simulations and extensive relative binding free energy calculations using thermodynamic integration were performed to elucidate the rank order of binding of monovalent cations, using x-ray crystal structures of the GluR5 kainate receptor dimers with bound cations from the alkali metal family. The simulations show good agreement with experiments and reveal that the underlying backbone structure of the binding site is one of the most rigid regions of the protein. A simplified model where the partial charge of coordinating oxygens was varied suggests that selectivity arises from the presence of two carboxylate groups. Furthermore, using a potential of mean force derived from umbrella sampling, we show that the presence of cations lower the energy barrier for anion approach and binding in the buried anion binding cavity.

Monovalent Cation Binding in the Minor Groove of DNA A-Tracts

The binding of five different monovalent cations to DNA oligomers containing A-tracts, runs of four or more contiguous adenine residues, has been assessed by capillary electrophoresis, using the Replacement Ion method. In this method, a nonbinding cation in the background electrolyte is gradually replaced by a binding cation, keeping the ionic strength of the solution constant. Monovalent cation binding reduces the effective charge of an A-tract-containing oligomer, decreasing its free solution mobility. The cations bind in the A-tract minor groove, because the binding site can be blocked by the minor groove binding drug netropsin. Li(+), NH(4)(+), and Tris(+) ions bind to A-tracts with similar affinities; the binding of Na(+) ions is weaker, and K(+) ion binding is highly variable. Each A-tract appears to bind one monovalent cation upon saturation of the binding site(s). For a given cation, the apparent dissociation constants depend on A-tract sequence and orientation, but not on the phasing of the A-tracts with respect to the helix repeat. Differences in the cooperativity of binding of the various cations to A-tracts with different sequences suggest that monovalent cation binding may be coupled with a conformational transition leading to the formation of the characteristic narrow minor groove A-tract structure.

DNA intercalation of methylene blue and quinacrine: new insights into base and sequence specificity from structural and thermodynamic studies with polynucleotides

The binding of the known DNA intercalators methylene blue and quinacrine with four sequence specific polynucleotides, viz. poly(dG-dC).poly(dG-dC), poly(dG).poly(dC), poly(dA-dT).poly(dA-dT) and poly(dA).poly(dT), have been compared using absorbance, fluorescence, competition dialysis and thermal melting and the thermodynamic aspects of the interaction studied. In all the cases, non-cooperative binding phenomena obeying neighbor exclusion principle was observed though the affinity was remarkably higher for quinacrine and the nature of the binding was characterized to be true intercalation. The data on the salt dependence of binding derived from the plot of log Kvs. log[Na(+)] revealed a slope of around 1.0, consistent with the values predicted by the theories for the binding of monovalent cations, and contained contributions from polyelectrolytic and non-polyelectrolytic forces. The bindings were characterized by strong stabilization of the polynucleotides against thermal strand separation in both optical melting as well as differential scanning calorimetry studies. The data analyzed from the thermal melting and isothermal titration calorimetry studies were in close proximity to those obtained from absorption spectral titration data. Isothermal titration calorimetry results revealed the bindings to poly(dG-dC).poly(dG-dC), poly(dG).poly(dC) and poly(dA-dT).poly(dA-dT) to be exothermic and favoured by both negative enthalpy and large favourable positive entropy changes, while that to poly(dA).poly(dT) was endothermic and entropy driven. The heat capacity changes obtained from temperature dependence of enthalpy gave negative values to all polynucleotides. New insights on the molecular aspects of interaction of these molecules to DNA have emerged from these studies.

DNA binding of benzophenanthridine compounds sanguinarine versus ethidium: Comparative binding and thermodynamic profile of intercalation

There is compelling evidence that cellular DNA is the target of many small molecule anticancer agents. Consequently, elucidation of the molecular nature governing the interaction of small molecules to DNA is paramount to the progression of the rational drug design strategies. In this study, we have compared the binding and thermodynamic aspects of two known DNA binding agents, ethidium and sanguinarine with calf thymus DNA. The study revealed non-cooperative binding phenomena for both the drugs to DNA with an affinity similar for ethidium and sanguinarine as observed from different techniques. The binding phenomena analyzed from isothermal titration calorimetry showed exothermic binding for both compounds that was favoured by negative enthalpy and positive entropy changes typical of intercalative binding. The binding of both the drugs was further characterized by strong stabilization of DNA against thermal strand separation in optical melting as well as differential scanning calorimetry studies. The data of the salt dependence of binding of sanguinarine and ethidium from the plot of log K versus log [Na +] revealed a slope of −0.711 and −0.875, respectively, consistent with the values predicted by the theories for the binding of monovalent cations and the binding free energy has been analyzed for contributions from polyelectrolytic and non-polyelectrolytic forces. The salt dependence of the binding was also evident from the conformational changes in the circular dichroism where both extrinsic and induced changes were lowered on increasing the salt concentration. The heat capacity changes obtained from temperature dependence of enthalpy change gave values of (−590 and −670) J · mol −1 · K −1, respectively for the binding of sanguinarine and ethidium to DNA. Overall the DNA binding of ethidium was slightly more favoured over sanguinarine.

Monovalent Cation Binding by Curved DNA Molecules Containing Variable Numbers of A-Tracts

Monovalent cation binding by DNA A-tracts, runs of four or more contiguous adenine or thymine residues, has been determined for two curved ∼200 basepair (bp) restriction fragments, one taken from the M13 origin of replication and the other from the VP1 gene of SV40. These two fragments have previously been shown to contain stable, centrally located bends of 44° and 46°, respectively, located within ∼60 bp “curvature modules” containing four or five irregularly spaced A-tracts. Transient electric birefringence measurements of these two fragments, sequence variants containing reduced numbers of A-tracts in the SV40 curvature module or changes in the residues flanking the A-tracts in the M13 curvature module, have been combined with the free solution electrophoretic mobilities of the same fragments using known equations to estimate the effective charge of each fragment. The effective charge is reduced, on average, by one-third charge for each A-tract in the curvature module, suggesting that each A-tract binds a monovalent cation approximately one-third of the time. Monovalent cation binding to two or more A-tracts is required to observe significant curvature of the DNA helix axis.