Transmission of binding information across lipid bilayers

A synthetic transmembrane receptor that is capable of transmitting binding information across a lipid bilayer membrane is reported. The binding event is based on aggregation of the receptor triggered by copper(II) complexation to ethylenediamine functionalities. By labelling the receptor with fluorescent dansyl groups, the copper(II) binding event could be monitored by measuring the extent of fluorescence quenching. Comparing the receptor with a control receptor lacking the transmembrane linkage revealed that the transmembrane receptor binds copper(II) ions more tightly than the non-spanning control receptor at low copper(II) concentrations. Since the intrinsic binding to copper(II) is the same for both receptors, this effect was attributed to synergy between the connected interior and exterior binding sides of the transmembrane receptor. Thus, this is the first reported artificial signalling event in which binding of a messenger on one side of the membrane leads to a cooperative binding event on the opposite side of the membrane, resembling biological signalling systems and helping us to get a better understanding of the requirements for more effective artificial signalling systems.     

Divalent binding of a bis(adamantyl)-functionalized calix[4]arene to beta-cyclodextrin-based hosts: an experimental and theoretical study on multivalent binding in solution and at self-assembled monolayers

The divalent binding of a bis(adamantyl)-functionalized calix[4]arene (1) to an EDTA-tethered beta-cyclodextrin (CD) dimer (2) in solution (1.2 x 10(7) M(-)(1)) was 3 orders of magnitude weaker than the binding constant ( approximately 10(10) M(-)(1)) for the interaction of 1 at CD self-assembled monolayers (SAMs) on gold. This difference in binding is rationalized using a theoretical model, which interprets the divalent binding as two consecutive monovalent binding events, i.e., an intermolecular interaction followed by an intramolecular binding event, the latter of which is associated with an effective concentration term accounting for the close proximity of the two interacting species. The methodology presented in the model is applicable to divalent binding both in solution and at SAMs and indicates that the difference in observed binding constants mainly stems from a difference in effective concentration.     

Dissecting the thermodynamics and cooperativity of ligand binding in cytochrome P450eryF

Conformational flexibility and cooperativity in ligand recognition are two key aspects of the catalytic diversity of cytochrome P450 enzymes. In this study, we dissect the ligand binding stoichiometry and energetics of the soluble bacterial P450eryF by isothermal titration calorimetry (ITC) using three allosteric and two non-allosteric ligands of diverse chemistry. Complementary spectral binding studies and sequential, two-ligand docking simulations were performed to help assign the binding sites. Binding of 4-phenylpyridine (4-PP) or 4-(4-chlorophenyl)imidazole (4-CPI) showed 1:1 stoichiometry in ITC, consistent with the lack of cooperativity observed in spectral binding studies. The larger ligands 9-aminophenanthrene (9-AP), 1-pyrenebutanol (1-PB), and alpha-naphthoflavone (ANF) show cooperative spectral binding and yielded 2:1 stoichiometry. The associated thermodynamic parameters for the sites were calculated using a sequential binding mechanism. The binding constant (KD) for the first site was almost two times lower than that of the second site for all three compounds. Ligand binding at site 1 was entropically favored, whereas binding at site 2 was weak and entropically unfavorable. Simulations showed that two molecules of 9-AP, ANF or 1-PB can be adequately docked to two individual sub-sites within a large binding pocket. The absence of hydrophobic tethering and ligand stacking are consistent with the single low affinity binding site observed for 4-CPI and 4-PP. Competitive binding studies with P450eryF preloaded with either 1-PB or ANF showed a decrease in the affinities for 9-AP at both the sites, with large entropy-enthalpy compensation, indicating the ability of the binding pocket to accommodate two ligands of diverse chemistry and enable cooperativity.     

Spectral and thermodynamic studies of the interaction of binding site probes with poly(vinylpyrrolidone)

The binding of two ionic azo dyes (4-phenylazo-1-naphthol mono-and disulfonate) and a fluorescent probe (2-p-toluidinonaphthalene-6-sulfonic acid, TNS) to poly(vinylpyrrolidone) (PVP) was studied to obtain information on the nature of the interaction, binding isotherm, and binding site. Sorption of the dyes followed a Langmuir isotherm only at low polymer saturation. Apparent cooperativity in binding was seen at higher saturation. The polymer had a higher intrinsic binding constant but lower binding capacity for the doubly charged dye than for the structurally similar singly charged dye. Both dyes consisted of tautomeric mixtures of hydrazone and azonaphthol forms in equilibrium in the bound and unbound state. The preferential binding of the azonaphthol tautomer of the disulfonate was highly exothermic and accompanied by an entropy decrease. The binding of the hydrazone form was less favored by 1.8 kcal/mol, was weakly exothermic, and accompained by an entropy increase. Increased preference for the azonaphthol tautomer accompanied chain extension from charging the polymer. Chain extension had no effect on the emission frequency of bound TNS. Large differences in binding capacities for similarly charged dyes indicated the existence of specific dye-site interactions. Arguments are presented against nonspecific hydrophobic interactions as predominant forces responsible for binding.     

Molecular interactions of nitrogen-containing bisphosphonates within farnesyl diphosphate synthase

Bisphosphonates, known for their effectiveness in the treatment of osteoporosis, inhibit bone resorption via mechanisms that involve binding to bone mineral and cellular effects on osteoclasts. The major molecular target of nitrogen-containing bisphosphonates (N-BPs) in osteoclasts is farnesyl diphosphate synthase (FPPS). N-BPs likely inhibit this enzyme by mimicking one or more of the natural isoprenoid lipid substrates (GPP/DMAPP and IPP) but the mode of inhibition is not established. The active site of FPPS comprises a subsite for each substrate. Kinetic studies with recombinant human FPPS indicate that both potent (risedronate) and weak (NE-58051) enzyme inhibitors compete with GPP for binding to FPPS, however, binding to this site does not completely explain the difference in potency of the two inhibitors, suggesting that a second binding site may also be a target of bisphosphonate inhibition. Using the docking software suite Autodock, we explored a dual inhibitor binding mode for recombinant human FPPS. Experimental support for dual binding is suggested by Dixon plots for the inhibitors. N-BPs may inhibit by binding to both the GPP and a second site with differences in potency at least partly arising from inhibition at the second site.     

Contributions of hydroxyethyl groups to the DNA binding affinities of anthracene probes

Contributions of hydroxyethyl functions to the DNA binding affinities of substituted anthracenes are evaluated by calorimetry and spectroscopy. Isothermal titration calorimetry indicated that binding of the ligands to calf thymus DNA (5 mM Tris buffer, 50 mM NaCl, pH 7.2, 25 degrees C) is exothermic. The binding constants increased from 1.5 x 10(4) to 1.7 x 10(6) M(-1) as a function of increase in the number of hydroxyethyl functions (0-4). DNA binding was accompanied by red-shifted absorption (approximately 630 cm(-1)), strong hypochromism (>65%), positive induced-circular dichroism bands, and negative linear dichroism signals. DNA binding, in general, increased the helix stabilities to a significant extent (DeltaT(m) approximately 7 degrees C, DeltaDeltaH approximately 3 kcal/mol, DeltaDeltaS approximately 6-20 cal/K.mol). The binding constants showed a strong correlation with the number of hydroxyethyl groups present on the anthracene ring system. Analysis of the binding data using the hydrophobicity parameter (Log P) showed a poor correlation between the binding affinity and hydrophobicity. This observation was also supported by a comparison of the affinities of probes carrying N-ethyl (Kb = 0.8 x 10(5) M(-1)) versus N-hydroxyethyl side chains (Kb = 5.5 x 10(5) M(-1)). These are the very first examples of a strong quantitative correlation between the DNA binding affinity of a probe and the number of hydroxyethyl groups present on the probe. These quantitative findings are useful in the rational design of new ligands for high-affinity binding to DNA.     

An Evaluation of Cellular Analyte-Receptor Binding Kinetics Utilizing Biosensors: A Fractal Analysis

A fractal analysis is presented for cellular analyte-receptor binding kinetics utilizing biosensors. Data taken from the literature can be modeled by using (a) a single-fractal analysis and (b) a single- and a dual-fractal analysis. Case (b) represents a change in the binding mechanism as the reaction progresses on the biosensor surface. Relationships are presented for the binding rate coefficient(s) as a function of the fractal dimension for the single-fractal analysis examples. In general, the binding rate coefficient is rather sensitive to the degree of heterogeneity that exists on the biosensor surface. For example, for the binding of mutagenized and back-mutagenized forms of peptide E1037 in solution to salivary agglutinin immobilized on a sensor chip, the order of dependence of the binding rate coefficient, k, on the fractal dimension, D(f), is 13.2. It is of interest to note that examples are presented where the binding coefficient (k) exhibits an increase as the fractal dimension (D(f)) or the degree of heterogeneity increases on the surface. The predictive relationships presented provide further physical insights into the binding reactions occurring on the surface. These should assist us in understanding the cellular binding reaction occurring on surfaces, even though the analysis presented is for the cases where the cellular "receptor" is actually immobilized on a biosensor or other surface. The analysis suggests possible modulations of cell surfaces in desired directions to help manipulate the binding rate coefficients (or affinities). In general, the technique presented is applicable for the most part to other reactions occurring on different types of biosensors or other surfaces. Copyright 2000 Academic Press.     

The Nature of the Binding of Au, Ag, and Pd to Benzene, Coronene, and Graphene: From Benchmark CCSD(T) Calculations to Plane-Wave DFT Calculations

The adsorption of Ag, Au, and Pd atoms on benzene, coronene, and graphene has been studied using post Hartree-Fock wave function theory (CCSD(T), MP2) and density functional theory (M06-2X, DFT-D3, PBE, vdW-DF) methods. The CCSD(T) benchmark binding energies for benzene-M (M = Pd, Au, Ag) complexes are 19.7, 4.2, and 2.3 kcal/mol, respectively. We found that the nature of binding of the three metals is different: While silver binds predominantly through dispersion interactions, the binding of palladium has a covalent character, and the binding of gold involves a subtle combination of charge transfer and dispersion interactions as well as relativistic effects. We demonstrate that the CCSD(T) benchmark binding energies for benzene-M complexes can be reproduced in plane-wave density functional theory calculations by including a fraction of the exact exchange and a nonempirical van der Waals correction (EE+vdW). Applying the EE+vdW method, we obtained binding energies for the graphene-M (M = Pd, Au, Ag) complexes of 17.4, 5.6, and 4.3 kcal/mol, respectively. The trends in binding energies found for the benzene-M complexes correspond to those in coronene and graphene complexes. DFT methods that use empirical corrections to account for the effects of vdW interactions significantly overestimate binding energies in some of the studied systems.     

Interaction of distamycin-oligopeptides with the self-complementary (d-CACAAGCTTGTG)2 duplex in solution

The complex formation of the DNA groove binding drugs netropsin, distamycin-3 and distamycin-5 with the self-complementary duplex (d-CACAAGCTTGTG)₂ has been investigated using CD titration measurements. Employing a general curve fitting program for data analysis, binding constants of complex formation and stoichiometry numbers were evaluated for the two distamycin-type oligopeptides. The binding model assumes simultaneous occurrence of two kinds of complexes characterized by (1:1) - and (1:2) -stoichiometry (duplex:ligand). The binding constants obtained for the (1:1)-complexes for distamycin-3 as well as for distamycin-5 are in full agreement with those calculated for the duplex sequence by means of the single-base specific binding constants of the methylpyrole carboxamide group of Gursky et al. [33]. Obtained binding results are explained by a possible interaction mechanism of distamycin with the particular dodecamer duplex in solution.     

Synthetic creatinine receptor: imprinting of a Lewis acidic zinc(II)cyclen binding site to shape its molecular recognition selectivity

Molecularly imprinted polymers (MIPs) from polymerizable Lewis acidic zinc(II)cyclen complexes and ethylene glycol dimethyl acrylate have been prepared. For the imprinting process the template molecule creatinine is reversibly coordinated to the zinc atom. The high strength of this interaction allows analyte binding to the MIP from aqueous solution with high affinity. Its pH dependence is used for controlled guest release with nearly quantitative analyte recovery rate. The binding capacity and selectivity profile of the MIP remains constant through several pH controlled binding and release cycles. MIPs missing a suitable metal binding site showed no significant affinity for thymine or creatinine. Flavin adsorbs nonspecifically to all polymers. The imprinting process reverses the binding selectivity of zinc(II)cyclen for creatinine and thymine from 1:34 in homogeneous solution to 3.5:1 in the MIP. Scatchard plot analysis of creatinine binding isotherms reveals uniform binding of the imprint, with fits indicating a one-site model; however, similar analysis for thymine indicate high and low affinity sites. This corresponds to unrestricted coordination sites freely accessible for thymine, e.g., at the polymer surface, and misshaped imprinted sites, which still can accommodate thymine. More than 50% of all binding sites exclusively bind creatinine and are not accessible to thymine. The binding properties of a copolymer of polymerizable zinc(II)cyclen and ethylene glycol dimethyl acrylate missing the creatinine template, which match the binding selectivity of the complex in solution, confirm that the origin of altered selectivities is the imprinting process. With binding ability at physiological pH, the MIPs are applicable for tasks in medicinal diagnostics or biotechnology. Imprinted zinc(II)cyclen complexes provide, like a metalloenzyme binding motif, high binding affinity by reversible coordination while the surrounding macromolecule determines binding selectivity.     

Analysis of recognition of fructose by imprinted polymers

Binding of fructose to the fructose imprinted polymer (MIP(Frc)) and pinacol imprinted polymer (control) were studied both in batch and a flow through mode. The influence of the cross-linkers ethylene glycol dimethacrylate (EDMA) and trimethylolpropane trimethacrylate (TRIM) on the binding characteristics was analysed. TRIM cross-linked MIPs showed a lower (unspecific) binding for the control polymer (pinacol imprinted) and higher binding of fructose as compared with the EDMA-MIPs. Furthermore interactions of a TRIM cross-linked molecularly imprinted polymer against fructose and its corresponding template were studied using a thermistor. Label-free detection of fructose was realised in the range of 0.5-10mM. The difference in enthalpy changes between specific binding of fructose to boronic acid moieties of the MIP and non-specific binding to the matrix leads to an 18-fold higher apparent imprinting factor than batch binding studies. Cross-reactivity studies using MIP sensor indicate that the interaction of fructose to MIP generates higher signal than disaccharides. The studies described in this paper demonstrate the potential of direct characterisation of molecular binding events.     

Adsorption of spillover hydrogen atoms on single-wall carbon nanotubes

Spillover of hydrogen on nanostructured carbons is a phenomenon that is critical to understand in order to produce efficient hydrogen storage adsorbents for fuel cell applications. The spillover and interaction of atomic hydrogen with single-walled carbon nanotubes (SWNTs) is the focus of this combined theoretical and experimental work. To understand the spillover mechanism, very low occupancies (i.e., 1 and 2 H atoms adsorbed) on (5,0), (7,0), (9,0) zigzag (semiconducting) SWNTs and a (5,5) armchair (metallic) SWNT, with corresponding diameters of 3.9, 5.5, 7.0, and 6.8 A, were investigated. The adsorption binding energy of H atoms depends on H occupancy, tube diameter, and helicity (or chirality), as well as endohedral (interior) vs exohedral (exterior) binding. Exohedral binding energies are substantially higher than endohedral binding energies due to easier sp(2)-sp(3) transition in hybridization of carbon on exterior walls upon binding. A binding energy as low as -8.9 kcal/mol is obtained for 2H atoms on the exterior wall of a (5, 0) SWNT. The binding energies of H atoms on the metallic SWNT are significantly weaker (about 23 kcal/mol weaker) than that on the semiconductor SWNT, for both endohedral and exohedral adsorption. The binding energy is generally higher on SWNTs of larger diameters, while its dependence on H occupancy is relatively weak except at very low occupancies. Experimental results at 298 K and for pressures up to 10 MPa with a carbon-bridged composite material containing SWNTs demonstrate the presence of multiple adsorption sites based on desorption hysteresis for the spiltover H on SWNTs, and the experimental results were in qualitative agreement with the molecular orbital calculation results.     

The binding of organic anions by polyvinylpyrrolidone: Determination of intrinsic binding constant and number of binding sites by competitive binding

The binding of 4′-dibutylaminoazobenzene-4-sulfonate (butyl orange, BO) and 1-amino-4-methylaminoanthraquinone-2-sulfonate (AQ) by crosslinked polyvinylpyrrolidone and the competition between BO and AQ for binding sites of the polymer were examined. The equilibrium data showed competitive binding. Thus the intrinsic binding constants and the number of binding sites can be evaluated easily and precisely by competitive binding. The thermodynamic parameters that accompained the binding were calculated from the intrinsic binding constant and its temperature-dependence. The thermodynamic data for BO showed that the binding process is athermal and stabilized entirely by the entropy term. The binding of BO to the polymer is entropically favorable as a result of the operation of the hydrophobic effect. In contrast, with AQ a favorable free energy for a dye-polymer complex formation is associated with a large negative enthalpy and a small entropy. Therefore it is likely that the binding of AQ occurs by energetic forces and that the large aromatic ring of the dye contributes to the binding energy. On the average, BO and AQ can bind to the crosslinked polymer to the extent of 1 dye/ca 73 basemol in 0.1M tris-acetate buffer, pH 7.0.     

A connection between the binding properties of imprinted and nonimprinted polymers: a change of perspective in molecular imprinting

In the current paradigm for molecular imprinting, the imprinted binding sites exist as a consequence of the polymerization process around templates, and the properties of nonimprinted polymers (NIPs) have largely been overlooked. Thus, nothing can be affirmed a priori concerning the binding properties of NIPs. We propose an alternative view where the imprinting effect is due to the presence of a template molecule that enhances the pre-existing binding properties of a polymer. If a NIP shows no binding properties toward a target molecule, the corresponding imprinted polymer (MIP) will show a weak imprinting effect. On the other hand, if a NIP shows binding properties toward a target molecule, the corresponding MIP will show a significant imprinting effect. To verify this hypothesis, we prepared a 96-member combinatorial polymeric library in the absence of any template molecule. This library was screened for several potential ligands, and with no exceptions, the composition of the best-binding NIP produced a MIP with excellent binding properties, whereas a low-binding NIP formulation produced a MIP with comparable low binding. To validate these results, the binding properties toward naproxen and ibuprofen were measured for two combinatorial libraries of polymers prepared in the presence (MIP library) and the absence (NIP library) of the template molecule. The experiment's results showed a correlation between the apparent affinity constants measured for the NIP and MIP libraries, confirming the proposed hypothesis. Moreover, for closely related molecules, it was shown that binding selectivity is an emergent property derived from the imprinting process and not a property of NIPs.     

Probing adsorbed fibronectin layer structure by kinetic analysis of monoclonal antibody binding

Adsorbed protein layers are often away from equilibrium and thus exhibit history dependent structures. We use the kinetics of monoclonal antibody binding, as measured using optical waveguide lightmode spectroscopy (OWLS), to investigate the structure of adsorbed fibronectin (Fn) layers formed under different kinetic paths. For all of the layers investigated, we find no difference between the apparent adsorption rate constants of (i) monoclonal antibodies specific to Fn's cell binding site (alpha-Fn) and (ii) monoclonal antibodies specific to cytochrome c (alpha-CC, as a control), indicating initial adsorption of antibodies to be non-specific. For certain layers, the saturation density and the initial projected area per antibody differ significantly between alpha-Fn and alpha-CC, suggesting specific binding to follow the initial non-specific attachment. The fraction of antibodies binding specifically to the Fn layer, and the number of Fn binding sites per specific binding event, are estimated in terms of the difference in initial projected areas between alpha-Fn and alpha-CC. For a Fn layer formed at a bulk concentration of 2 microg/mL, we find a decrease in specific binding with an increase in Fn layer formation time, suggesting post-adsorption structural changes of a lower density adsorbed layer diminish binding site availability. Conversely, for a Fn layer formed at a bulk concentration of 40 microg/mL, we find an increase in specific binding with an increase in the aging time of the Fn layer, implying post-adsorption structural changes reveal binding sites for a higher density adsorbed layer.     

Determining thermodynamic parameters from isothermal calorimetric isotherms of the binding of macromolecules to metal cations originally chelated by a weak ligand

An accurate data analysis method for determining stoichiometry and thermodynamic parameters from isothermal titration calorimetry data for the binding of macromolecules to metal cations that are solubilized through an association with a weak ligand is presented. This approach is applied to determine the binding constant for the association of Cu(II) to the first 16 residues of the Alzheimer's amyloid beta peptide, Abeta(1-16) under conditions where Cu(II) is rendered soluble through weak binding to glycine. At pH 7.2 and 37 degrees C, a binding constant of 1.5 x 10(9) M-1 (Kd = 0.7 nM) is determined for the association of Cu(II) with Abeta(1-16).     

Binding of warfarin influences the acid-base equilibrium of H242 in sudlow site I of human serum albumin

Sudlow Site I of human serum albumin (HSA) is located in subdomain IIA of the protein and serves as a binding cavity for a variety of ligands. In this study, the binding of warfarin (W) is examined using computational techniques and isothermal titration calorimetry (ITC). The structure of the docked warfarin anion (W-) to Site I is similar to that revealed by X-ray crystallography, with a calculated binding constant of 5.8 x 10(5) M(-1). ITC experiments (pH 7.13 and I = 0.1) carried out in three different buffers (MOPs, phosphate and Tris) reveal binding of W- is accompanied by uptake of 0.30+/-0.02 protons from the solvent. This measurement suggests that the binding of W- is stabilized by an ion-pair interaction between protonated H242 and the phenoxide group of W-.     

Synthesis, optical properties, and LFER analysis of solvent-dependent binding constants of Hamilton-receptor-connected merocyanine chromophores

A merocyanine dye equipped with a Hamilton-receptor unit has been synthesized that enables strong noncovalent binding of other merocyanine dyes bearing barbituric acid acceptor groups by six hydrogen bonds. NMR and UV/vis titration experiments in toluene, chloroform, dichloromethane, dioxane, and THF provide evidence for the formation of 1:1 complexes even in the dipolar solvents. An enhanced binding strength is observed for the more dipolar merocyanine dyes in the head-to-tail assembly structure with binding constants up to >10 (8) M (-1) in toluene. In the present bimolecular complexes two merocyanine chromophores are assembled in a head-to-tail fashion that affords increased dipole moments as demanded for efficient electric field induced poling processes in nonlinear optical and photorefractive polymeric hosts. The solvent dependency of the binding constants for various barbituric acid dye-Hamilton receptor complexes as well as a perylene imide-melamine complex reveals linear free energy relationships (LFER) that allow for an estimation of binding constants larger than 10 (12) M (-1) for Hamilton receptor organized head-to-tail merocyanine bimolecular complexes in aliphatic solvents. It is suggested that such LFER are valuable tools for the estimation of binding constants in solvents where experimental binding constants cannot be determined because of solubility or spectroscopic problems.     

A Single and a Dual-Fractal Analysis of Analyte-Receptor Binding Kinetics for Surface Plasmon Resonance Biosensor Applications

The diffusion-limited binding kinetics of analyte in solution to either a receptor immobilized on a surface or to a receptorless surface is analyzed within a fractal framework for a surface plasmon resonance biosensor. The data is adequately described by a single- or a dual-fractal analysis. Initially, the data was modeled by a single-fractal analysis. If an inadequate fit was obtained then a dual-fractal analysis was utilized. The regression analysis provided by Sigmaplot (32) was used to determine if a single fractal analysis is sufficient or if a dual-fractal analysis is required. In general, it is of interest to note that the binding rate coefficient and the fractal dimension exhibit changes in the same direction (except for a single example) for the analyte-receptor systems analyzed. Binding rate coefficient expressions as a function of the fractal dimension developed for the analyte-receptor binding systems indicate, in general, the high sensitivity of the binding rate coefficient on the fractal dimension when both a single- and a dual-fractal analysis is used. For example, for a single-fractal analysis and for the binding of human endothelin-1 (ET-1) antibody in solution to ET-115-21.BSA immobilized on a surface plasmon resonance (SPR) surface (33), the order of dependence of the binding rate coefficient, k, on the fractal dimension, Df, is 6.4405. Similarly, for a dual-fractal analysis and for the binding of 10(-6) to 10(-4) M bSA in solution to a receptorless surface (direct binding to SPR surface) (41) the order of dependence of k1 and k2 on Df1 and Df2 were -2.356 and 6.241, respectively. Binding rate coefficient expressions are also developed as a function of the analyte concentration in solution. The binding rate coefficient expressions developed as a function of the fractal dimension(s) are of particular value since they provide a means to better control SPR biosensor performance by linking it to the degree of heterogeneity that exists on the SPR biosensor surface. Copyright 1999 Academic Press.