Dissecting the cholera toxin-ganglioside GM1 interaction by isothermal titration calorimetry

The complex of cholera toxin and ganglioside GM1 is one of the highest affinity protein-carbohydrate interactions known. Herein, the GM1 pentasaccharide is dissected into smaller fragments to determine the contribution of each of the key monosaccharide residues to the overall binding affinity. Displacement isothermal titration calorimetry (ITC) has allowed the measurement of all of the key thermodynamic parameters for even the lowest affinity fragment ligands. Analysis of the standard free energy changes using Jencks' concept of intrinsic free energies reveals that the terminal galactose and sialic acid residues contribute 54% and 44% of the intrinsic binding energy, respectively, despite the latter ligand having little appreciable affinity for the toxin. This analysis also provides an estimate of 25.8 kJ mol(-1) for the loss of independent translational and rotational degrees of freedom on complexation and presents evidence for an alternative binding mode for ganglioside GM2. The high affinity and selectivity of the GM1-cholera toxin interaction originates principally from the conformational preorganization of the branched pentasaccharide rather than through the effect of cooperativity, which is also reinvestigated by ITC.     

Binding isotherm determination by isothermal titration calorimetry

Journal of Thermal Analysis and Calorimetry - A simple method for determination of binding isotherm in the protein-ligand interaction was introduced using isothermal titration calorimetric data....     

Nucleotide coordination with 14 lanthanides studied by isothermal titration calorimetry

ITC reveals the increasingly importance of entropy for heavier lanthanides binding to nucleotides. The phosphate group forming chelating effect with purine bases but not with pyrimidines.     

Dicynthaurin (ala) monomer interaction with phospholipid bilayers studied by fluorescence leakage and isothermal titration calorimetry

The interaction of the antimicrobial peptide dicynthaurin (ala) monomer with model membranes of zwitterionic and negatively charged lipids and mixtures thereof was studied by means of isothermal titration calorimetry (ITC), fluorescent leakage, and dynamic light scattering (DLS) measurements. For the ITC analysis, we have applied the surface partitioning equilibrium model which shows that the interaction is predominately driven by hydrophobic effects (Kb between 2 x 10(4) and 1 x 10(5) M(-1)). Under low salt conditions, the enhanced electrostatic interaction leads to larger peptide concentrations immediately above the vesicle surface, which initiates the insertion of the peptide into the bilayer more effectively. Fluorescent leakage measurements have shown a fast leakage of the fluorescent dye within seconds after peptide addition. The analysis of the leakage kinetics was performed in terms of an initial pore formation model (up to t = 1000 s) that takes the reversible surface aggregation of bound peptide monomers into account. From this analysis, a minimum aggregation number of n = 7 +/- 2 per pore is obtained.     

Hydrophobic interaction chromatography of proteins: Studies of unfolding upon adsorption by isothermal titration calorimetry

Heat of adsorption is an excellent measure for adsorption strength and, therefore, very useful to study the influence of salt and temperature in hydrophobic interaction chromatography. The adsorption of bovine serum albumin and β‐lactoglobulin to Toyopearl Butyl‐650 M was studied with isothermal titration calorimetry to follow the unfolding of proteins on hydrophobic surfaces. Isothermal titration calorimetry is established as an experimental method to track conformational changes of proteins on stationary phases. Experiments were carried out at two different salt concentrations and five different temperatures. Protein unfolding, as indicated by large changes of molar enthalpy of adsorption Δh_ads, was observed to be dependent on temperature and salt concentration. Δh_ads were significantly higher for bovine serum albumin and ranged from 578 (288 K) to 811 (308 K) kJ/mol for 1.2 mol/kg ammonium sulfate. Δh_ads for β‐lactoglobulin ranged from 129 kJ/mol (288 K) to 186 kJ/mol (308 K). For both proteins, Δh_ads increased with increasing temperature. The influence of salt concentration on Δh_ads was also more pronounced for bovine serum albumin than for β‐lactoglobulin. The comparison of retention analysis evaluated by the van't Hoff algorithm shows that beyond adsorption other processes occur simultaneously. Further interpretation such as unfolding upon adsorption needs other in situ techniques.     

A thermodynamic study on the interaction of nickel ion with myelin basic protein by isothermal titration calorimetry

The interaction of myelin basic protein (MBP) from the bovine central nervous system with divalent nickel ion was studied by isothermal titration calorimetry at 37 and 47 °C in Tris buffer solution at pH = 7. The new solvation model was used to reproduce the heats of MBP + Ni²⁺ interaction over the whole Ni²⁺ concentrations. It was found that MBP has three identical and independent binding sites for Ni²⁺ ions. The intrinsic dissociation equilibrium constant and the molar enthalpy of binding are 89.953 μM, −14.403 kJ mol⁻¹ and 106.978 μM, −14.026 kJ mol⁻¹ at 37 and 47 °C, respectively. The binding parameters recovered from the new solvation model were correlated to the structural changes of MBP due to its interaction with nickel ion interaction. It was found that in the low and high concentrations of the nickel ions, the MBP structure was destabilized.     

Binding of 8-anilinonaphthalene sulfonate to dimeric and tetrameric concanavalin A: energetics and its implications on saccharide binding studied by isothermal titration calorimetry and spectroscopy

The binding of 8-anilinonaphthalene sulfonate to concanavalin A has been investigated. Isothermal titration calorimetry (ITC) and circular dichroism studies have been performed under different experimental conditions to understand the binding quantitatively and evaluate contributions of different forces responsible for it. Isothermal titration calorimetric results of concanavalin A with ANS at pH 5.2 and 2.5, where it exists as a dimer, indicated binding heterogeneity and two classes of noninteracting sites. Enhancement of the binding constants from native to pH 2.5 suggests that the ANS binding is strongly influenced by the protein charge and the favorable alteration in the structure of concanavalin A as suggested by near-UV CD results. No binding was observed with the tetrameric form of concanavalin A, indicating shielding of sites due to dimerization of canonical dimers. The results have also demonstrated existence of a hydrophobic binding site that is distinct from the saccharide binding site.     

Thermodynamic study of the dimerization of 8-anilino-1-naphthalenesulfonic acid by isothermal titration calorimetry

Dimerization of the fluorescence probe, 8-anilino-1-naphthalenesulfonic acid (ANS) in aqueous media have been studied by isothermal titration calorimetry (ITC). ITC experiments carried out at different pHs show that dimerization constants are highly pH dependent, decreasing their values with increasing pH. No dimerization is detected over pH 7. Analyzing the dependence of K_dim on pH, using a model that only considers dimers between zwitterionic molecules of ANS, a value of 5.6 for the pK of anilinium moiety is obtained. It is in agreement with the pK determined spectrophotometrically. The dimerization process is enthalpically disfavored and entropically driven at all pH and temperatures studied, indicating that hydrophobic effect has an important role on the formation of dimers. Although dimerization constants are low, dimerization equilibria must be taken into account when the energetics of the interaction of ANS to a protein is studied at pH below 7.     

Affinity capillary electrophoresis and isothermal titration calorimetry for the determination of fatty acid binding with beta-cyclodextrin

Affinity capillary electrophoresis (ACE) and isothermal titration calorimetry (ITC) were used to investigate the binding interaction between several fatty acids (FAs) and beta-cyclodextrin (beta-CD). Within each method, steps taken to obtain accurate binding constants are discussed. The stoichiometry of interaction was revealed to be 1:1 regardless of FA chain length. The binding constants obtained using ACE were: octanoate, 6.4x10(2); 2-octenoate, 4.7x10(2); decanoate, 3.7x10(3); 9-decenoate, 1.8x10(3) and dodecanote, 1.4x10(4). The binding constants obtained from ITC were of the same order of magnitude, but were consistently greater than those from ACE. Thermodynamic data obtained using ITC are used to explain the observed trends in binding strength.     

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.     

A review on the ligand binding studies by isothermal titration calorimetry

Thermodynamics of biomacromolecule ligand interaction is very important to understand the structure function relationship in proteins. One of the most powerful techniques useful to obtain additional information about the structure of proteins in biophysical chemistry field is isothermal titration calorimetry (ITC). An ITC experiment is a titration of a biomacromolecule solution by a solution containing a reactant (ligand) at constant temperature to obtain the exchanged heat of the reaction. The total concentration of ligand is the independent variable under experimental control. There are many reports on data analysis for ITC to find the number of binding sites (g), the equilibrium constant (K), the Gibbs free energy of binding process (ΔG), the enthalpy of binding (ΔH) and the entropy of binding (ΔS). Moreover, ITC gives information about the type of reaction, electrostatic and hydrophobic interactions, including determination of cooperativity characterization in binding process by calculating the Hill coefficient (n). A double reciprocal plot and a graphical fitting method are two simple methods used in the enzyme inhibition and metal binding to a protein. Determination of a binding isotherm needs more ITC experiments and more complex data analysis. Protein denaturation by ligand includes two processes of binding and denaturation so that ITC data analysis are more complex. However, the enthalpy of denaturation process obtained by ITC help to understand the fine structure of a protein.     

Applications of isothermal titration calorimetry in protein folding and molecular recognition

During the past decade, isothermal titration calorimetry (ITC) has developed from a specialist method to a major, commercially available tool in the arsenal directed at understanding molecular interactions. At present, ITC is used to study all types of binding reactions, including protein-protein, protein-ligand, DNA-drug, DNA-protein, receptor-target, and enzyme kinetics, and it is becoming the method of choice for the determination of the thermodynamic parameters associated with the structure transformation of one molecule or non-covalent interaction of two (or more) molecules. Here, the new applications of ITC in protein folding/unfolding and misfolding, as well as its traditional application in molecular interaction/recognition are reviewed, providing an overview of what can be achieved in these fields using this method and what developments are likely to occur in the near future.     

New methods for data analysis of isothermal titration calorimetry

Heat divided by ligand concentration vs. heat, similar to the Scatchard plot, was introduced to obtain the equilibrium constant (K) and the enthalpy of binding (DH) using isothermal titration calorimetry data. Values of K and DH obtained by this linear pseudo-Scatchard plot for a system with a set of independent binding sites (such as binding fluoride ions on urease and monosaccharide methyl a-D-mannopyranoside on concavalin A) were remarkably like that obtained from a normal fitting Wiseman method and other our technical methods. On applying this graphical method to study the binding of copper ion on myelin basic protein (MBP), a concave downward curve obtained was consistent with the positive cooperativity in the binding. A graphical fitting by simple method for determination of thermodynamic parameters was also introduced. This method is general, without any assumption and restriction made in previous method. This general method was applied to the product inhibition study of adenosine deaminase. This revised version was published online in July 2006 with corrections to the Cover Date.     

Monitoring lipid membrane translocation of sodium dodecyl sulfate by isothermal titration calorimetry

We establish high-sensitivity isothermal titration calorimetry (ITC) as a fast, reliable, and versatile tool for assessing membrane translocation of charged compounds. A combination of ITC uptake and release titrations can discriminate between the two extreme cases of half-sided binding and complete transbilayer equilibration on the experimental time scale. To this end, we derive a general fit function for both assays that allows for incorporation of different membrane partitioning models. Electrostatic effects are taken into account with the aid of Gouy-Chapman theory, thus rendering uptake and release experiments amenable to the investigation of charged solutes. This is exemplified for the flip-flop of the anionic detergent sodium dodecyl sulfate (SDS) across the membranes of 100-nm-diameter unilamellar vesicles composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) in aqueous solution (10 mM phosphate buffer, 154 mM NaCl, pH 7.4). If repulsive electrostatic forces are accounted for adequately, SDS binding to POPC membranes can be evaluated on the basis of ideal mixing in all phases. At 25 degrees C, the intrinsic partition coefficient between the interfacial aqueous phase and the membrane amounts to 3.5 x 10(6); however, detergent flip-flop is negligibly slow under these conditions. Raising the temperature to 65 degrees C lowers the intrinsic partition coefficient to 1.4 x 10(6) but enables rapid transbilayer distribution of the detergent and, therefore, binding to or desorption from both membrane leaflets. Thus, combining a surface partition equilibrium with simple electrostatic theory appears highly useful in monitoring transmembrane movement of ionic compounds by ITC, thereby eliminating the need for specific reporter groups.     

Complex formation of large-ring cyclodextrins with iodine in aqueous solution as revealed by isothermal titration calorimetry

Complex formation of large cyclodextrins(CDs) having DP 21–32 with iodine in aqueous KI solution was studied by isothermal titration calorimetry. The curves obtained for the titration of the CDs with iodine cannot be analyzed by a model based on a single set of identical sites, but, rather, by a model assuming 1 : 2 complex formation with identical interacting sites. For the two identical interacting sites, the binding constants K₁ and K₂ (K₁ < K₂), defined relative to the progress of saturation, lie in the range 0.7 to 7.3×10³ M^–1 and 3.0 to 62.6×10³ M^–1, respectively. The values of ΔH₂ and T ΔS₂ lie in the range –34.9 to –136.4 kJ·mol^–1 and –15.5 to –112.8 kJ·mol^–1, respectively. The largest values of –T ΔS₂ obtained for a CD of DP 26 can, in part, be attributed to a large decrease in conformational flexibility of the CD which occurs during complex formation.     

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.