DNA-surfactant interactions: coupled cooperativity in ligand binding leads to duplex stabilization.

The cooperative nature of interaction of cationic surfactants with short oligonucleotides leading to eventual stabilization of DNA duplexes is demonstrated. At submicellar concentrations and DNA:surfactant charge ratios of 0.2 to 0.8, the association of single chain (CTAB) and double chain (DOTAP) surfactants to oligonucleotides is initiated by electrostatic interaction of cationic ligands with polyanionic DNA that aligns the surfactant molecules on the DNA template. This is followed by binding of new surfactant ligands to the initial complex, driven cooperatively by the hydrophobic forces, leading to in situ formation of surfactant-bound and bare duplexes as separate species. These exhibit independent melting behaviour characterised by double transition in thermal UV profiles, with a higher T(m) for surfactant-DNA complexes. Understanding the cooperative binding of the cationic surfactants to the DNA described here may have implications for rational design of DNA binding drugs and DNA delivery systems.     

Thermodynamic model of cooperativity in a dimeric protein: unique and independent parameters formulation.

A model of the cooperative interaction of ligand binding to a dimeric protein is presented based upon the unique and independent parameters (UIP) thermodynamic formulation (Gutheil and McKenna, Biophys. Chem. 45 (1992) 171-179). The analysis is developed from an initial model which includes coupled conformational and ligand binding equilibria. This completely general model is then restricted to focus on conformationally mediated cooperative interactions between the ligands and the expressions for the apparent ligand binding constant and the apparent ligand-ligand interaction constant are derived. The conditions under which there is no cooperative interaction between the ligands are found as roots to a polynomial equation. Consideration of the distribution of species among the various conformational states in this general model leads to a set of inequalities which can be represented as a two dimensional plot of boundaries. By superimposing a contour plot of the value of the apparent ligand-ligand interaction constant over the plot of boundaries a complete graphical representation of this system is achieved similar to a phase diagram. It is found that the parameter space homologous to Koshland-Nemethy-Filmer type of model is most consistent with both positive and negative cooperativity in this model. The maximal amount of positive and negative cooperativity are found to be simple functions of Kc, the equilibrium constant associated with the change of a subunit and ligand from the unligated to ligated conformation. It is shown that under certain limiting conditions the apparent allosteric interaction between ligands is equal to the conformational interaction between subunits. The methods presented are generally applicable to the theoretical analysis of thermodynamic interactions in complex systems.     

EQUIL: Simulation and data analysis of binding reactions with arbitrary chemical models

We have developed an algorithm for simulation and analysis of arbitrary chemical systems in equilibrium, with emphasis on ligand binding reactions. The program EQUIL can treat reactions involving multiple ligands, multiple binding sites, ternary complex models, allosteric effectors, competitive and noncompetitive binding, conformational changes, cooperativity, and generally any scheme that can be represented as a set of chemical equations. EQUIL is based on a general thermodynamic model of chemical equilibria; it does not involve nonlinear transformation of experimental data, but it does require the user to define the model of interaction between ligands and receptors by writing down the appropriate chemical reactions. EQUIL contains features of particular importance to ligand binding experiments: variable binding capacities, nonspecific binding, and the ability to simultaneously analyze data from different types of experiments. Furthermore, the simulation feature of EQUIL allows the user to investigate the feasibility of experiments that could possibly distinguish between different reaction models. We illustrate the use of this program on personal computers to analyze and simulate simple and complicated interactions between ligands and receptors.     

Ligand-receptor interactions and membrane structure investigated by AFM and time-resolved fluorescence microscopy

The atomic force microscope (AFM) and the associated dynamic force spectroscopy technique have been exploited to quantitatively assess the interaction between proteins and their binding to specific ligands and membrane surfaces. In particular, we have studied the specific interaction between lung surfactant protein D and various carbohydrates. In addition, we have used scanning AFM and time-resolved fluorescence microscopy to image the lateral structure of different lipid bilayers and their morphological changes as a function of time. The various systems studied illustrate the potential of modern AFM techniques for application to biomedical research, specifically within immunology and liposome-based drug delivery.     

Co-evolution of proteins with their interaction partners

The divergent evolution of proteins in cellular signaling pathways requires ligands and their receptors to co-evolve, creating new pathways when a new receptor is activated by a new ligand. However, information about the evolution of binding specificity in ligand-receptor systems is difficult to glean from sequences alone. We have used phosphoglycerate kinase (PGK), an enzyme that forms its active site between its two domains, to develop a standard for measuring the co-evolution of interacting proteins. The N-terminal and C-terminal domains of PGK form the active site at their interface and are covalently linked. Therefore, they must have co-evolved to preserve enzyme function. By building two phylogenetic trees from multiple sequence alignments of each of the two domains of PGK, we have calculated a correlation coefficient for the two trees that quantifies the co-evolution of the two domains. The correlation coefficient for the trees of the two domains of PGK is 0. 79, which establishes an upper bound for the co-evolution of a protein domain with its binding partner. The analysis is extended to ligands and their receptors, using the chemokines as a model. We show that the correlation between the chemokine ligand and receptor trees' distances is 0.57. The chemokine family of protein ligands and their G-protein coupled receptors have co-evolved so that each subgroup of chemokine ligands has a matching subgroup of chemokine receptors. The matching subfamilies of ligands and their receptors create a framework within which the ligands of orphan chemokine receptors can be more easily determined. This approach can be applied to a variety of ligand and receptor systems.     

Electrophoretic and spectroscopic characterization of the protein patterns formed in different surfactant solutions

The complexations between catalase and the sodium perfluorooctanoate/sodium octanoate and sodium perfluorooctanoate/sodium dodecanoate systems have been studied by a combination of electrophoresis and spectroscopy measurements. The numbers of adsorption sites on the protein were determined from the observed increases of the zeta-potential as a function of surfactant concentration in the regions where the adsorption was a consequence of the hydrophobic effect. The Gibbs energies of adsorption of the surfactants onto the protein were evaluated and the results show that for all systems, Gibbs energies are negative and larger, in absolute values, at low values of surfactant concentration where binding to the high energy sites takes place, and become less negative as more surfactant molecules bind, suggesting a saturation process. The role of hydrophobic interactions in these systems has been demonstrated to be the predominant. Spectroscopy measurements suggest conformational changes on catalase depending on the surfactant mixture as well as the mixed ratio. No isosbestic point or shifts have been found showing that catalase has spectrophotometrically one kind of binding site for these surfactant mixtures.     

Shear-driven rolling of DNA-adhesive microspheres

Many biologically important cell binding processes, such as the rolling of leukocytes in the vasculature, are multivalent, being mediated by large numbers of weak binding ligands. Quantitative agreement between experiments and models of rolling has been elusive and often limited by the poor understanding of the binding and unbinding kinetics of the ligands involved. Here, we present a cell-free experimental model for such rolling, consisting of polymer microspheres whose adhesion to a glass surface is mediated by ligands with well-understood force-dependent binding free energy—short complementary DNA strands. We observe robust rolling activity for certain values of the shear rate and the grafted DNA strands’ binding free energy and force sensitivity. The simulation framework developed to model leukocyte rolling, adhesive dynamics, quantitatively captures the mean rolling velocity and lateral diffusivity of the experimental particles using known values of the experimental parameters. Moreover, our model captures the velocity variations seen within the trajectories of single particles. Particle-to-particle variations can be attributed to small, plausible differences in particle characteristics. Overall, our findings confirm that state-of-the-art adhesive dynamics simulations are able to capture the complex physics of particle rolling, boding well for their extension to modeling more complex systems of rolling cells.     

Theoretical aspects of DNA-protein interactions: co-operative and non-co-operative binding of large ligands to a one-dimensional homogeneous lattice

The interaction of proteins binding non-specifically to DNA, as well as the properties of many other interacting ligand-lattice systems important in molecular biology, requires a fundamentally different type of theoretical analysis than that provided by the classical Scatchard independent-binding-site treatment. Exact and relatively simple equations describing the binding of both non-interacting and interacting (co-operative) ligands to a homogeneous one-dimensional lattice are derived in terms of ligand site size, intrinsic binding constant and ligand-ligand co-operativity (equations (10) and (15) in the text). The mathematical approach is based on simple conditional probabilities, and reveals some largely unrecognized characteristics of such lattice binding systems. The results indicate that the binding of any non-interacting ligand covering more than one lattice residue results in non-linear (convex downward) Scatchard plots. The introduction of positive ligand-ligand co-operativity antagonizes this non-linearity, and eventually leads to plots of the opposite curvature. The maxima, limiting slopes, and intercepts of such plots can be used to estimate the required binding parameters. The method can be extended to systems involving heterogeneous ligands, and some types of heterogeneous lattices. Procedures for applying the method to a variety of interacting systems are presented, and a preliminary analysis is carried out for some selected sets of data from the literature.     

Influence of targeting ligand flexibility on receptor binding of particulate drug delivery systems

Receptor-mediated drug targeting via nanoengineered particulate delivery systems is an emerging field. However, little is known about how such magic bullets should be assembled to yield optimal targeting efficiency. Here we investigated the influence of targeting ligand flexibility on binding of ligand-coated microparticles to cell surface receptors. Using the ganglioside G(M1)-binding B subunit of cholera toxin as ligand and fluorescent microparticles as a model delivery system, conjugates with different numbers of linkages between ligand and particle were prepared and tested for their efficiency to bind to live fibroblast monolayers. Our results show that multiple bonds between ligand and particle reduce the targeting rate by up to 50% compared to constructs where ligands are attached via single aliphatic chains. Thus, for maximum performance, targeted particulate drug delivery systems should be assembled such that ligands are attached via single sigma bonds only, allowing the ligand molecules to adopt an optimal binding conformation.     

Influence of quercetin on the interaction of gliclazide with human serum albumin – spectroscopic and docking approaches

Protein‐binding interactions are displacement reactions which have been implicated as the causative mechanisms in many drug–drug interactions. Thus, the aim of presented study was to analyse human serum albumin‐binding displacement interaction between two ligands, hypoglycaemic drug gliclazide and widely distributed plant flavonoid quercetin. Fluorescence analysis was used in order to investigate the effect of substances on intrinsic fluorescence of human serum albumin (HSA) and to define binding and quenching properties of ligand–albumin complexes in binary and ternary systems, respectively. Both ligands showed the ability to bind to HSA, although to a different extent. The displacement effect of one ligand from HSA by the other one has been described on the basis of the quenching curves and binding constants comparison for the binary and ternary systems. According to the fluorescence data analysis, gliclazide presents a substance with a lower binding capacity towards HSA compared with quercetin. Results also showed that the presence of quercetin hindered the interaction between HSA and gliclazide, as the binding constant for gliclazide in the ternary system was remarkably lower compared with the binary system. This finding indicates a possibility for an increase in the non‐bound fraction of gliclazide which can lead to its more significant hypoglycaemic effect. Additionally, secondary and tertiary structure conformational alterations of HSA upon binding of both ligands were investigated using synchronous fluorescence, circular dichroism and FT‐IR. Experimental data were complemented with molecular docking studies. Obtained results provide beneficial information about possible interference upon simultaneous co‐administration of the food/dietary supplement and drug.     

Reconciliation of classical and reacted‐site probability approaches to allowance for ligand multivalence in binding studies

The objective of this investigation is to engender greater confidence in the validity of binding equations derived for multivalent ligands on the basis of reacted‐site probability theory. To that end, a demonstration of the theoretical interconnection between expressions derived by the classical stepwise equilibria and reacted‐site probability approaches for univalent ligands is followed by the use of the traditional stepwise procedure to derive binding equations for bivalent and trivalent ligands. As well as demonstrating the unwieldy nature of the classical binding equation for multivalent ligand systems, that exercise has allowed numerical simulation to be used to illustrate the equivalence of binding curves generated by the two approaches. The advantages of employing a redefined binding function for multivalent ligands are also confirmed by subjecting the simulated results to a published analytical procedure that has long been overlooked. Copyright © 2013 John Wiley & Sons, Ltd.     

Substrate-Modulated Thermal Fluctuations Affect Long-Range Allosteric Signaling in Protein Homodimers: Exemplified in CAP

The role of conformational dynamics in allosteric signaling of proteins is increasingly recognized as an important and subtle aspect of this ubiquitous phenomenon. Cooperative binding is commonly observed in proteins with twofold symmetry that bind two identical ligands. We construct a coarse-grained model of an allosteric coupled dimer and show how the signal can be propagated between the distant binding sites via change in slow global vibrational modes alone. We demonstrate that modulation on substrate binding of as few as 5-10 slow modes can give rise to cooperativity observed in biological systems and that the type of cooperativity is given by change of interaction between the two monomers upon ligand binding. To illustrate the application of the model, we apply it to a challenging test case: the catabolite activator protein (CAP). CAP displays negative cooperativity upon association with two identical ligands. The conformation of CAP is not affected by the binding, but its vibrational spectrum undergoes a strong modification. Intriguingly, the first binding enhances thermal fluctuations, yet the second quenches them. We show that this counterintuitive behavior is, in fact, necessary for an optimal anticooperative system, and captured within a well-defined region of the model's parameter space. From analyzing the experimental results, we conclude that fast local modes take an active part in the allostery of CAP, coupled to the more-global slow modes. By including them into the model, we elucidate the role of the modes on different timescales. We conclude that such dynamic control of allostery in homodimers may be a general phenomenon and that our model framework can be used for extended interpretation of thermodynamic parameters in other systems.     

Towards a model of non-equilibrium binding of metal ions in biological systems

We have used a systems biology approach to address the hitherto insoluble problem of the quantitative analysis of non-equilibrium binding of aqueous metal ions by competitive ligands in heterogeneous media. To-date, the relative proportions of different metal complexes in aqueous media has only been modelled at chemical equilibrium and there are no quantitative analyses of the approach to equilibrium. While these models have improved our understanding of how metals are used in biological systems they cannot account for the influence of kinetic factors in metal binding, transport and fate. Here we have modelled the binding of aluminium, Al(III), in blood serum by the iron transport protein transferrin (Tf) as it is widely accepted that the biological fate of this non-essential metal is not adequately described by experiments, invitro and insilico, which have consistently demonstrated that at equilibrium 90% of serum Al(III) is bound by Tf. We have coined this paradox ‘the blood-aluminium problem’ and herein applied a systems biology approach which utilised well-found assumptions to pare away the complexities of the problem such that it was defined by a comparatively simple set of computational rules and, importantly, its solution assumed significant predictive capabilities. Here we show that our novel computational model successfully described the binding of Al(III) by Tf both at equilibrium and as equilibrium for Al_Tf was approached. The model predicted significant non-equilibrium binding of Al by ligands in competition with Tf and, thereby, provided an explanation of why the distribution of Al(III) in the body cannot be adequately described by its binding and transport by Tf alone. Generically the model highlighted the significance of kinetic in addition to thermodynamic constraints in defining the fate of metal ions in biological systems.     

The cooperative binding of large ligands to a one-dimensional lattice: the steric hindrance effect

The cooperative binding of monomeric ligands to a long lattice of a linear polymer with complete or partial steric hindrance is treated using a matrix method. Results and typical calculations of the model are represented. Non-saturated cooperative binding as well as two-step (biphasic) binding isotherms can be interpreted by the steric hindrance model. This is applicable to the analysis of the binding of surfactants to polymer. The usefulness and the limitation also are discussed.     

Mapping the importance of four factors in creating monovalent ion selectivity in biological molecules

The ability of macrocycles, enzymes, ion channels, transporters, and DNA to differentiate among ion types is often crucial to their function. Using molecular dynamics simulations on both detailed systems and simple models, we quantify the importance of several factors which affect the ion selectivity of such molecules, including the number of coordinating ligands, their dipole moment, and their vibrational motion. The information resulting from our model systems is distilled into a series of selectivity maps that can be used to read off the relative free energy associated with binding of different ions, and to provide an estimate of the importance of the various factors. Although our maps cannot capture all elements of real systems, it is remarkable that they produce differential site-binding energies that are in line with experiment and more-detailed simulations for a variety of systems—making them useful for understanding the origins of selective binding and transport. The chemical nature of the coordinating ligands is essential for creating thermodynamic ion selectivity in flexible molecules (such as 18c6), but as the binding site becomes more rigid, the number of ligands (as in ion channels) and the reduction of thermal fluctuations (as in amino-acid transporters) can become important. In the future, our maps could aid in the determination of the local structure from binding energies and assist in the design of novel ion selective molecules.     

Prediction,docking study and molecular simulation of 3D DNA aptamers to their targets of endocrine disrupting chemicals

Abstract

Typical endocrine disrupting chemicals, including BPA (Bisphenol A), E₂ (17-β-Estradiol) and PCB 72 (polychlorinated biphenyl 72), are commonly and widely present in the environment with good chemical stability that are difficult to decompose in vitro and in vivo. Most of the high-qualified antibodies are required as the key biomaterials to fabricate the immunosensor for capturing and detecting. As an ideal alternative, the short-chain oligonucleotides (aptamer) are essentially and effectively employed with the advantages of small size, chemical stability and high effectiveness for monitoring these environmental contaminants. However, the molecular interaction, acting site and mode are still not well understood. In this work, we explored the binding features of the aptamers with their targeting ligands. The molecular dynamics simulations were performed on the aptamer–ligand complex systems. The stability of each simulation system was evaluated based on its root-mean-square deviation. The affinities of these proposed ligands and the predicted binding sites are analyzed. According to the binding energy analysis, the affinities between ligands and aptamers and the stability of the systems are BPA?>?PCB 72 >E₂. Trajectory analysis for these three complexes indicated that these three ligands were able to steadily bind with aptamers at docking site from 0 to 50?ns and contributed to alteration of conformation of aptamers.     

Metal sulfides in oxygenated aquatic systems: implications for the biotic ligand model

The Biotic Ligand Model (BLM) attempts to predict metal toxicity to aquatic organisms on the basis of metal speciation and effects at the cell surface. Current versions of the BLM for silver and copper consider metal binding by inorganic ligands, dissolved organic matter (DOM) and also competition at the cell surface from calcium and protons (pH). Recent studies reported in the geochemical and ecotoxicological literature have indicated the importance of sulfide as a ligand, even in fully oxygenated aquatic systems. Speciation calculations for oxygenated waters do not currently include reduced sulfur as a ligand and as a consequence, no version of the BLM model has been published including reduced sulfur. This reflects the limitations on our knowledge regarding reduced sulfur in aquatic systems. In this paper we highlight the need to include reduced sulfur in the Biotic Ligand Model, with the interaction between silver and inorganic metal sulfides as a specific example. The geochemical importance of metal sulfides as ligands for silver and the effect of 'dissolved' metal sulfide and other ligands on metal toxicity and accumulation are described and reviewed. Recommendations are made for future work needed to incorporate sulfide ligands into the BLM's modeling framework.     

Proton magnetic resonance studies of the binding of an anionic surfactant with a benzene ring to a protein polypeptide with special reference to SDS-polyacrylamide gel electrophoresis.

The binding of an anionic surfactant to a protein polypeptide has been studied by the proton magnetic resonance (PMR) technique to form a part of our studies on the principles of SDS-polyacrylamide gel electrophoresis. Sodium 4-(p-butylphenyl) butane-1-sulfonate (CH3-(CH2)3-0-(CH2)4-SO3-Na+) was employed as an anionic surfactant, and reduced and carbosyamidomethylated (RCAM) bovine serum albumin as a typical protein polypeptide. The binding isotherm of the surfactant to RCAM bovine serum albumin was similar to that of sodium dodecyl sulfate (SDS). The surfactant could replace SDS in SDS-polyacrylamide gel electrophoresis without affecting the wellknown mode of spearation of protein bands. These results gave a sound basis for the assumption that the investigation of the complex between a surfactant with a benzene ring and RCAM bovine serum albumin would provide useful knowledge concerning the principles of SDS-polyacrylamide gel electrophoresis. Aggregation of the aromatic surfactant necessarily brings benzene rings together. A benzene ring is a strong source of the ring current effect on chemical shifts in nuclear magnetic resonance (NMR). Chemical shifts of the surfactant in NMR are, therefore, sensitive to whether the surfactant molecules are single-molecularly dissolved or aggregated. Full advantage was taken of the above fact in the present PMR study of the binding of the surfactant to RCAM bovine serum albumin. The chemical shifts of the phenyl and methyl protons both for the single-molecular and micellar aggregated states were estimated from measurements of the shifts as a function of the surfactant concentration. They shifted to a higher magnetic field on micelle formation, due to the increase of the ring current effect. Corresponding measurements for the complex between the surfactant and RCAM bovine serum albumin gave estimates of the chemical shifts of the phenyl and methyl groups of the surfactant bound to the protein polypeptide. They were found to shift to a magnetic field somewhat higher than that for the micellar state throughout the concentration range of the surfactant examined. These results strongly suggest that the surfactant molecules bind to the protein polypeptide in the form of micelle-like clusters, and that PMR of the groups are further influenced by the diagmagnetic effect of the protein polypeptide present as a core. No appreciable change in the mode of binding, corresponding to the steep increase in the amount of binding in the binding isotherm, was observed from the PMR studies. Taking the observed similarity between SDS and the aromatic surfactant in the binding and the gel electrophoresis into consideration, the present results strongly suggest that SDS also binds to protein polypeptides in the form of micelle-like clusters under the conditions of SDS-polyacrylamide gel electrophoreses, and support our "necklace model".