A dynamic dialysis method for studying protein-ligand binding using chromatographic theory

A new dynamic dialysis method has been developed for studying protein-ligand binding phenomena. The method depends on analysis of the elution pattern of ligand in a single dialyzing process where the ligand concentration in the sample compartment changes greatly with time. The dialyzer is composed of a long, narrow chamber (the sample compartment) between two sheets of semipermeable membrane and two outside chambers (the sink compartment) connected as a single path. Eluting buffer flows in the sink compartment to exchange the ligand with the solution in the sample compartment. Therefore, the ligand concentration gradient in the sink compartment is in the longitudinal direction. The mathematical expressions to analyze the experimental data were derived from a modified theory of chromatography. Examination of the binding of sulfanilamide to bovine serum albumin using this method shows that these equations are valid for use in studying protein-ligand binding.     

Error analysis in equilibrium dialysis: evaluation of adsorption phenomena

Various sources of error in equilibrium dialysis may lead to inaccurate results of binding experiments: (i) the finite time of dialysis; (ii) the Donnan effects; (iii) the adsorption of ligand to the membrane; and (iv) release of contaminating material from dialysis casings. These errors were analyzed for a polynucleotide-oligopeptide model system with particular regard to adsorption phenomena and the underlying mechanisms. Adsorption data were treated according to Freundlich and Langmuir isotherms. The latter turned out to be more appropriate for the consideration of adsorption phenomena with respect to a minimum error propagation. Furthermore, it was shown that the degree of adsorption varies with ionic strength and temperature and could be interpreted in terms of polyelectrolyte theory. The kinetics of both adsorption and of the ligand distribution between the polymer and buffer compartments follow first order at the beginning of dialysis which is in line with a simple diffusion process. After 13-15 h data deviate from first order kinetics indicating an alteration in the transport mechanism. The effects of errors on binding parameters were determined and a detailed protocol for correction is presented allowing one to obtain binding data from equilibrium dialysis experiments with the required degree of accuracy. The fundamental principles and results for the system under investigation generally apply to all protein-ligand systems.     

92 Novel interpretation of the isotherms of multimodal ligands binding with DNA

The binding of ligands with DNA is a key moment in a whole range of cellular processes that provide not only the normal cell vital activity but also the development of some pathological processes. Depending on ligand type, structure of DNA adsorption centers, and physical–chemical conditions of the surrounding, the ligand may bind to DNA by several modes [1]. Particularly, adsorption isotherm of multimodal ligands binding to DNA in Scatchard’s coordinates has a concave shape with two brightly expressed linear areas in the region of small fillings. The analysis of such type of adsorption isotherm for determining of important binding parameters such as binding constant and number of adsorption centers (the part of DNA polymer with which one ligand molecule binds) presents difficulties. Practically in all cases, the analysis of such adsorption isotherm is carried out by linear parts of curves. Such analysis mode of experimental points is approximate method, since all registered of experimental points are roughly divided into two groups and they are treated by linear binding isotherm and therefore the binding parameters are determined. In the present work, the non-linear adsorption isotherm in Scatchard‘s coordinates is obtained which allowed, provided, the more precise treatment of all experimental points by unique curve which includes linear regions as well. Such mode of treatment of experimental points makes more precise the determination of not only binding constant and number of adsorption centers that correspond to the one ligand molecule binding, but also additional binding parameter – a proportion of adsorption centers of each binding to DNA type of multimodal ligand.     

Equilibrium and kinetic studies of the binding of tri- and tetra-anionic ligands to bovine serum albumin.

The binding of tri- and tetra-anionic azo dyes (Amaranth, Ponceau 4R, and Ponceau 6R) to bovine serum albumin (BSA) at pH = 7.0 and 25 degrees C has been studied by equilibrium dialysis, spectrophotometry, and by stopped-flow and temperature-jump methods. Equilibrium dialysis revealed that BSA has one primary binding site and about two secondary sites for each dye. The values of the binding constant for the primary site show that the stability of the complex at the primary site progressively increases with an increase in the number and the density of anionic charges on ligand. Kinetic data have been found to be consistent with a scheme in which a rapid bimolecular binding is followed by two isomerizations of the complex (in the case of Amaranth) or by one isomerization (in the cases of Ponceau 4R and Ponceau 6R). Equilibrium and rate constants for each step of the scheme were determined. From the results it was found that the increment of the number and the density of anionic charges on ligand accelerates the forward process of the final isomerization step but retards the backward one of it, resulting in the enhancement of the stability of the complex at the primary site. On the basis of these results and the structure of the ligands, the detailed binding mechanism has been discussed in the light of the electrostatic interaction between the ligands and the binding site on BSA.     

An approach based on diffusion to study ligand-macromolecule interaction

A new approach has been developed to study binding of a ligand to a macromolecule based on the diffusion process. In terms of the Fick's first law, the concentration of free ligand in the presence of a protein can be determined by the measurement of those ligands which are diffused out. This method is applied to the study of binding of methyl-orange to lysozyme in phosphate buffer of pH 6.2, at 30 degrees C. The binding isotherm was determined initially, followed by application of the Hill equation to the data obtained, then binding constant and binding capacity were estimated.     

An evaluation of ways of using equilibrium dialysis to quantify the binding of ligand to macromolecule.

1. The effect of systematic error (loss of ligand, complex or macromolecule) on three of the experimental designs by which equilibrium dialysis may be used to quantify the interaction of ligand and macromolecule is examined theoretically, and the design that is least sensitive to systematic error is identified. 2. Thirteen methods for fitting the binding isotherm to experimental data are compared by using them to analyse simulated data containing random error, and the most reliable method is identified.     

An efficient computational method for calculating ligand binding affinities

Virtual compound screening using molecular docking is widely used in the discovery of new lead compounds for drug design. However, the docking scores are not sufficiently precise to represent the protein-ligand binding affinity. Here, we developed an efficient computational method for calculating protein-ligand binding affinity, which is based on molecular mechanics generalized Born/surface area (MM-GBSA) calculations and Jarzynski identity. Jarzynski identity is an exact relation between free energy differences and the work done through non-equilibrium process, and MM-GBSA is a semimacroscopic approach to calculate the potential energy. To calculate the work distribution when a ligand is pulled out of its binding site, multiple protein-ligand conformations are randomly generated as an alternative to performing an explicit single-molecule pulling simulation. We assessed the new method, multiple random conformation/MM-GBSA (MRC-MMGBSA), by evaluating ligand-binding affinities (scores) for four target proteins, and comparing these scores with experimental data. The calculated scores were qualitatively in good agreement with the experimental binding affinities, and the optimal docking structure could be determined by ranking the scores of the multiple docking poses obtained by the molecular docking process. Furthermore, the scores showed a strong linear response to experimental binding free energies, so that the free energy difference of the ligand binding (ΔΔG) could be calculated by linear scaling of the scores. The error of calculated ΔΔG was within ≈±1.5 kcal•mol⁻¹ of the experimental values. Particularly, in the case of flexible target proteins, the MRC-MMGBSA scores were more effective in ranking ligands than those generated by the MM-GBSA method using a single protein-ligand conformation. The results suggest that, owing to its lower computational costs and greater accuracy, the MRC-MMGBSA offers efficient means to rank the ligands, in the post-docking process, according to their binding affinities, and to compare these directly with the experimental values.     

The Binding of Benzopyranes to Human Serum Albumin. A Structure–Affinity Study

The binding of several benzopyranes to serum albumin was studied by equilibrium dialysis at pH7.4 in a 67mM sodium phosphate buffer at 37°C. The equilibrium data were analyzed using a computer program for curve fitting. The binding isotherm for warfarin, 4-hydroxycoumarin, 4-chromanol, coumarin, 3-acetylcoumarin, and benzoic acid can be described by two stoichiometric dissociation constants. Elimination of the 4-hydroxyl group in the coumarin chemical structures decreases the binding affinity of the compounds on the primary binding site of serum albumin, with 4-chromanol the smallest ligand which binds to seroalbumin with high affinity. Thus, the affinity of 4-benzopyranol and the 4-hydroxybenzopyranones greater than that of benzopyranones. On the other hand, elimination of the 2-oxo group in the benzopyranone chemical structures decreases affinity for the secondary binding site.     

Rapid affinity measurement of protein-protein interactions in a microfluidic platform

A rapid screening method has been developed to determine binding affinities for protein-ligand interactions using the Gyrolab workstation, a commercial microfluidic platform developed to accurately and precisely quantify proteins in solution. This method was particularly suited for assessing the high-affinity interactions that have become typical of therapeutic antibody-antigen systems. Five different commercially available antibodies that bind digoxin and a digoxin-bovine serum albumin (BSA) conjugate with high affinity were rigorously evaluated by this method and by the more conventional kinetic exclusion assay (KinExA) method. Binding parameter values obtained using Gyrolab were similar to those recovered from KinExA. However, the total experimental time for 20 binding affinity titrations, with each titration covering 12 data points in duplicate, took approximately 4h by the Gyrolab method, which reduced the experimental duration by more than 10-fold when compared with the KinExA method. This rapid binding analysis method has significant applications in the screening and affinity ranking selection of antibodies from a very large pool of candidates spanning a wide range of binding affinities from the low pM to μM range.     

Proton exchange coupled to the specific binding of alkylsulfonates to serum albumins

We have applied isothermal titration calorimetry to investigate the linkage between ligand binding and the uptake or release of protons by human serum albumin (HSA) and bovine serum albumin (BSA). The ligands were sodium decyl sulfate (SDeS) and sodium dodecyl sulfate (SDS). Within a certain temperature range, the binding isotherm could be clearly resolved into two classes of sites (high affinity and low affinity) and modeled assuming independence and thermodynamic equivalence of the sites within each class. Measurements at pH 7.0 in different buffer systems revealed that the binding of SDS to the high affinity sites did not couple to any exchange of protons in either of the proteins. Saturation of the 6-8 low affinity sites for SDS, on the other hand, brought about the release of two protons from both HSA and BSA. In addition to elucidating the pH dependence of ligand binding, this analysis stressed that binding enthalpies for the low affinity sites measured by calorimetry must be corrected for effects due to the concomitant protonation of the buffer. The shorter ligand SDeS bound to HSA with a comparable stoichiometry but with four times lower affinity. Interestingly, no proton linkage was observed for the binding of SDeS. An empirical structural analysis suggested that His 242 in site 7 (of HSA) is a likely candidate for one of the proton donors.     

Prediction of ligand binding using an approach designed to accommodate diversity in protein-ligand interactions

Computational determination of protein-ligand interaction potential is important for many biological applications including virtual screening for therapeutic drugs. The novel internal consensus scoring strategy is an empirical approach with an extended set of 9 binding terms combined with a neural network capable of analysis of diverse complexes. Like conventional consensus methods, internal consensus is capable of maintaining multiple distinct representations of protein-ligand interactions. In a typical use the method was trained using ligand classification data (binding/no binding) for a single receptor. The internal consensus analyses successfully distinguished protein-ligand complexes from decoys (r2, 0.895 for a series of typical proteins). Results are superior to other tested empirical methods. In virtual screening experiments, internal consensus analyses provide consistent enrichment as determined by ROC-AUC and pROC metrics.     

Method dependence of apparent stoichiometry in the binding of salicylate ion to human serum albumin: a comparison between equilibrium dialysis and fluorescence titration.

The binding of salicylate ion to human serum albumin (HSA) was studied in 100 mM potassium phosphate buffer (pH 7.4, 25 degrees C), using equilibrium dialysis and fluorescence titration methods. The protein samples tested were (a) dialyzed human plasma and (b) a commercial preparation of HSA, essentially free of globulin and fatty acids. Independent of the analytical method used, Scatchard and nonlinear regression analyses of the data pointed to a single class of high-affinity salicylate binding sites. On the other hand, the binding parameters were found to be method dependent. K(d) ranged between 25 +/- 2.4 and 62 +/- 15 microM in equilibrium dialysis and between 10 +/- 1.3 and 40 +/- 3.0 microM in fluorescence titration. (The higher limits refer to plasma samples at high [HSA]). Following the same pattern, the apparent stoichiometry of binding (though independent of sample identity and concentration) was higher in equilibrium dialysis (n(app) = 3.2 +/- 0.10) than in fluorescence titration (n(app) 1.9 +/- 0.30). The difference between the two methods could be reconciled by invoking two distinct classes of binding sites (I and II), which had identical (or marginally different) K(d) values, while differing in the magnitude of the fluorescence signal (Deltaf) generated upon ligand binding (Deltaf, PL(I) = Deltaf(I); Deltaf, PL(II) = 0). Further, it was assumed that the state of occupation of class II sites affected the fluorescence efficiency of class I sites, such that Deltaf, PL(I,II) = betaDeltaf(I) (beta = interaction factor). A random binding scheme involving P, PL(I), PL(II), and PL(I,II) was formulated. The model adequately predicted the behavior of the system when monitored through the change in protein fluorescence: Taking K(d) = 25 microM and n(T) = 3, the interaction factor beta was found to be 0.62 +/- 0.10. It was concluded that the correct parameters for the binding of salicylate ion to HSA are K(d) = 25 +/- 2.4 microM and n(T) = 3.2 +/- 0.10, as indicated by equilibrium dialysis of purified HSA. Besides updating information relating to the salicylate binding potential of HSA, this study serves to illustrate a likely complication in the study of protein-ligand interactions by fluorometric methods.     

Molecular imprinting of proteins and other macromolecules resulting in new adsorbents

When the model protein bovine serum albumin (BSA) was dissolved in a concentrated aqueous solution of the multifunctional ligand L-malic acid, the solution was lyophilized, and the solid residue thoroughly washed with tetrahydrofuran to extract malic acid, then the resultant ("imprinted") protein was capable of binding 26.4 +/-0.9 mol equivalents of the ligand in anhydrous ethyl acetate. The nonimprinted BSA (i.e., that prepared in the same manner apart from the absence of malic acid) bound less then one-tenth of that amount under identical conditions. Furthermore, both imprinted and nonimprinted BSA exhibited little binding of L-malic acid in water. The imprinted BSA retained its "memory" for the ligand in ethyl acetate even after a prolonged incubation under vacuum; dissolution in water, however, eliminated the imprinted protein's binding capacity. The BSA imprinted with L-malic acid displayed affinity for this ligand not only in ethyl acetate but also in many other anhydrous solvents. It was found that the higher the solvent's propensity to form hydrogen bonds, the lower the protein-ligand binding in it, thus pointing to hydrogen bonds as the driving force of this binding. Studies with completely or partially cleaved BSA, with other globular proteins, glutathione, and poly(L-aspartic acid) revealed that the critical requirement for the imprintability is the presence of a sufficiently long polymeric chain. Moreover, many hydrogen-bond-forming macromolecules other than proteins, such as dextrans and their derivatives, partially hydrolyzed starch, and poly(methacrylic acid), also could be imprinted for subsequent binding in ethyl acetate. The mechanism of imprinting and binding inferred from these experiments involves a multipoint hydrogen bonding in water of each ligand molecule with two or more sites on the polymeric chain, thereby folding a segment of the latter into a cavity around the ligand; following lyophilization and extraction of the ligand, the cavities remain in organic solvents (but not in water) and give rise to ligand binding. This conclusion is supported by the results of binding of numerous malic acid analogs and related ligands to BSA imprinted with L-malic acid. Finally, BSA imprinted with malic acid was used as a selective adsorbent for a chromatographic separation of an equimolar mixture of maleic and acrylic acids in ethyl acetate.     

Estimation of protein-ligand binding parameters from contiuous ultrafiltration results.

A method of analyzing the result of continuous ultrafiltration experiments to obtain protein-ligand binding parameters is presented. This method employs a nonlinear least-squares regression algorithm coupled with a model of protein-ligand binding which alloww the computation of free ligand concentrations, and a second-order Runge-Kutta method to integrate free concentrations with respect to collected ultrafiltrate. The approach is general and effectively removes the constraints on maximum fraction size imposed by other methods.     

Energetic contributions of amino acid residues and its cross-talk to delineate ligand-binding mechanism

Receptor-based QSAR approaches can enumerate the energetic contributions of amino acid residues toward ligand binding only when experimental binding affinity is associated. The structural data of protein-ligand complexes are witnessing a tremendous growth in the Protein Data Bank deposited with a few entries on binding affinity. We present here a new approach to compute the E nergetic CONT ributions of A mino acid residues and its possible C ross-T alk (ECONTACT) to study ligand binding using per-residue energy decomposition, molecular dynamics simulations and rescoring method without the need for experimental binding affinity. This approach recognizes potential cross-talks among amino acid residues imparting a nonadditive effect to the binding affinity with evidence of correlative motions in the dynamics simulations. The protein-ligand interaction energies deduced from multiple structures are decomposed into per-residue energy terms, which are employed as variables to principal component analysis and generated cross-terms. Out of 16 cross-talks derived from eight datasets of protein-ligand systems, the ECONTACT approach is able to associate 10 potential cross-talks with site-directed mutagenesis, free energy, and dynamics simulations data strongly. We modeled these key determinants of ligand binding using joint probability density function (jPDF) to identify cross-talks in protein structures. The top two cross-talks identified by ECONTACT approach corroborated with the experimental findings. Furthermore, virtual screening exercise using ECONTACT models better discriminated known inhibitors from decoy molecules. This approach proposes the jPDF metric to estimate the probability of observing cross-talks in any protein-ligand complex. The source code and related resources to perform ECONTACT modeling is available freely at https://www.gujaratuniversity.ac.in/econtact /.     

Study of the binding of thyroxine by thyroxine-binding prealbumin by a dialysis method using a fixed free concentration of ligand (author's transl)]

In order to study ligand-protein binding in solution, a dialysis method was used in which the free concentration of ligand can be controlled. The method has certain advantages and was applied to the binding of thyroxine by thyroxine-binding prealbumin, a system about which the results found in the literature are not in good agreement. From the isotherm drawn according to the Scatchard plot, it was found that thyroxine-binding prealbumin only presents a single binding site for thyroxine per molecule, the association constant being 1.7 . 10(8) M-1.     

NMR spectroscopy in structure-based drug design

NMR methods for the study of motion in proteins continue to improve, and a number of studies of protein-ligand complexes relevant to drug design have been reported over the past year, for example, studies of fatty-acid-binding protein and SH2 and SH3 domains. These studies have begun to give a picture of the structural dynamics of protein-ligand complexes and to relate the changes in dynamics on ligand binding to the origins of specificity. NMR is also valuable in locating binding sites, both qualitatively from changes in chemical shift and more precisely from distances measured from relaxation effects. The conformation of the bound ligand can provide useful information for drug design, and over the past year improvements in methods have made it easier to obtain quantitative information from transferred nuclear Overhauser effect experiments.     

Further consideration of systematic errors present in protein-ligand interactions

Relatively minor systematic errors present during measurements of protein-ligand interaction can lead to large inaccuracies in the calculated values of the equilibrium dissociation constant and the total concentration of the binding protein. These errors, which include binding of the ligand to low affinity material and underestimation of bound ligand, cause the calculation of the concentration of free ligand at equilibrium to be overestimated. We report herein a model of ligandprotein binding which incorporates these errors into the mathematical formulation of the equilibrium binding equation. The effect of these errors on the Scatchard plot is presented.     

Interactions in affinity partition studied using fluorescence spectroscopy.

Fluorescence titration has been used to determine the binding constant and number of binding sites for the textile triazine dye Procion Yellow HE-3G to lactate dehydrogenase from rabbit muscle (E.C. 1.1.1.27). Triazine dye was either free in solution or attached to one of the polymer carriers, polyethylene glycol or dextran. Titrations were performed in solutions of buffer, dextran, and polyethylene glycol. Aqueous two-phase systems composed of polyethylene glycol and dextran were prepared and the binding constant and number of binding sites for ligand polyethylene glycol-Procion Yellow to lactate dehydrogenase were determined in both upper and lower phases of these systems. Affinity partition of lactate dehydrogenase in a PEG-dextran system was also performed using PEG-Procion Yellow as ligand, and partition coefficients of lactate dehydrogenase showed good agreement with theoretical partition coefficients calculated from the binding constant and number of binding sites obtained from fluorescence titration. The advantage of using fluorescence titration to determine affinity of a polymer ligand for a protein is that measurement of binding strength can be made in the actual environment encountered by protein-ligand complex during the purification process.     

The role of backbone motions in ligand binding to the c-Src SH3 domain.

The Src homology 3 (SH3) domain of pp60(c-src) (Src) plays dual roles in signal transduction, through stabilizing the repressed form of the Src kinase and through mediating the formation of activated signaling complexes. Transition of the Src SH3 domain between a variety of binding partners during progression through the cell cycle requires adjustment of a delicate free energy balance. Although numerous structural and functional studies of SH3 have provided an in-depth understanding of structural determinants for binding, the origins of binding energy in SH3-ligand interactions are not fully understood. Considering only the protein-ligand interface, the observed favorable change in standard enthalpy (DeltaH=-9.1 kcal/mol) and unfavorable change in standard entropy (TDeltaS=-2.7 kcal/mol) upon binding the proline-rich ligand RLP2 (RALPPLPRY) are inconsistent with the predominantly hydrophobic interaction surface. To investigate possible origins of ligand binding energy, backbone dynamics of free and RLP2-bound SH3 were performed via (15)N NMR relaxation and hydrogen-deuterium (H/(2)H) exchange measurements. On the ps-ns time scale, assuming uncorrelated motions, ligand binding results in a significant reduction in backbone entropy (-1.5(+/-0.6) kcal/mol). Binding also suppresses motions on the micros-ms time scale, which may additionally contribute to an unfavorable change in entropy. A large increase in protection from H/(2)H exchange is observed upon ligand binding, providing evidence for entropy loss due to motions on longer time scales, and supporting the notion that stabilization of pre-existing conformations within a native state ensemble is a fundamental paradigm for ligand binding. Observed changes in motion on all three time scales occur at locations both near and remote from the protein-ligand interface. The propagation of ligand binding interactions across the SH3 domain has potential consequences in target selection through altering both free energy and geometry in intact Src, and suggests that looking beyond the protein-ligand interface is essential in understanding ligand binding energetics.