Multiple Multidentate Halogen Bonding in Solution,in the Solid State,and in the (Calculated) Gas Phase

The binding properties of neutral halogen‐bond donors (XB donors) bearing two multidentate Lewis acidic motifs toward halides were investigated. Employing polyfluorinated and polyiodinated terphenyl and quaterphenyl derivatives as anion receptors, we obtained X‐ray crystallographic data of the adducts of three structurally related XB donors with tetraalkylammonium chloride, bromide, and iodide. The stability of these XB complexes in solution was determined by isothermal titration calorimetry (ITC), and the results were compared to X‐ray analyses as well as to calculated binding patterns in the gas phase. Density functional theory (DFT) calculations on the gas‐phase complexes indicated that the experimentally observed distortion of the XB donors during multiple multidentate binding can be reproduced in 1:1 complexes with halides, whereas adducts with two halides show a symmetric binding pattern in the gas phase that is markedly different from the solid state structures. Overall, this study demonstrates the limitations in the transferability of binding data between solid state, solution, and gas phase in the study of complex multidentate XB donors.     

Study on the interaction of amino phosphine ester derivatives with DNA by spectroscopy, modeling and calorimetry

The binding properties of amino phosphate ester derivatives, compound 1 and 2 with calf thymus DNA (CT-DNA) were investigated by UV spectra, fluorescence spectra, molecular modeling and isothermal titration calorimetry (ITC). The intrinsic binding constants K_b of compound 1 and 2 with CT-DNA were determined by fluorescence spectroscopy and ITC, respectively. The results indicated that the two compounds bind to CT-DNA with different binding affinity, which is in the order of compound 1 > compound 2. At the same time, fluorescence spectra suggested that the mechanism of the binding of the two compounds to CT-DNA is a static enhancing type. According to the ethidium bromide displacement experiments, UV spectra, molecular modeling and ITC studies, it can be concluded that compound 1 and 2 are intercalators that can slide into the G–C rich region of CT-DNA. Furthermore, ITC data showed that compound/DNA binding is enthalpy controlled.     

Thermodynamics of formation of host-guest supramolecular polymers

The interactions between three beta-cyclodextrin hosts (having 1-3 binding sites) and two adamantyl guests (having 1-2 binding sites) have been studied by ITC, ROESY, static and dynamic light scattering (SLS and DLS), and AFM and TEM techniques. The enthalpy and free energy values (determined from ITC experiments) evidence that the single interaction between one binding site of the guest and one binding site of the host is independent of the number of binding sites of the interacting species. The average values are deltaH degrees = -26.6 +/- 2.3 kJ mol(-1) and deltaG degrees = -30.4 +/- 3.2 kJ mol(-1), indicating that the process is mainly enthalpy driven. In all cases, the experimental molar ratio (from ITC experiments) agrees with the expected one from the number of binding sites of both the host and guest. The formation of polymer-like entities was demonstrated by SLS, DLS, AFM, and TEM measurements. The structure of polymers is linear when both the host and the guest are ditopic entities and dendritic (or Cayley tree type) when the host and the guest have three and two binding sites, respectively.     

Binding of chitosan to phospholipid vesicles studied with isothermal titration calorimetry

We thermodynamically characterize the interaction of chitosan with small liposomes and the binding and organization of the polysaccharide on the membrane of the vesicles. By means of isothermal titration calorimetry (ITC), we obtain the enthalpy variations arising from binding of the positively ionized chitosan to neutral and negatively charged liposomes. The strong electrostatic interaction of the polysaccharide with the negative charges at the membrane gives rise to highly exothermic signal until charge compensation is reached. The equilibrium constant, the interaction stoichiometry, and the molar enthalpy of binding chitosan monomers to phospholipids from the external leaflet of the vesicle membrane are obtained from the isotherm curve fitting assuming independent binding sites. The strong exothermic signal indicates that the electrostatically driven binding of chitosan to the membrane is energetically favored, leading to further stabilization of the vesicle suspension. The higher the net negative charge of the vesicles, the more pronounced the adsorption of chitosan is, leading to weaker chain organization of the adsorbed chitosan at the membrane. At the point of charge saturation, vesicle aggregation takes place and we show that this behavior does not always lead to charge reversal at the membrane. Models for the binding behavior and structural organization of chitosan are proposed based on the experimental results from ITC, ζ-potential, and dynamic light scattering.     

An isothermal titration calorimetry study of phytate binding to lysozyme

Isothermal titration calorimetry (ITC) was used to detect phytate binding to the protein lysozyme. This binding interaction was driven by electrostatic interaction between the positively charged protein and negatively charged phytate. When two phytate molecules bind to the protein, the charge on the protein is neutralised and no further binding occurs. The stoichiometry of binding provided evidence of phytate–lysozyme complex formation that was temperature dependent, being most extensive at lower temperatures. The initial stage of phytate binding to lysozyme was less exothermic than later injections and had a stoichiometry of 0.5 at 313 K, which was interpreted as phytate crosslinking two lysozyme molecules with corresponding water displacement. ITC could make a valuable in vitro assay to understanding binding interactions and complex formation that normally occur in the stomach of monogastric animals and the relevance of drinking water temperature on the extent of phytate–protein interaction. Interpretation of ITC data in terms of cooperativity is also discussed.     

Multivalent gold glycoclusters: high affinity molecular recognition by bacterial lectin PA-IL

Multivalent protein-carbohydrate interactions are involved in the initial stages of many fundamental biological and pathological processes through lectin-carbohydrate binding. The design of high affinity ligands is therefore necessary to study, inhibit and control the processes governed through carbohydrate recognition by their lectin receptors. Carbohydrate-functionalised gold nanoclusters (glyconanoparticles, GNPs) show promising potential as multivalent tools for studies in fundamental glycobiology research as well as biomedical applications. Here we present the synthesis and characterisation of galactose functionalised GNPs and their effectiveness as binding partners for PA-IL lectin from Pseudomonas aeruginosa. Interactions were evaluated by hemagglutination inhibition (HIA), surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC) assays. Results show that the gold nanoparticle platform displays a significant cluster glycoside effect for presenting carbohydrate ligands with almost a 3000-fold increase in binding compared with a monovalent reference probe in free solution. The most effective GNP exhibited a dissociation constant (K(d)) of 50 nM per monosaccharide, the most effective ligand of PA-IL measured to date; another demonstration of the potential of glyco-nanotechnology towards multivalent tools and potent anti-adhesives for the prevention of pathogen invasion. The influence of ligand presentation density on their recognition by protein receptors is also demonstrated.     

Calorimetric and spectroscopic studies on the interaction of anticancer drug mitoxantrone with human serum albumin

Binding of the anticancer drug mitoxantrone with the protein human serum albumin (HSA) has been studied by using isothermal titration calorimetry (ITC), in combination with fluorescence, UV–visible, and circular dichroism spectroscopy. The thermodynamic parameters of binding have been evaluated from ITC and spectroscopic results and compared. The ITC results demonstrate that the binding of mitoxantrone with HSA occurs according to two sets of binding sites on the protein as opposed to the fluorescence and UV–visible spectroscopic results. Blockage of one binding site on HSA for mitoxantrone in the presence of NaCl indicates strong involvement of electrostatic interactions in the binding of the drug with the protein. An insignificant temperature dependence of the association constant observed in fluorescence measurements suggests a very low enthalpy of binding which is in close agreement with the results obtained from ITC measurements. Fluorescence life time measurements suggest formation of a static complex between mitoxantrone and HSA. The discrepancies in the ITC and fluorescence results suggest that one of the binding sites on the protein for mitoxantrone does not contain tryptophan residue in its immediate vicinity. The calorimetric and spectroscopic results have provided quantitative information on the binding of mitoxantrone with HSA and suggest that the binding is dominated by electrostatic interactions.     

Studies into the thermodynamic origin of negative cooperativity in ion-pairing molecular recognition

The association of synthetic receptors to target guests often proceeds through the cooperative action of multiple binding forces. An investigation into the thermodynamic origin of cooperativity in ion-pairing host-guest binding in water is described. The binding affinities of 1,2,3,4-butanetetracarboxylate, tricarballate, glutarate, and acetate to a C(3)(v) symmetric metallo-host (1) are characterized in terms of the binding constants (K(a)) and the thermodynamic parameters deltaG degrees, deltaH degrees, and deltaS degrees, as determined by isothermal titration calorimetry (ITC). These values are used to determine the individual contributions of the binding interaction to the overall binding. Several ways to view the combination of the individual binding events that make up the whole are analyzed, all of which lead to the conclusion of negative cooperativity. Combined, the data were used to evaluate the thermodynamic origin of negative cooperativity for this series of guests, revealing that entropy is the largest contributing factor. An interpretation of this result focuses upon differences in the number of water molecules displaced upon binding.     

Van der Waals interactions dominate ligand-protein association in a protein binding site occluded from solvent water

In the present study we examine the enthalpy of binding of 2-methoxy-3-isobutylpyrazine (IBMP) to the mouse major urinary protein (MUP), using a combination of isothermal titration calorimetry (ITC), NMR, X-ray crystallography, all-atom molecular dynamics simulations, and site-directed mutagenesis. Global thermodynamics data derived from ITC indicate that binding is driven by favorable enthalpic contributions, rather than a classical entropy-driven signature that might be expected given that the binding pocket of MUP-1 is very hydrophobic. The only ligand-protein hydrogen bond is formed between the side-chain hydroxyl of Tyr120 and the ring nitrogen of the ligand in the wild-type protein. ITC measurements on the binding of IBMP to the Y120F mutant demonstrate a reduced enthalpy of binding, but nonetheless binding is still enthalpy dominated. A combination of solvent isotopic substitution ITC measurements and all-atom molecular dynamics simulations with explicit inclusion of solvent water suggests that solvation is not a major contributor to the overall binding enthalpy. Moreover, hydrogen/deuterium exchange measurements suggest that there is no significant contribution to the enthalpy of binding derived from "tightening" of the protein structure. Data are consistent with binding thermodynamics dominated by favorable dispersion interactions, arising from the inequality of solvent-solute dispersion interactions before complexation versus solute-solute dispersion interactions after complexation, by virtue of poor solvation of the binding pocket.     

Linear Precision Glycomacromolecules with Varying Interligand Spacing and Linker Functionalities Binding to Concanavalin A and the Bacterial Lectin FimH

A series of precision glycomacromolecules is prepared following previously established solid phase synthesis allowing for controlled variations of interligand spacing and the overall number of carbohydrate ligands. In addition, now also different linkers are installed between the carbohydrate ligand and the macromolecular scaffold. The lectin binding behavior of these glycomacromolecules is then evaluated in isothermal titration calorimetry (ITC) and kinITC experiments using the lectin Concanavalin A (Con A) in its dimeric and tetrameric form. The results indicate that both sterical and statistical effects impact lectin binding of precision glycomacromolecules. Moreover, ITC results show that highest affinity toward Con A can be achieved with an ethyl phenyl linker, which parallels earlier findings with the bacterial lectin FimH. In this way, a first set of glycomacromolecule structures is selected for testing in a bacterial adhesion–inhibition study. Here, the findings point to a one‐sugar binding mode mainly affected by sterical restraints of the nonbinding parts of the respective glycomacromolecule.     

Thermodynamics of binding of 2-methoxy-3-isopropylpyrazine and 2-methoxy-3-isobutylpyrazine to the major urinary protein

In the present study we examine the thermodynamics of binding of two related pyrazine-derived ligands to the major urinary protein, MUP-I, using a combination of isothermal titration calorimetry (ITC), X-ray crystallography, and NMR backbone (15)N and methyl side-chain (2)H relaxation measurements. Global thermodynamics data derived from ITC indicate that binding is driven by favorable enthalpic contributions, rather than the classical entropy-driven hydrophobic effect. Unfavorable entropic contributions from the protein backbone and side-chain residues in the vicinity of the binding pocket are partially offset by favorable entropic contributions at adjacent positions, suggesting a "conformational relay" mechanism whereby increased rigidity of residues on ligand binding are accompanied by increased conformational freedom of side chains in adjacent positions. The principal driving force governing ligand affinity and specificity can be attributed to solvent-driven enthalpic effects from desolvation of the protein binding pocket.     

Native ESI Mass Spectrometry Can Help to Avoid Wrong Interpretations from Isothermal Titration Calorimetry in Difficult Situations

We studied by native ESI-MS the binding of various DNA-polymerase-derived peptides onto DNA-polymerase processivity rings from Escherichia coli, Pseudomonas aeruginosa, and Mycobacterium tuberculosis. These homodimeric rings present two equivalent specific binding sites, which leads to successive formation during a titration experiment of singly- and doubly occupied rings. By using the ESI-MS free-ring spectrum as a ruler, we derived by robust linear regression the fractions of the different ring species at each step of a titration experiment. These results led to accurate K_d values (from 0.03 to 0.5 μM) along with the probability of peptide loss due to gas phase dissociation (GPD). We show that this good quality is due to the increased information content of a titration experiment with a homodimer. Isothermal titration calorimetry (ITC) led with the same binding model to K_d(ITC) values systematically higher than their ESI-MS counterparts and, often, to poor fit of the ITC curves. A processing with two competing modes of binding on the same site requiring determination of two (K_d, ΔH) pairs greatly improved the fits and yielded a second K_d(ITC) close to K_d(ESI-MS). The striking features are: (1) ITC detected a minor binding mode (~20%) of ‘low-affinity’ that did not appear with ESI-MS; (2) the simplest processing of ITC data with only one (K_d, ΔH) pair led wrongly to the Kd of the low-affinity binding mode but to the ΔH of the high-affinity binding mode. Analogous misleading results might well exist in published data based on ITC experiments.

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Graphical Abstract ?

     

Effective disruption of phosphoprotein-protein surface interaction using Zn(II) dipicolylamine-based artificial receptors via two-point interaction

Protein phosphorylation is ubiquitously involved in living cells, and it is one of the key events controlling protein-protein surface interactions, which are essential in signal transduction cascades. We now report that the small molecular receptors bearing binuclear Zn(II)-Dpa can strongly bind to a bis-phosphorylated peptide in a cross-linking manner under neutral aqueous conditions when the distance between the two Zn(II) centers can appropriately fit in that of the two phosphate groups of the phosphorylated peptide. The binding property was quantitatively determined by ITC (isothermal titration calorimetry), induced CD (circular dichroism), and NMR. On the basis of these findings, we demonstrated that these types of small molecules were able to effectively disrupt the phosphoprotein-protein interaction in a phosphorylated CTD peptide and the Pin1 WW domain, a phosphoprotein binding domain, at a micromolar level. The strategy based on a small molecular disruptor that directly interacts with phosphoprotein is unique and should be promising in developing a designer inhibitor for phosphoprotein-protein interaction.     

Electrophoretic affinity measurements on microchip. Determination of binding affinities between diketopiperazine receptors and peptide ligands

ACE on a microchip (MC-ACE) is introduced as a fast and reliable method to determine binding affinities. It is based on monitoring the change in the ionic mobility of a receptor upon binding to a ligand, or vice versa. The method is complementary to other standard methods for binding affinity determinations, like isothermal titration calorimetry (ITC), NMR, UV-Vis spectroscopy, etc. and allows for affinity studies of weak to strong binding interactions. The method is attractive since it principally allows for the analysis of the binding affinity of multiple receptors to a given ligand and requires comparatively small quantities of the binding partners (particularly in comparison to affinity measurements on capillary). We demonstrate the applicability of MC-ACE for the determination of the binding affinities between acid-rich diketopiperazine receptors and basic tripeptides in aqueous solution.     

钌(II)配合物与酵母tRNA相互作用的等温滴定量热研究

The interaction of metal complex with RNA has been studied by isothermal titration calorimetry (ITC) for the first time. ITC experiments show that complex [Ru(phen)2MPIP]^2 {phen= 1,10-phenanthroline, MP[P-2-(4-methylphenyl)imidazo[4,5-f]-1, 10-phenanthroline} interacts with yeast tRNA in terms of a model for a singleset of identical sites through intercalation, which is consistent with our previous observation obtained from spectroscopic methods, and this binding process was driven by a moderately favorable enthalpy decrease in combination with a moderately favorable entropy increase, suggesting that ITC is an effective method for deep studying the interactions of metal complexes with RNA.     

Interactions of poly(amidoamine) dendrimers with the surfactants SDS, DTAB, and C12EO6: an equilibrium and structural study using a SDS selective electrode, isothermal titration calorimetry, and small angle neutron scattering

Interactions in aqueous solutions of different generations of poly(amidoamine) (PAMAM) dendrimers containing amine, hydroxyl, or delta-glucolactone functional groups at the periphery with the anionic surfactant sodium dodecyl sulfate (SDS) were investigated. We used a SDS-specific electrode (EMF) for SDS monomer concentration monitoring, isothermal titration calorimetry (ITC) for binding information, and small angle neutron scattering (SANS) for structural studies. ITC experiments monitoring the interaction of the dendrimers with cationic dodecyltrimethylammonium bromide (DTAB) and nonionic hexaethylene glycol mono-n-dodecyl ether (C12EO6) showed no significant binding effects. In contrast, SDS binds to all of the above dendrimers. EMF and ITC data demonstrated a regular trend for both the onset of binding and binding saturation as the generation in each family of dendrimers increased. In addition, generation G6 exhibited a noncooperative binding process at very low SDS concentrations. Furthermore, the onset of cooperative binding in the EMF experiments started at lower concentrations as the weight % (w/v), the size, and the numbers of the internal or surface groups increased. On the other hand, the binding capacity of the dendrimers showed only a small dependence on the above parameters. At SDS concentrations approaching the binding limit and also at selective concentrations within the binding range, SANS measurements indicated that in all cases the bound surfactant is in the micellar form. From the electromotive force (EMF) measurements, ITC data, and SANS data, the stoichiometry of the supramolecular complexes was determined.     

Multivalent Gold Glycoclusters: High Affinity Molecular Recognition by Bacterial Lectin PA‐IL

Multivalent protein‐carbohydrate interactions are involved in the initial stages of many fundamental biological and pathological processes through lectin–carbohydrate binding. The design of high affinity ligands is therefore necessary to study, inhibit and control the processes governed through carbohydrate recognition by their lectin receptors. Carbohydrate‐functionalised gold nanoclusters (glyconanoparticles, GNPs) show promising potential as multivalent tools for studies in fundamental glycobiology research as well as biomedical applications. Here we present the synthesis and characterisation of galactose functionalised GNPs and their effectiveness as binding partners for PA‐IL lectin from Pseudomonas aeruginosa. Interactions were evaluated by hemagglutination inhibition (HIA), surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC) assays. Results show that the gold nanoparticle platform displays a significant cluster glycoside effect for presenting carbohydrate ligands with almost a 3000‐fold increase in binding compared with a monovalent reference probe in free solution. The most effective GNP exhibited a dissociation constant (K_d) of 50 nM per monosaccharide, the most effective ligand of PA‐IL measured to date; another demonstration of the potential of glyco‐nanotechnology towards multivalent tools and potent anti‐adhesives for the prevention of pathogen invasion. The influence of ligand presentation density on their recognition by protein receptors is also demonstrated.     

Investigation of the interaction between peanut agglutinin and synthetic glycopolymeric multivalent ligands

The interaction between synthetic glycoplymers bearing beta-D-galactose side groups and the lectin peanut agglutinin (PNA) was investigated by UV-difference spectroscopy and isothermal titration calorimetry (ITC). UV-difference spectroscopy indicated that the polymer-lectin interaction was stronger than that between PNA and either the corresponding monomer, D-galactose or D-lactose. The thermodynamics of binding (K, DeltaG, DeltaH, DeltaS and n) were determined from ITC data by fitting with a two-site, non-cooperative binding model. It was found that the glycopolymer displayed around a 50 times greater affinity for the lectin than the parent carbohydrate, and around 10 times greater than the monomer, on a valency-corrected basis. Binding was found to be entropically driven, and was accompanied by aggregation and precipitation of protein molecules. Furthermore, interesting differences between polymers prepared either from deacetylated monomers, or by deacetylation of pre-formed polymers, were found.