Free Ligand In Solution Research Articles

α‐Cyclodextrin Host–Guest Binding: A Computational Study of the Different Driving Forces

AbstractFree‐energy differences govern the equilibrium between bound and unbound states of a host and its guest molecules. The understanding of the underlying entropic and enthalpic contributions, and their complex interplay are crucial for the design of new drugs and inhibitors. In this study, molecular dynamics (MD) simulations were performed with inclusion complexes of α‐cyclodextrin (αCD) and three monosubstituted benzene derivatives to investigate host–guest binding. αCD Complexes are an ideal model system, which is experimentally and computationally well‐known. Thermodynamic integration (TI) simulations were carried out under various conditions for the free ligands in solution and bound to αCD. The two possible orientations of the ligand inside the cavity were investigated. Agreement with experimental data was only found for the more stable orientation, where the substituent resides inside the cavity. The better stability of this conformation results from stronger Van der Waals interactions and a favorable antiparallel host–guest dipole–dipole alignment. To estimate the entropic contributions, simulations were performed at three different temperatures (250, 300, and 350 K) and using positional restraints for the host. The system was found to be insensitive to both factors, due to the large and symmetric cavity of αCD, and the nondirectional nature of the host–guest interactions.

Photogenerated carbohydrate microarrays to study carbohydrate–protein interactions using surface plasmon resonance imaging

A photochemical strategy to generate carbohydrate microarrays on flat sensor surfaces, and to study the protein-binding effects of these arrays by surface plasmon resonance imaging is described. The approach was validated using a panel of carbohydrate-binding proteins. The coupling agents, thiol-functionalized perfluorophenyl azides, allow the covalent attachment of underivatized carbohydrates to gold surfaces by a fast photochemical reaction. Carbohydrate microarrays composed of 3,6-di- O-(α- d-mannopyranosyl)- d-mannopyranose (Man3), 2- O-α- d-mannopyranosyl- d-mannopyranose (Man2), d-mannose (Man), d-glucose (Glc), and d-galactose (Gal) were constructed, and the binding studies were carried out in real-time using surface plasmon resonance imaging. Results showed that the immobilized carbohydrate ligands retained their binding affinities with lectins, the rank order of which was consistent with that of the free ligands in solution. The detection limit of Man3, Man2, Man, and Glc with the lectin Concanavalin A was measured to be 0.29 nM, 0.18 nM, 0.61 nM, and 3.1 nM, respectively. In addition, soybean agglutinin and Griffonia simplicifolia lectin II were tested on the array, and the results were consistent with the binding selectivity of these lectins with the carbohydrate ligands.

Glyconanomaterials: synthesis, characterization, and ligand presentation.

Glyconanomaterials, nanomaterials carrying surface-tethered carbohydrate ligands, have emerged and demonstrated increasing potential in biomedical imaging, therapeutics, and diagnostics. These materials combine the unique properties of nanometer-scale objects with the ability to present multiple copies of carbohydrate ligands, greatly enhancing the weak affinity of individual ligands to their binding partners. Critical to the performance of glyconanomaterials is the proper display of carbohydrate ligands, taking into consideration of the coupling chemistry, the type and length of the spacer linkage, and the ligand density. This article provides an overview of the coupling chemistry for attaching carbohydrate ligands to nanomaterials, and discusses the need for thorough characterization of glyconanomaterials, especially quantitative analyses of the ligand density and binding affinities. Using glyconanoparticles synthesized by a versatile photocoupling chemistry, methods for determining the ligand density by colorimetry and the binding affinity with lectins by a fluorescence competition assay are determined. The results show that the multivalent presentation of carbohydrate ligands significantly enhances the binding affinity by several orders of magnitude in comparison to the free ligands in solution. The effect is sizeable even at low surface ligand density. The type and length of the spacer linkage also affect the binding affinity, with the longer linkage promoting the association of bound ligands with the corresponding lectins.

Binding studies of adhesion/growth-regulatory galectins with glycoconjugates monitored by surface plasmon resonance and NMR spectroscopy

Functionalized fluorescent glycans have the potential to act as tools to detect and analyze protein-carbohydrate interactions. We present here a facile strategy for immobilization of functionalized lactose as a model disaccharide. Bioactivity was tested with three members of the adhesion/growth-regulatory galectins family in different types of assay, i.e. matrix in surface plasmon resonance (SPR), free ligand in solution by STD/trNOESY and docking measurements. In all cases, the activity of the disaccharide was maintained. The attachment of this new fluorescent glycoconjugate to the surface results in a well-defined interface, enabling desired orientational flexibility and enhanced access of binding partners. The results indicate that this new glycoconjugate exhibits binding affinity to galectin-1, 3 and CG-16. Kinetic analysis of the interaction between these galectins and immobilized glycoconjugate by SPR yielded a K(D) of 1.01 mM for galectin-1, 83.5 microM for galectin-3 and 0.28 mM for CG-16. No major contacts to the aglyconic part were detected, which might compromise the specificity of the binding process with other headgroups. Thus, testing these proteins offers the potential for medical applications to detect these endogenous effectors or further derivatives and characterize their carbohydrate specificity.

A photochemically initiated chemistry for coupling underivatized carbohydrates to gold nanoparticles.

The sensitive optoelectronic properties of metal nanoparticles make nanoparticle-based materials a powerful tool to study fundamental biorecognition processes. Here we present a new and versatile method for coupling underivatized carbohydrates to gold nanoparticles (Au NPs) via the photochemically induced reaction of perfluorophenylazide (PFPA). A one-pot procedure was developed where Au NPs were synthesized and functionalized with PFPA by a ligand-exchange reaction. Carbohydrates were subsequently immobilized on the NPs by a fast light activation. The coupling reaction was efficient, resulting in high coupling yield as well as high ligand surface coverage. A colorimetric system based on the carbohydrate-modified Au NPs was used for the sensitive detection of carbohydrate-protein interactions. Binding and cross-reactivity studies were carried out between carbohydrate-functionalized Au NPs and lectins. Results showed that the surface-bound carbohydrates not only retained their binding affinities towards the corresponding lectin, but also exhibited affinity ranking consistent with that of the free ligands in solution.

Postsynthetic Modification of Gold Nanoparticles with Calix[4]arene Enantiomers: Origin of Chiral Surface Plasmon Resonance

Gold nanoparticles postsynthetically modified with chiral 1,3-disubstituted diamino calix[4]arene ligands 2a and 2b are shown to exhibit a circular dichroism (CD)-active surface plasmon resonance absorption (SPR) band. Electronic communication between adsorbed ligand and the gold nanoparticle surface is evidenced in an almost 10-fold increase in the ligand molar ellipticity in the pi-pi* transition spectral range when bound to the gold surface relative to free solution. The footprint of ligand on the gold nanoparticle surface at saturation is measured to be 91 A(2) via monitoring the red shift in the SPR band accompanying ligand adsorption. At ligand concentrations above that required for surface saturation, the ellipticity of the band in the SPR spectral range plateaus to a constant value, whereas the ellipticity of the band in the pi-pi* transition spectral range continues to increase in a manner that corresponds to free ligand in solution. This critical observation correlates ligand adsorption and the onset of the CD-active SPR band. On the basis of the packing characteristics of the bulky calixarene ligand, which are controlled by achiral tert-butyl groups, and the postsynthetic nature of nanoaprticle surface modification of 4.7 nm gold cores used in this study, which precludes synthesis of chiral arrangements of gold atoms, a mechanism responsible for the CD-active SPR bands is proposed, which is based upon the influence of the asymmetric center of the chiral adsorbate on the electronic states of the metal nanoparticle core-an explanation supported by the observed interactions between the gold surface and adsorbed ligand.

An Exchange Method To Investigate the Kinetics of Cd Complexation in Soil Solutions

Knowledge of the dynamic speciation of metals in soil solution is necessary for soil-plant transfer models. The lability of a metal in a given context partly depends on the kinetics of interconversion between the free ion (M) and one complex (ML), governed by the association (c(k)a) and dissociation (c(k)d) rate constants of the reaction of formation of ML If only one single complex is assumed to be present in solution, a single kinetic experiment will enable estimation of ckd and cka', that is, the product of cka by the concentration of free ligand in solution. The method consists of the measurements over time of the radioactive metal remaining in solution after the labeling of a Ca resin--solution system in balanceforthe nonlabeled metal initially present. The method is described for Cd and was applied on four soil solution extracts. The order of magnitude of the estimated c(k)a'- and c(k)d-values was about 10(-3) s(-1). The advantages and drawbacks of the method are discussed.

How to determine free Gd and free ligand in solution of Gd chelates. A technical note

Gd(III) chelates are often used as reporting probes in magnetic resonance imaging applications. Their use relies on the assumption that their high stability ensures against the release of free ligand and free Gd(3+) ions, which are both highly toxic to living systems. The presence of free Gd(3+) ions may have a profound effect on the contrast in the MR image as they may form highly hydrated, macromolecular complexes endowed with very high relaxivity thus providing erroneous information on the extent of the contrast effect induced by the given reporting probe. Although the stability of a given complex may be high enough, the complexation step could not have been completed. Therefore any Gd complex preparation has to be carefully checked for the content of free ligand and free metal ions. Herein the currently used procedures that allow an assessment of the amounts of free metal ions and free ligand in a solution of a given Gd complex are described in detail.

N-Biphenyl thioureas as carboxylate receptors. Effect of the ligand substituents on the geometry of the complexes

Six new biphenyl thiourea derivatives have been prepared to be used in carboxylate sensing. Experiments carried out with these ligands have demonstrated that the type of interaction with TBA carboxylates is strongly dependent on the substituents in the thiourea moiety. These interactions go from the formation of 1:1 hydrogen-bonded complexes to acid–base reactions. In addition, different geometries have been observed for the complexes being dependent on the conformations of the free ligands in solution.

Controlling protein transport in ultrafiltration using small charged ligands

Previous studies have demonstrated that protein transport during ultrafiltration can be strongly influenced by solution pH and ionic strength. The objective of this study was to examine the possibility of controlling protein transmission using a small, highly charged ligand that selectively binds to the protein of interest. Experiments were performed using bovine serum albumin and the dye Cibacron Blue. Protein sieving data were obtained with essentially neutral and negatively charged versions of a composite regenerated cellulose membrane to examine the effects of electrostatic interactions. The addition of only 1 g/L of Cibacron Blue to an 8 g/L BSA solution reduced the BSA sieving coefficient through the negatively-charged membrane by more than two orders of magnitude, with this effect being largely eliminated at high salt and with the neutral membrane. Protein sieving data were in good agreement with model calculations based on the partitioning of a charged sphere in a charged pore accounting for the change in net protein charge due to ligand binding and the increase in solution ionic strength due to the free ligand in solution.

Dimeric Galectin-1 Binds with High Affinity to α2,3-Sialylated and Non-sialylated Terminal N-Acetyllactosamine Units on Surface-bound Extended Glycans

Galectin-1 is a member of the galectin family of glycan-binding proteins and occurs as an approximately 29.5-kDa noncovalent homodimer (dGal-1) that is widely expressed in many tissues. Here, we report that human recombinant dGal-1 bound preferentially and with high affinity (apparent K(d) approximately 2-4 microM) to immobilized extended glycans containing terminal N-acetyllactosamine (LN; Galbeta1-4GlcNAc) sequences on poly-N-acetyllactosamine (PL; (-3Galbeta1-4GlcNAcbeta1-)(n)) sequences, complex-type biantennary N-glycans, or novel chitin-derived glycans modified to contain terminal LN. Although terminal Gal residues are important for dGal-1 recognition, dGal-1 bound similarly to alpha3-sialylated and alpha2-fucosylated terminal LN, but not to alpha6-sialylated and alpha3-fucosylated terminal LN. The binding specificity of human recombinant dGal-1 was similar to that observed with purified bovine heart-derived dGal-1. Unexpectedly, dGal-1 bound free ligands in solution with relatively low affinity and displayed no preference for extended glycans, indicating that dGal-1 preferentially recognizes extended glycans only when they are surface-bound, such as found on cell surfaces. Human dGal-1 also bound to both native and desialylated human promyelocytic HL-60 cells with similar affinity as observed for immobilized long chain PL. Binding to these cells was reduced upon treatment with endo-beta-galactosidase, which cleaves PL sequences, indicating that cell-surface PLs are ligands. To test the role of dimerization in dGal-1 binding, we examined the binding of a mutated form of dGal-1 that weakly dimerizes (monomeric Gal-1 (mGal-1)) and a covalently dimerized (chemically cross-linked) form of mGal-1 (cd-mGal-1). dGal-1 and cd-mGal-1 had similar affinities that were both approximately 3.5-fold higher for immobilized PL than observed for mGal-1, suggesting that dGal-1 acts as a dimer to cross-link terminal LN units on immobilized PL. These results indicate that dGal-1 functions as a dimer to recognize LN units on extended PLs on cell surfaces.

Determination of Ligand−Protein Dissociation Constants by Electrospray Mass Spectrometry-Based Diffusion Measurements

A novel approach for the quantification of ligand-protein interactions is presented. Electrospray ionization mass spectrometry (ESI-MS) is used to monitor the diffusion behavior of noncovalent ligands in the presence of their protein receptors. These data allow the fraction of free ligand in solution to be determined, such that the corresponding dissociation constants can be calculated. A set of conditions is developed that provides an "allowable range" of concentrations for this type of assay. The method is tested by applying it to two different inhibitor-enzyme systems. The dissociation constants measured for benzamidine-trypsin and for N,N',N' '-triacetylchitotriose-lysozyme are (50 +/- 10) and (6 +/- 1) mM, respectively. Both of these results are in good agreement with previous data from the literature. In contrast to traditional ESI-MS-based methods, the approach used in this work does not rely on the preservation of specific solution-type noncovalent interactions in the gas phase. It is shown that this method allows an accurate determination of dissociation constants, even in cases in which the ion abundance ratio of free to ligand-bound protein in ESI-MS does not reflect the corresponding concentration ratio in solution.

Binding of the natural substrates and products to KDO8P synthase: 31P and 13C solution NMR characterization

Proton decoupled 31P and 13C solution NMR experiments were applied to mixtures of 3-deoxy- d- manno-2-octulosonate-8-phosphate (KDO8P) synthase, with each of its natural substrates, phosphoenolpyruvate and arabinose-5-phosphate (ASP), and product KDO8P to identify the formation of the enzyme–substrate and enzyme–product complexes. Effects arising from ligand interactions with the enzyme are reported via chemical shifts and line broadening with respect to those of the free ligands in solution, depending on the strength and dynamics of binding under thermodynamic equilibrium conditions. The characterization was done both at low and high field spectrometers, 200 and 500 MHz ( 1H frequencies), and in cases of 31P NMR measurements, it was demonstrated that only the low field spectrometer is capable of providing direct experimental evidence on the enzyme–ligand interactions. Since both the substrate A5P and the product KDO8P exhibit multiple anomeric forms in solution, evidence for the preference of recognition and binding of particular forms is sought.

A sterically-hindered tricyclic biimidazoline ligand which is also a precursor to a novel tetramethyl-cyclen

The synthesis of a tricyclic biimidazoline L featuring gem-dimethyl-substituted carbons α to the imino-nitrogens is described. A tris-chelate cadmium complex [Cd L 3](ClO 4) 2 has been prepared and fully characterized. Unlike the parent ligand's cadmium(II) tris-chelate complex, this [Cd L 3] 2+ does not undergo facile exchange with free ligand in solution at ambient temperature. Ligand L is a convenient precursor to a novel bis( gem-dimethyl) derivative of cyclen: 2,2,9,9-tetramethyl-1,4,7,10-tetraazacyclododecane (2,2,9,9-Me 4-Cyclen). Crystal structures of the L 2·dithiooxamide adduct, the [Cd L 3](ClO 4) 2 complex, as well as 2,2,9,9-Me 4-Cyclen have been determined.

31P-CP–MAS NMR studies on TPP + bound to the ion-coupled multidrug transport protein EmrE

The binding of tetraphenylphosphonium (TPP +) to EmrE, a membrane-bound, 110 residue Escherichia coli multidrug transport protein, has been observed by 31P cross-polarisation–magic-angle spinning nuclear magnetic resonance spectroscopy (CP–MAS NMR). EmrE has been reconstituted into dimyristoyl phosphatidylcholine bilayers. CP–MAS could selectively distinguish binding of TPP + to EmrE in the fluid membrane. A population of bound ligand appears shifted 4 ppm to lower frequency compared to free ligand in solution, which suggests a rather direct and specific type of interaction of the ligand with the protein. This is also supported by the observed restricted motion of the bound ligand. The observation of another weakly bound substrate population arises from ligand binding to negatively charged residues in the protein loop regions.

Structure-based thermodynamic analysis of the dissociation of protein phosphatase-1 catalytic subunit and microcystin-LR docked complexes.

The relationship between the structure of a free ligand in solution and the structure of its bound form in a complex is of great importance to the understanding of the energetics and mechanism of molecular recognition and complex formation. In this study, we use a structure-based thermodynamic approach to study the dissociation of the complex between the toxin microcystin-LR (MLR) and the catalytic domain of protein phosphatase-1 (PP-1c) for which the crystal structure of the complex is known. We have calculated the thermodynamic parameters (enthalpy, entropy, heat capacity, and free energy) for the dissociation of the complex from its X-ray structure and found the calculated dissociation constant (4.0 x 10(-11)) to be in excellent agreement with the reported inhibitory constant (3.9 x 10(-11)). We have also calculated the thermodynamic parameters for the dissociation of 47 PP-1c:MLR complexes generated by docking an ensemble of NMR solution structures of MLR onto the crystal structure of PP-1c. In general, we observe that the lower the root-mean-square deviation (RMSD) of the docked complex (compared to the X-ray complex) the closer its free energy of dissociation (deltaGd(o)) is to that calculated from the X-ray complex. On the other hand, we note a significant scatter between the deltaGd(o) and the RMSD of the docked complexes. We have identified a group of seven docked complexes with deltaGd(o) values very close to the one calculated from the X-ray complex but with significantly dissimilar structures. The analysis of the corresponding enthalpy and entropy of dissociation shows a compensation effect suggesting that MLR molecules with significant structural variability can bind PP-1c and that substantial conformational flexibility in the PP-1c:MLR complex may exist in solution.

Kinetic and mechanistic aspects of iron(II) coordination to bipyridyl-based hydrogel pofymer membranes

A range of hydrophilic membranes composed of copolymers of 4-methyl-4′-vinyl-2, 2′-bipyridyl with 2-hydroxyethyl methacrylate have been synthesized. These membranes readily coordinate iron(II) from aqueous solution to form the tris(2,2′-bipyridyl)iron(II) species, but at a rate very much slower than that of the free ligand in solution. Kinetic studies on the rate of development of these colour centres have shown the process to be anion-dominated and pseudo-first order for iron(II) sulfate and second order for the chloride and perchlorate. The extent of coordination within the membrane is also dependent on the salt used, as the anion influences the concentration of FeII partitioned within the gel matrix, and ultimately the position of the equilibrium established between the mono and tris complexes. Mechanisms are proposed incorporating the known water-structuring effects that anions impose on this type of hydrogel environment and accounting for the presence of ion pairs and their effect on the molecular reorganisations that are necessary in order for tris complexation to occur.

Influence of the matrix on the complexation of metal ions by chelating ion exchangers

Chelating ion exchangers show distribution coefficients that are considerably lower than those expected from complexation equilibria with the corresponding free ligands in solution. Furthermore, dependence of distribution coefficients on the concentration of exchanging ions in solution is observed. Both effects are due to the influence of the surrounding matrix on the anchor groups. The distribution coefficients of Fe3+, Ni2+, Cu2+, Pb2+ and UO2+2 on two cellulose exchangers carrying different anchor groups are investigated as function of the concentration in solution. In case of the anchor group of high flexibility a strong increase of the distribution coefficients with decreasing concentration in solution is measured. This is explained by the fact that the anchor groups located at the surface are less affected by interactions with the matrix, with the result of higher distribution coefficients. The explanation is in agreement with the sorption isotherm.

Notes. Complexation of gold(I) thiomalate (‘myocrisin’) with 1,3-diazinane-2-thione in aqueous solution followed by13C nuclear magnetic resonance spectroscopy

The interaction of gold(I) thiomalate (‘Myocrisin’; an anti-arthritic drug)[Au(tm)] with 1,3-diazinane-2-thione (L) has been studied in aqueous solution at pH * 7.2 by using 13C n.m.r. spectroscopy. It is found that [Au(tm)] forms a 1:1 complex of tm–Au–L. In the presence of excess thiomalate, L is displaced as a free ligand in solution suggesting that a thiol binds to gold(I) more strongly than does a thione.