Binding of ionic ligands to polyelectrolytes.

Ionic ligands can bind to polyelectrolytes such as DNA or charged polysaccharides. We develop a Poisson-Boltzmann treatment to compute binding constants as a function of ligand charge and salt concentration in the limit of low ligand concentration. For flexible chain ligands, such as oligopeptides, we treat their conformations using lattice statistics. The theory predicts the salt dependence and binding free energies, of Mg(2+) ions to polynucleotides, of hexamine cobalt(III) to calf thymus DNA, of polyamines to T7 DNA, of oligolysines to poly(U) and poly(a), and of tripeptides to heparin, a charged polysaccharide. One parameter is required to obtain absolute binding constants, the distance of closest separation of the ligand to the polyion. Some, but not all, of the binding entropies and enthalpies are also predicted accurately by the model.     

A new radiochemical method to investigate ion binding with polyelectrolytes

A new method for investigating the binding of ions with polyelectrolytes has been developed. This method, based on Donnan equilibrium and an isotope exchange between the electrolyte and polyelectrolyte, can distinguish territorial from specific binding of ions and can determine fractions of ions bound with the polyion. This method can determine ion binding with polyelectrolytes in a wide range of polyelectrolyte concentrations in multicomponent solutions. The method was tested with radioactive tracers 22Na+, 36Cl- and heparin sodium salt. The influence of the ionic strength on the Na+ binding with heparin was investigated at 310 K. In the limit of zero ionic strength, all Na+ ions are bound to heparin, but only 45% of them are exchangeable. Thus Na+ ions can be bound both territorially and specifically. The fraction of bound ions decreases rapidly with increasing ionic strength. The fraction of the specifically bound ions becomes negligible when the ionic strength exceeds 0.01 M, whereas the fraction of territorially bound ions can be neglected at ionic strengths higher than 0.45 M.     

Critical polyelectrolyte adsorption under confinement: planar slit, cylindrical pore, and spherical cavity

We explore the properties of adsorption of flexible polyelectrolyte chains in confined spaces between the oppositely charged surfaces in three basic geometries. A method of approximate uniformly valid solutions for the Green function equation for the eigenfunctions of polymer density distributions is developed to rationalize the critical adsorption conditions. The same approach was implemented in our recent study for the "inverse" problem of polyelectrolyte adsorption onto a planar surface, and on the outer surface of rod-like and spherical obstacles. For the three adsorption geometries investigated, the theory yields simple scaling relations for the minimal surface charge density that triggers the chain adsorption, as a function of the Debye screening length and surface curvature. The encapsulation of polyelectrolytes is governed by interplay of the electrostatic attraction energy toward the adsorbing surface and entropic repulsion of the chain squeezed into a thin slit or small cavities. Under the conditions of surface-mediated confinement, substantially larger polymer linear charge densities are required to adsorb a polyelectrolyte inside a charged spherical cavity, relative to a cylindrical pore and to a planar slit (at the same interfacial surface charge density). Possible biological implications are discussed briefly in the end.     

Spectroscopic studies of the structural domains of mammalian DNA beta-polymerase

The 8- and 31-kDa fragments of beta-polymerase, prepared by controlled proteolysis as described (Kumar, A., Widen, S. G., Williams, K. R., Kedar, P., Karpel, R. L., and Wilson, S. H. (1990) J. Biol. Chem. 265, 2124-2131), constitute domains that are structurally and functionally dissimilar. There is little disruption of secondary structure upon proteolysis of the intact enzyme, as suggested from CD spectra of the fragments. beta-Polymerase is capable of binding both single- and double-stranded nucleic acids: the 8-kDa fragment binds specifically to single-stranded lattices, whereas the 31-kDa domain displays affinity exclusively for double-stranded polynucleotides. These domains are connected by a highly flexible protease-hypersensitive segment that may allow the coordinate functioning of the two binding activities in the intact protein. beta-Polymerase binds to poly(ethenoadenylic acid) with higher affinity, similar cooperativity, but lesser salt dependence than the 8-kDa fragment. Under physiological conditions, the intact enzyme displays greater binding free energy for single-stranded polynucleotides than the 8-kDa fragment, suggesting that the latter may carry a truncated binding site. Binding of double-stranded calf thymus DNA brings about a moderate quenching of the Tyr and Trp fluorescence emission of both the 31-kDa fragment and beta-polymerase and induces a 6-nm blue shift in the Trp emission maximum of the intact enzyme, but not in the fragment. This latter result is likely due to a change in the relative orientation of the 8- and 31-kDa domains in the intact protein upon interaction with double-stranded DNA; alternatively, the binding mode of intact protein may differ from that of the fragment. Simultaneous interaction of both domains with polynucleotides most likely does not occur since double-stranded DNA binding to the 31-kDa domain of intact beta-polymerase induces the displacement of single-stranded polynucleotides from the 8-kDa domain. These results are evaluated in light of the role of beta-polymerase in DNA repair.     

Comparison of polyelectrolyte theories of the binding of cations to DNA.

Predictions of the binding of counterions to DNA made using the counterion condensation theory developed by Manning are compared with those made using the Poisson-Boltzmann equation, solved numerically by the Runge-Kutta procedure. Ions are defined as territorially or atmospherically bound if they fall within a given distance, defined by counterion condensation theory, from the DNA surface. Two types of experimental situations are considered. The first is the delocalized binding of a single type of counterion to DNA. In this case the Poisson-Boltzmann treatment predicts somewhat lower extents of binding TO DNA, modeled as a 10-A radius cylinder, than does Manning theory. The two theories converge as the radius decreases. The second type of experiment is the competition of ions of different valence for binding to DNA. The theories are compared with literature values of binding constants of divalent ions in the presence of monovalent ions, and of spermidine 3+ in the presence of Na+ or Mg2+. Both predict with fair accuracy the salt dependence of the equilibrium constants.     

Cooperative, excluded-site binding and its dynamics for the interaction of gene 5 protein with polynucleotides

The binding of gene 5 protein to various single-stranded polynucleotides is investigated by fluorescence titrations and stopped-flow measurements. The association state of gene 5 protein itself is analyzed by equilibrium sedimentation: the monomer-dimer equilibrium found in the micromolar concentration range is described by a stability constant of 8 X 10(5) M-1. The fluorescence quenching upon binding to polynucleotides, studied over a broad concentration range and analyzed in terms of a cooperative excluded-site binding model, provides binding constants for "isolated" and for "cooperative" sites. The cooperativity for various ribo- and deoxyribopolymers is between 400 and 800 and is virtually independent of the ionic strength. The binding to isolated sites is strongly dependent upon the ionic strength; analysis in terms of polyelectrolyte theory indicates the compensation of 4 +/- 0.5 charges upon complex formation. The number of nucleotide residues covered by one protein molecule is also found to be 4 +/- 0.5 units. The affinity of gene 5 protein for polynucleotides increases in the series poly(C) less than poly(dA) less than poly(A) less than poly(U) much less than poly(dT); the binding constant for poly(dT) is roughly a factor of 1000 higher than that for the other polymers. Model studies with Lys-Tyr-Lys and Lys-Trp-Lys suggest that the preferential interaction with poly(dT) is not simply due to enhanced stacking interactions between the aromatic amino acids and the thymine residues. Stopped-flow reaction curves obtained by mixing of gene 5 protein with poly(dT) in the micromolar concentration range show three relaxation processes with time constants between 1 ms and 1 s.(ABSTRACT TRUNCATED AT 250 WORDS)     

Flexible polymer-induced condensation and bundle formation of DNA and F-actin filaments

A simple semi-empirical theory is developed for the ionic strength dependence of the flexible polymer-induced condensation of semiflexible polyelectrolytes such as DNA and F-actin filaments. Critical concentrations of flexible polymer needed for condensation are calculated by comparing the free energies of inserting the semiflexible polyelectrolytes in a solution of flexible polymers, respectively, in their free state, and in their condensed state. Predictions of the theory are compared to experimental data on the condensation of DNA and F-actin filaments induced by the flexible polymer poly(ethylene oxide). The theory also predicts that reentrant decollapse is possible at low ionic strength and high concentrations of flexible polymer, as observed for DNA.     

Binding of Mg2+ to single-stranded polynucleotides: hydration and optical studies

The binding of Mg(2+) to single-stranded ribo- and deoxy-polynucleotides, poly(rA), poly(rU), poly(dA) and poly(dT), has been investigated in dilute aqueous solutions at pH 7.5 and 20 degrees C. A combination of ultrasound velocimetry, density, UV and CD spectroscopy have been employed to study hydration and spectral effects of Mg(2+) binding to the polynucleotides. Volume and compressibility effects of Mg(2+) binding to random-coiled poly(rU) and poly(dT) correspond to two coordination bonds probably between the adjacent phosphate groups. The same parameters for poly(rA)+Mg(2+) correspond to an inner-sphere complex with three-four direct contacts. However, almost no hydration effects are arising in binding to its deoxy analog, poly(dA), indicating mostly a delocalized binding mode. In agreement with hydration studies, optical investigations revealed almost no influence of Mg(2+) on poly(dA) properties, while it stabilizes and aggregates poly(rA) single-helix. The evidence presented here indicates that Mg(2+) are able to bind specifically to single-stranded polynucleotides, and recognize their composition and backbone conformation.     

Specific interactions versus counterion condensation. 2. Theoretical treatment within the counterion condensation theory

Polyuronates such as pectate and alginate are very well-known examples of biological polyelectrolytes undergoing, upon addition of divalent cations, an interchain association that acts as the junction of an eventually formed stable hydrogel. In the present paper, a thermodynamic model based on the counterion condensation theory has been developed to account for this cation-induced chain pairing of negatively charged polyelectrolytes. The strong interactions between cross-linking ions and uronate moieties in the specific binding site have been described in terms of chemical bonding, with complete charge annihilation between the two species. The chain-pairing process is depicted as progressively increasing with the concentration of cross-linking counterions and is thermodynamically defined by the fraction of each species. On these bases, the total Gibbs energy of the system has been expressed as the sum of the contributions of the Gibbs energy of the (single) chain stretches and of the (associated) dimers, weighted by their respective fractions 1 - theta and theta. In addition, the model assumes that the condensed divalent counterions exhibit an affinity free-energy for the chain, G(C)(aff,0), and the junction, G(D)(aff,0), respectively. Moreover, a specific Gibbs energy of chemical bonding, G(bond,0), has been introduced as the driving force for the formation of dimers. The model provides the mathematical formalism for calculating the fraction, theta, of chain dimers formed and the amount of ions condensed and bound onto the polyelectrolyte when two different types of counterions (of equal or different valence) are present. The effect of the parameter G(bond,0) has been investigated and, in particular, its difference from G(C,D)(aff,0) was found to be crucial in determining the distribution of the ions into territorial condensation and chemical bonding, respectively. Finally, the effect of the variation of the molar ratio between cross-linking ions and uronic groups in the specific binding sites, sigma0, was evaluated. In particular, a remarkable decrease in the amount of condensed counterions has been pointed out in the case of sigma0 = 1/3, with respect to the value of sigma0 = 1/4, characterizing the traditional "egg-box" structure, as a result of the drop of the charge density of the polyelectrolyte induced by complete charge annihilation.     

Electrooptical study of the electric polarizability of rodlike fragments of DNA

The anisotropy of electrical polarizability of rodlike fragments of DNA has been studied by a number of electro-optical methods: Kerr effect (combined with flow birefringence), light scattering, diehroism, and fluorescence in an electric field. The most sensitive technique (Kerr effect) has been used to study the variation of the polarizability with the nature and concentration of counteroins. DNA fragments constitute a truly rigid polyelectrolyte of known structure. The value obtained can then be quantitatively compared to the predictions of those of the theories of the longitudinal polarizability of rigid polyelectrolytes which are based on true molecular parameters. The comparison emphasizes the role of the counterion–counterion repulsion. Oosawa's theory seems to represent the best approach but fails to explain the differences observed between monovalent and divalent ions.     

Interactions des ions métalliques avec le DNA. I. Comportement de polyélectrolyte et liaison des ions compensateurs

The theory of polyelectrolyte solution of Alexandrowicz: and Katchalsky is used to calculate the electrostatic potential of single stranded polynucleotides for different ionic strength. We have considered the potential of double stranded DNA as the superposition of the different potentials produced by each chain, provided the average distance between the strands is higher than an ionic strength-dependent parameter b. For ionic strength lower than 5 × 10^?2M, the assumption is no longer valid, and a cylindrical model with a uniform charge density must be used. The continuity between the two models was tested, and thus we can calculate the electrical potential at the vicinity of a phosphate group in the whole range of ionic-strength where the double helix is stable. It was therefore possible to determine the theoretical number of ions bound electrostatically to DNA and we found an increase of ion binding with a decrease of ionic strength. Such a model was further applied to the change of specific volume in different salt solutions. Comparison is made with recent pycnometric data on Na^? and Cs^? salts of DNA. Agreement is good in the case of Cs⁺, but for Na⁺, cation binding is likely to be accompanied by a change of the hydration of DNA, which depends on ionic strength. With the same model we can see easily the ion-trapping properties of DNA which play an important role in any formation of complex between heavy ions and bases.     

Light scattering,CD, and ligand binding studies of ferrihemoglobin–polyelectrolyte complexes

Quasi‐elastic light scattering (QELS), electrophoretic light scattering (ELS), CD spectroscopy, and azide binding titrations were used to study the complexation at pH 6.8 between ferrihemoglobin and three polyelectrolytes that varied in charge density and sign. Both QELS and ELS show that the structure of the soluble complex formed between ferrihemoglobin and poly(diallyldimethylammonium chloride) [PDADMAC] varies with protein concentration. At fixed 1.0 mg/mL polyelectrolyte concentration, protein addition increases complex size and decreases complex mobility in a tightly correlated manner. At 1.0 mg/mL or greater protein concentration, a stable complex is formed between one polyelectrolyte chain and many protein molecules (i.e., an intra‐polymer complex) with apparent diameter approximately 2.5 times that of the protein‐free polyelectrolyte. Under conditions of excess polyelectrolyte, each of the three ferrihemoglobin–polyelectrolyte solutions exhibits a single diffusion mode in QELS, which indicates that all protein molecules are complexed. CD spectra suggest little or no structural disruption of ferrihemoglobin upon complexation. Azide binding to the ferrihemoglobin–poly(2‐acrylamide‐2‐methylpropanesulfonate) [PAMPS] complex is substantially altered relative to the polyelectrolyte‐free protein, but minimal change is induced by complexation with an AMPS‐based copolymer of reduced linear charge density. The change in azide binding induced by PDADMAC is intermediate between that of PAMPS and its copolymer. © 1999 John Wiley & Sons, Inc. Biopoly 50: 153–161, 1999     

Base specificity of polyamine binding to synthetic polynucleotides.

The binding of polyamines and magnesium to synthetic polynucleotides has been studied by gel filtration on a Sephadex G-50 column. Among the single-stranded polynucleotides examined [poly(A), poly(C), and poly(U)], polyamines were found to bind to poly(C) and poly(U) preferentially, while the binding of Mg2+ was greatest with poly(A). Spermine bound to poly(U) was displaced completely by NH4+ but incompletely by Mg2+, while Mg2+ bound to poly(A) was displaced completely be spermine but incompletely by NH4+. The optimal pH for the binding of spermine to poly(U) was found to be about 7.9, while Mg2+ could bind to poly(A) over a broad pH range (7.1--8.7).     

Absorption and fluorescence studies of the binding of the recA gene product from E. coli to single-stranded and double-stranded DNA. Ionic strength dependence

The binding of the recA gene product from E. coli to double-stranded and single-stranded nucleic acids has been investigated by following the change in melting temperature of duplex DNA and the fluorescence of single-stranded DNA or poly(dA) modified by reaction with chloroacetaldehyde. At low ionic strength, in the absence of Mg2+ ions, RecA protein binds preferentially to duplex DNA or poly(dA-dT). This leads to an increase of the DNA melting temperature. Stabilization of duplex DNA decreases when ionic strength or pH increases. In the presence of Mg2+ ions, preferential binding to single-stranded polynucleotides is observed. Precipitation occurs when duplex DNA begins to melt in the presence of RecA protein. From competition experiments, different single-stranded and double-stranded polydeoxynucleotides can be ranked according to their ability to bind RecA protein. Structural changes induced in nucleic acids upon RecA binding are discussed together with conformational changes induced in RecA protein upon magnesium binding.     

Stoichiometry and specificity of binding of Rauscher oncovirus 10,000-dalton (p10) structural protein to nucleic acids.

A structural protein of Rauscher oncovirus of about 8,000 to 10,000 daltons (p10), encoded by the gag gene, has been purified in high yield to apparent homogeneity by a simple three-step procedure. The purified protein was highly basic, with an isoelectric point of more than 9.0, and its immunological antigenicity was chiefly group specific. A distinctive property of the protein was the binding to nucleic acids. The stoichiometry of p10 binding to Rauscher virus RNA was analyzed using both 125I-labeled p10 and 3H-labeled RNA. The protein-RNA complex, cross-linked by formaldehyde, was separated from free RNA and free protein by velocity sedimentation and density gradient centrifugation. A maximum of about 140 mol of p10 was bound per mol of 35S RNA, or about one molecule of p10 per 70 nucleotides. This protein-RNA complex banded at a density of about 1.55 g/ml. The number of nucleic acid sites bound and the affinity of p10 binding differed significantly among the other polynucleotides tested. The protein bound to both RNA and DNA with a preference for single-stranded molecules. Rauscher virus RNA and single-stranded phage fd DNA contained the highest number of binding sites. Binding to fd DNA was saturated with about 30 mol of p10 per mol of fd DNA, an average of about one p10 molecule per 180 nucleotides. The apparent binding constant was 7.3 X 10(7) M(-1). The properties of the p10 place it in a category with other nucleic acid binding proteins that achieve a greater binding density on single-stranded than on double-stranded molecules and appear to act by facilitating changes in polynucleotide conformation.     

NMR and density study of Co site binding by polyelectrolytes

The changes of density and chemical shifts of the water proton upon addition of CoCl₂ to aqueous solutions of tetramethylammonium salts of seven polyelectrolytes (polyphosphate, maleic acid-methylvinylether alternated copolymer, polyacrylic acid, carboxymethylcellulose of substitution degree 0.98, 1.3, 2.1, 2.65) have been measured. Assuming a negligible contribution of pseudo-contact interaction to the water proton chemical shift and a constant hyperfine constant upon displacement of water molecules by other ligands, has permitted the calculation of (i) the number of water molecules released by Co²⁺ ions upon binding and the approximate fraction of Co²⁺ ions bound with loss of water, and (ii) the total volume change upon binding and the individual contributions of counterions and polyelectrolyte charged sites to this volume change. Our results are generally in agreement with those obtained using other methods.     

A model for the complex between the helix destabilizing protein GP32 of bacteriophage T4 and single-stranded DNA

A model for the structure of the complex between the helix-destabilizing protein of bacteriophage T4, GP32, and single-stranded DNA is proposed. In this model the bases are arranged in a helix, that is characterized by a relatively large distance between successive bases, a substantial base tilt, in combination with a small rotation per base. This helix is further organized into a tertiary structure, possibly a superhelix, of which the corresponding protein shell corresponds to the relatively rigid and rod-like structure that is observed in hydrodynamic experiments. It is proposed that similar structural features apply to other single-stranded DNA binding proteins in complex with polynucleotides.     

Structure and dynamics of the complex of single stranded DNA binding protein of Escherichia coli with circular single stranded DNA of filamentous phages

We have analyzed the equilibrium and nonequilibrium properties of the complex of the single stranded DNA binding protein of Escherichia coli (EcoSSB) and circular single stranded DNA of filamentous phages M13mp8 and F1 using static and dynamic light scattering, analytical ultracentrifugation and electron microscopy. Upon binding to the single stranded DNA the EcoSSB tetramer replaces an equivalent volume of water trapped within the coiled single stranded DNA and hinders the folding of the single stranded DNA into secondary structures at all salt concentrations. The salt dependent compaction of the stoichiometric complex can be described assuming a flexible polyelectrolyte chain. The solution structure of the macromolecular complex is a random coil and in the electron microscope a beaded flexible structure of the complex with a bead diameter of 6 nm appears at all salt concentrations used. The internal motions of the stoichiometric complex can be described by the Rouse-Zimm model of polymer dynamics. The segmental mobility of the complex can be correlated with changes in the binding site size of the EcoSSB tetramer; it indicates the presence of interactions between EcoSSB tetramers bound to single stranded DNA.     

Effect of polyelectrolyte adsorption on lateral distribution and dynamics of anionic lipids: a Monte Carlo study of a coarse-grain model

We employ Monte Carlo simulations to investigate the interaction between an adsorbing linear flexible cationic polyelectrolyte and a ternary mixed fluid membrane containing neutral (phosphatidylcholine, PC), monovalent (phosphatidylserine, PS), and multivalent (phosphatidylinositol, PIP₂) anionic lipids. We systematically explore the influences of polyelectrolyte chain length, polyelectrolyte charge density, polyelectrolyte total charge amount, and salt solution ionic strength on the static and dynamic properties of different anionic lipid species. Our results show that the multivalent PIP₂ lipids dominate the polyelectrolyte–membrane interaction and competitively inhibit polyelectrolyte–PS binding. When the total charge amount of the polyelectrolyte is less than that of the local oppositely charged PIP₂ lipids, the polyelectrolyte can drag the bound multivalent lipids to diffuse on the membrane, but cannot interact with the PS lipids. Under this condition, the diffusion behaviors of the polyelectrolyte closely follow the prediction of the Rouse model, and the polyelectrolyte chain properties determine the adsorption amount, concentration gradients, and hierarchical mobility of the bound PIP₂ lipids. However, when the total charge amount of the polyelectrolyte is larger than that of the local PIP₂ lipids, the polyelectrolyte further binds the PS lipids around the polyelectrolyte–PIP₂ complex to achieve local electrical neutrality. In this condition, parts of the polyelectrolyte desorb from the membrane and show faster mobility, and the bound PS presents much faster mobility than the segregated PIP₂. This work provides an explanation for heterogeneity formation in different anionic lipids induced by polyelectrolyte adsorption.     

Cooperative disordering of single-stranded polynucleotides through copper crosslinking

The thermal transitions of single-stranded polynucleotides are noncooperative. In contrast, Cu(II) cooperatively disorders the single-stranded helical structures of poly(A) and poly(C), as demonstrated by ORD and UV spectral changes as a function of the Cu²⁺ activity, and by a dramatic chain-length dependence of the spectral changes. Equilibrium dialysis binding studies indicate that the cooperative disordering is paralleled by a somewhat less cooperative binding process. The difference between the thermal- and Cu(II)-induced transition is explained by the following mechanism. (1) Cu(II) initially binds in a noncooperative fashion to phosphate. (2) The Cu(II) so bound forms a second bond to a nonadjacent base site on the same polymer strand or another strand. These intramolecular and intermolecular crosslinks to the bases are responsible for the disordering. (3) The initial crosslinks formed provide nuclei for the cooperative formation of additional crosslinks, producing cooperative spectral changes paralleled by cooperative binding. A comparison of the spectral and binding transitions indicates that there is appreciable noncooperative binding of copper to phosphate, which produces no spectral changes in the presence of added electrolyte. This comparison also indicates that each copper crosslink disorders several bases. The formation of intermolecular crosslinks is demonstrated by a polymer concentration dependence of the disordering. The formation of intramolecular crosslinks can be deduced from the fact that the “cooperative unit” required to explain the differences between the hexamer, which does not readily form intramolecular crosslinks, and the polymer is considerably larger than the cooperative unit determined from the polymer results. The poly(A) disordering transition is less symmetrical than that of poly(C), particularly at low polymer concentrations. These results, together with other phenomena, are explained by a greater flexibility of poly(A), which favors the formation of small intramolecular loops.