Multithermal titration calorimetry: a rapid method to determine binding heat capacities

Herein a new method that allows binding DeltaCp to be determined with a single experiment is presented. Multithermal titration calorimetry (MTC) is a simple extension of isothermal titration calorimetry (ITC) that explicitly takes into account the thermal dependences of DeltaH and the binding constant. Experimentally, this is accomplished by performing a single stepwise titration with ITC equipment, allowing temperature re-adjustments of the system at intermediate states of the titration process. Thus, from the resulting multitherm, DeltaCp can also be determined. The experimental feasibility of MTC was tested by using the well-characterized lysozyme-chitotriose complex as a model system.     

Isothermal titration calorimetry of metal ions binding to proteins: An overview of recent studies

Isothermal titration calorimetry (ITC) is a technique that is capable of quantifying the stoichiometry, equilibrium constants and thermodynamics of molecular binding events. Thus, important information about the interaction of metal ions with biological macromolecules can be obtained with ITC measurements. This review highlights many of the recent studies of metal ions binding to proteins that have used ITC to quantify the thermodynamics of metal-protein interactions.     

Recent trends and some applications of isothermal titration calorimetry in biotechnology

Isothermal titration calorimeters (ITCs) are thermodynamic instruments used for the determination of enthalpy changes in any physical/chemical reaction. This can be applied in various fields of biotechnology. This review explains ITC applications, especially in bioseparation, drug development and cell metabolism. In liquid chromatography, the separation/purification of specific proteins or polypeptides in a mixture is usually achieved by varying the adsorption affinities of the different proteins/polypeptides for the adsorbent under different mobile-phase conditions and temperatures. Using ITC analysis, the binding mechanism of proteins with adsorbent solid material is derived by elucidating enthalpy and entropy changes, which offer valuable guidelines for designing experimental conditions in chromatographic separation. The binding affinity of a drug with its target is studied by deriving binding enthalpy and binding entropy. To improve the binding affinity, suitable lead compounds for a drug can be identified and their affinity tested by ITC. Recently ITC has also been used in studying cell metabolism. The heat produced by animal cells in culture can be used as a primary indicator of the kinetics of cell metabolism, which provides key information for drug bioactivity and operation parameters for process cell culture.     

Isothermal titration calorimetry uncovers substrate promiscuity of bicupin oxalate oxidase from Ceriporiopsis subvermispora

Isothermal titration calorimetry (ITC) may be used to determine the kinetic parameters of enzyme-catalyzed reactions when neither products nor reactants are spectrophotometrically visible and when the reaction products are unknown. We report here the use of the multiple injection method of ITC to characterize the catalytic properties of oxalate oxidase (OxOx) from Ceriporiopsis subvermispora (CsOxOx), a manganese dependent enzyme that catalyzes the oxygen-dependent oxidation of oxalate to carbon dioxide in a reaction coupled with the formation of hydrogen peroxide. CsOxOx is the first bicupin enzyme identified that catalyzes this reaction. The multiple injection ITC method of measuring OxOx activity involves continuous, real-time detection of the amount of heat generated (dQ) during catalysis, which is equal to the number of moles of product produced times the enthalpy of the reaction (ΔH_app). Steady-state kinetic constants using oxalate as the substrate determined by multiple injection ITC are comparable to those obtained by a continuous spectrophotometric assay in which H₂O₂ production is coupled to the horseradish peroxidase-catalyzed oxidation of 2,2′-azinobis-(3-ethylbenzthiazoline-6-sulfonic acid) and by membrane inlet mass spectrometry. Additionally, we used multiple injection ITC to identify mesoxalate as a substrate for the CsOxOx-catalyzed reaction, with a kinetic parameters comparable to that of oxalate, and to identify a number of small molecule carboxylic acid compounds that also serve as substrates for the enzyme.     

Explicit formulation of titration models for isothermal titration calorimetry

Isothermal titration calorimetry (ITC) produces a differential heat signal with respect to the total titrant concentration. This feature gives ITC excellent sensitivity for studying the thermodynamics of complex biomolecular interactions in solution. Currently, numerical methods for data fitting are based primarily on indirect approaches rooted in the usual practice of formulating biochemical models in terms of integrated variables. Here, a direct approach is presented wherein ITC models are formulated and solved as numerical initial value problems for data fitting and simulation purposes. To do so, the ITC signal is cast explicitly as a first-order ordinary differential equation (ODE) with total titrant concentration as independent variable and the concentration of a bound or free ligand species as dependent variable. This approach was applied to four ligand-receptor binding and homotropic dissociation models. Qualitative analysis of the explicit ODEs offers insights into the behavior of the models that would be inaccessible to indirect methods of analysis. Numerical ODEs are also highly compatible with regression analysis. Since solutions to numerical initial value problems are straightforward to implement on common computing platforms in the biochemical laboratory, this method is expected to facilitate the development of ITC models tailored to any experimental system of interest.     

Isothermal titration calorimetry for drug design: Precision of the enthalpy and binding constant measurements and comparison of the instruments

Isothermal titration calorimetry (ITC) is one of the most robust label- and immobilization-free techniques used to measure protein – small molecule interactions in drug design for the simultaneous determination of the binding affinity (ΔG) and the enthalpy (ΔH), both of which are important parameters for structure-thermodynamics correlations. It is important to evaluate the precision of the method and of various ITC instrument models by performing a single well-characterized reaction. The binding between carbonic anhydrase II and acetazolamide was measured by four ITC instruments – PEAQ-ITC, iTC200, VP-ITC, and MCS-ITC and the standard deviation of ΔG and ΔH was determined. Furthermore, the limit of an approach to reduce the protein concentration was studied for a high-affinity reaction (K_d = 0.3 nM), too tight to be measured by direct (non-displacement) ITC. Chemical validation of the enthalpy measurements is discussed.     

Isothermal titration calorimetry — A new method for the quantification of microbial degradation of trace pollutants

The environmental fate and, in particular, biodegradation rates of hydrophobic organic compounds (HOC) are of high interest due to the ubiquity, persistence, and potential health effects of these compounds. HOC tend to interact with bioreactor materials and sampling devices and are frequently volatile, so that conventionally derived degradation parameters are often biased. We report on the development and validation of a novel calorimetric approach that serves to gain real time information on the kinetics and the physiology of HOC bioconversion in aqueous systems while overcoming weaknesses of conventional biodegradation experiments. Soil bacteria Mycobacterium frederiksbergense LB501T, Rhodococcus erythropolis K2-3 and Pseudomonas putida G7 were exposed to pulsed titrations of dissolved anthracene, 4-(2,4-dichlorophenoxy)butyric acid or naphthalene, respectively, and the thermal responses were monitored. The combinations of strains and pollutants were selected as examples for complete and partial biodegradation and complete degradation with storage product formation, respectively. Heat production signals were interpreted thermodynamically and in terms of Michaelis-Menten kinetics. The half-saturation constant k_D and the degradation rate r_D^Max were derived. Comparison with conventional methods shows the suitability to extract kinetic degradation parameters of organic trace pollutants from simple ITC experiments, while thermodynamic interpretation provided further information about the metabolic fate of HOC compounds.     

Structural energetics of protein-carbohydrate interactions: Insights derived from the study of lysozyme binding to its natural saccharide inhibitors

High-sensitivity isothermal titration calorimetry was used to characterize the binding of the glycohydrolitic enzyme hen egg-white lysozyme to its natural saccharide inhibitors, chitobiose and chitrotriose. Measurements were done at a pH of 4.7, in the 15 degrees C -45 degrees C temperature range. Using a structural-energetic parameterization derived previously for lectin-carbohydrate associations, both binding enthalpies and entropies for the present systems and for the complex of chitobiose with turkey egg-white lysozyme from the literature were correctly accounted for. These observations suggest that both lysozymes and lectins follow the same structural-energetic behavior in the binding to their ligands. From the analysis of lysozyme data in conjunction with other binding data reported in the literature, an ad hoc parameterization of DeltaCp for protein-carbohydrate complexes was derived for the first time. The novel parameters for both polar and apolar surface areas differed significantly from correlations obtained previously from model compounds and protein-folding data. As DeltaCp is extremely sensitive to changes in solvent structure, this finding indicates that protein-carbohydrate complexes have distinctive hydration properties. According to our analysis, the dehydration of polar groups is the major cause for the observed decrease in DeltaCp, which implies that these groups behave hydrophobically. The contribution of apolar surface areas was found of the expected sign, but their specific weight is much smaller than those obtained in other correlations. This small contribution to DeltaCp is consistent with Lemieux's hypothesis of a low degree of hydration of apolar surfaces on carbohydrates.     

Conformational dynamics promote binding diversity of dynein light chain LC8

A highly conserved and ubiquitous protein known as LC8 binds over twenty different partners, characteristic of a molecular hub (Barbar, 2008 Biochemistry, 47, 503-508). Structural studies of LC8 complexes with binding partners having diverse recognition sequences show that the same binding groove of LC8 accommodates the various binding motifs. Here we use thermodynamics and dynamics measurements of LC8 complexes to group LC8 binding partners in two categories: those whose binding is enthalpically driven and those that are entropically favored. Peptides that are enthalpically driven completely silence the millisecond-microsecond relaxation signal, suggesting a significant rigidifying of the binding groove, while peptides in the entropically favored group exhibit the same conformational dynamics as the free protein, suggesting that the peptide sits loosely in the binding groove and so retains flexibility of the groove, and presumably of the bound peptide. The inherent disorder in the LC8 binding groove and in LC8 binding partners allows both types of binding, accounts for the lack of a conserved recognition consensus motif and underlies the binding specificity and broad selectivity observed in LC8 binding.     

New insights into the mechanism of dihydrodipicolinate synthase using isothermal titration calorimetry

Thermodynamic binding information, obtained via isothermal titration calorimetry (ITC), provides new insights into the binding of substrates, and of allosteric inhibitor interactions of dihydrodipicolinate synthase (DHDPS) from Escherichia coli. DHDPS catalyses the first committed step in (S)-lysine biosynthesis: the Schiff-base mediated aldol condensation of pyruvate with (S)-aspartate semi-aldehyde. Binding studies indicate that pyruvate is a weak binder (0.023 mM) but that (S)-ASA does not interact with the enzyme in the absence of a Schiff-base with pyruvate. These results support the assignment of a ping pong catalytic mechanism in which enthalpically driven Schiff-base formation (ΔH = −44.5 ± 0.1 kJ mol⁻¹) provides the thermodynamic impetus for pyruvate association. The second substrate, (S)-ASA, was observed to bind to a Schiff-base mimic (ΔH = −2.8 ± 0.1 kJ mol⁻¹) formed through the reduction of the intermediate pyruvyl–Schiff-base complex.     

Mapping of suramin binding sites on the group IIA human secreted phospholipase A2

Suramin is a polysulphonated napthylurea used as an antiprotozoal/anthelminitic drug, which also inhibits a broad range of enzymes. Suramin binding to recombinant human secreted group IIA phospholipase A₂ (hsPLA₂GIIA) was investigated by molecular dynamics simulations (MD) and isothermal titration calorimetry (ITC). MD indicated two possible bound suramin conformations mediated by hydrophobic and electrostatic interactions with amino-acids in three regions of the protein, namely the active-site and residues located in the N- and C-termini, respectively. All three binding sites are located on the phospholipid membrane recognition surface, suggesting that suramin may inhibit the enzyme, and indeed a 90% reduction in hydrolytic activity was observed in the presence of 100 nM suramin. These results correlated with ITC data, which demonstrated 2.7 suramin binding sites on the hsPLA₂GIIA, and indicates that suramin represents a novel class of phospholipase A₂ inhibitor.     

Effect of metal ion substitutions in anticoagulation factor I from the venom of Agkistrodon acutus on the binding of activated coagulation factor X and on structural stability

Anticoagulation factor I (ACF I) isolated from the venom of Agkistrodon acutus is an activated coagulation factor X (FXa)-binding protein that binds in a Ca²⁺-dependent fashion with marked anticoagulant activity. The thermodynamics of the binding of alkaline earth metal ions to ACF I and the effects of alkaline earth metal ions on the guanidine hydrochloride (GdnHCl)-induced unfolding of ACF I and the binding of ACF I to FXa were studied by isothermal titration calorimetry, fluorescence, circular dichroism, and surface plasmon resonance, respectively. The results indicate that the ionic radii of the cations occupying Ca²⁺-binding sites in ACF I crucially affect the binding affinity of ACF I for alkaline earth metal ions as well as the structural stability of ACF I against GdnHCl denaturation. Sr²⁺ and Ba²⁺, with ionic radii larger than the ionic radius of Ca²⁺, can bind to Ca²⁺-free ACF I (apo-ACF I), while Mg²⁺, with an ionic radius smaller than that of Ca²⁺, shows significantly low affinity for the binding to apo-ACF I. All bindings of Ca²⁺, Sr²⁺, and Ba²⁺ ions in two sites of ACF I are mainly enthalpy-driven and the entropy is unfavorable for them. Sr²⁺-stabilized ACF I exhibits slightly lower resistance to GdnHCl denaturation than Ca²⁺–ACF I, while Ba²⁺-stabilized ACF I exhibits much lower resistance to GdnHCl denaturation than Ca²⁺–ACF I. Mg²⁺ and Sr²⁺, with ionic radii close to that of Ca²⁺, can bind to FXa and therefore also induce the binding of ACF I to FXa, whereas Ba²⁺, with a much larger ionic radius than Ca²⁺, cannot support the binding of ACF I with FXa. Our observations suggest that bindings of Ca²⁺, Sr²⁺, and Ba²⁺ ions in two sites of ACF I increase the structural stability of ACF I, but these bindings are not essential for the binding of ACF I with FXa, and that the binding of Mg²⁺, Ca²⁺, and Sr²⁺ ions to FXa may be essential for the recognition between FXa and ACF I.     

Isothermal chemical denaturation to determine binding affinity of small molecules to G-protein coupled receptors

The determination of accurate binding affinities is critical in drug discovery and development. Several techniques are available for characterizing the binding of small molecules to soluble proteins. The situation is different for integral membrane proteins. Isothermal chemical denaturation has been shown to be a valuable biophysical method to determine, in a direct and label-free fashion, the binding of ligands to soluble proteins. In this study, the application of isothermal chemical denaturation was applied to an integral membrane protein, the A2a G-protein coupled receptor. Binding affinities for a set of 19 small molecule agonists/antagonists of the A2a receptor were determined and found to be in agreement with data from surface plasmon resonance and radioligand binding assays previously reported in the literature. Therefore, isothermal chemical denaturation expands the available toolkit of biophysical techniques to characterize and study ligand binding to integral membrane proteins, specifically G-protein coupled receptors in vitro.     

Energetic differences between the specific binding of a 40 bp DNA duplex and the lac promoter to lac repressor protein

The energetics of LRP binding to a 104 bp lac promoter determined from ITC measurements were compared to the energetics of binding to a shorter 40 bp DNA duplex with the 21 bp promoter binding site sequence. The promoter binding affinity of 2.47 +/- 0.0 1x 10(7) M(-1) was higher than the DNA binding affinity of 1.81 +/- 0.67 x 10(7) M(-1) while the binding enthalpy of -804 +/- 41 kJ mol(-1) was lower than that of the DNA binding enthalpy of -145 +/- 16 kJ mol(-1) at 298.15 K. Both the promoter and DNA binding reactions were exothermic in phosphate buffer but endothermic in Tris buffer that showed the transfer of four protons to LRP in the former reaction but only two in the latter. A more complicated dependence of these parameters on temperature was observed for promoter binding. These energetic differences are attributable to additional LRP-promoter interactions from wrapping of the promoter around the LRP.     

Effect of the phosphate substrate on drug-inhibitor binding to human purine nucleoside phosphorylase

The thermodynamics of the drug-inhibitors acyclovir, ganciclovir, and 9-benzylguanine binding to human purine nucleoside phosphorylase (hsPNP) were determined from isothermal titration calorimetry as a function of the substrate phosphate ion (Pi) concentration from 0 to 0.125 M and temperature from 15 °C to 35 °C. At 25 °C and with an increase in the Pi concentration from 0 to 50 mM, acyclovir binding becomes more entropically-driven and ganciclovir binding becomes more enthalpically-driven. At 25 °C, the tighter 9-benzylguanine binding reaction goes from an enthalpically-driven reaction in the absence of Pi to an entropically-driven reaction at 10 mM Pi, and the enthalpically-driven nature of the binding reaction is restored at 75 mM Pi. Since the dependencies of the driving-nature of the binding reactions on Pi concentration can be simulated by Pi binding to its catalytic site, it is believed that bound Pi affects the interactions of the side-chains with the ribose catalytic site. However, the binding constants are unaffected by change in the bound Pi concentration because of enthalpy-entropy compensation. The enzymatic activity of hsPNP was determined by an ITC-based assay employing 7-methylguanosine and Pi as the substrates. The heat of reaction determined from the assay increased by 7.5 kJ mol⁻¹ with increase in Pi concentration from 50 to 100 mM and is attributed to weak binding of the Pi to a secondary regulatory site. Although the binding constants of acyclovir and ganciclovir at 20 μM hsPNP were in agreement with the inverse inhibition constants determined from the ITC enzyme inhibition assays at 60 nM, the binding constant of 9-benzylguanine, which interacts with Phe159 from an adjacent subunit, decreased from 5.62 × 10⁵ M⁻¹ to 1.14 × 10⁵ M⁻¹. This reduction in the 9-benzylguanine binding affinity along with a 7-fold increase in the specific activity of hsPNP at 14.5 nM results from partial dissociation of the hsPNP trimer into monomers below the 60 nM level.     

Effects of organic solvents on the enzyme activity of Trypanosoma cruzi glyceraldehyde-3-phosphate dehydrogenase in calorimetric assays

In drug discovery programs, dimethyl sulfoxide (DMSO) is a standard solvent widely used in biochemical assays. Despite the extensive use and study of enzymes in the presence of organic solvents, for some enzymes the effect of organic solvent is unknown. Macromolecular targets may be affected by the presence of different solvents in such a way that conformational changes perturb their active site structure accompanied by dramatic variations in activity when performing biochemical screenings. To address this issue, in this work we studied the effects of two organic solvents, DMSO and methanol (MeOH), in the isothermal titration calorimetry (ITC) kinetic assays for the catalyzed reaction of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from Trypanosoma cruzi. The solvent effects on T. cruzi GAPDH had not yet been studied. This enzyme was shown here to be affected by the organic solvents content up to 5.0% for MeOH and up to 7.5% for DMSO. The results show that when GAPDH is assayed in the presence of DMSO (5%, v/v) using the ITC experiment, the enzyme exhibits approximately twofold higher activity than that of GAPDH with no cosolvent added. When MeOH (5%, v/v) is the cosolvent, the GAPDH activity is sixfold higher. The favorable effects of the organic solvents on the Michaelis-Menten enzyme-substrate complex formation ensure the consistency of the biological assays, structural integrity of the protein, and reproducibility over the measurement time. The reaction was also kinetically monitored by standard spectrophotometric assays to establish a behavioral performance of T. cruzi GAPDH when used for screening of potential inhibitors.     

Advantages of isothermal titration calorimetry for xylanase kinetics in comparison to chemical-reducing-end assays

In lignocellulosic raw materials for biomass conversion, hemicelluloses constitute a substantial fraction, with xylan being the primary part. Although many pretreatments reduce the amount or change the distribution of xylan, it is important to degrade residual xylan so as to improve the overall yield. Typically, xylanase reaction rates are measured in stopped assays by chemical quantification of the reducing ends. With isothermal titration calorimetry (ITC), the heat flow of the hydrolysis can be measured in continuous fashion, with the reaction rate being directly proportional to the heat flow. Reaction enthalpies for carbohydrate hydrolysis are typically below 5 kJ/mol, which is the limiting factor for straight forward calorimetric quantification of enzymatic reaction rates using current ITC technology. To increase the apparent reaction enthalpy, we employed a subsequent oxidation of hydrolysis products by carbohydrate oxidase and catalase. Here we show that the coupled assay with carbohydrate oxidase and catalase can be used to measure enzyme kinetics of a GH10 xylanase from Aspergillus aculeatus on birch xylan and wheat arabinoxylan. Results are discussed in the light of a critical analysis of the sensitivity of four chemical-reducing-end quantification methods using well-characterized substrates.     

Importance of oligomerisation on Pseudomonas aeruginosaLectin-II binding affinity. In silico and in vitro mutagenesis

The effect of terminal GLY114* deletion on the binding affinity of the PA-IIL lectin toward l-fucose was investigated. Both experimental (isothermal titration calorimetry) and computational (molecular dynamics simulations) methods have shown that the deletion mutation decreases the L-fucose affinity. It implies that the PA-IIL saccharide binding affinity is influenced by the dimerization of the lectin. A detailed analysis of computational data confirms the key role of electrostatic interactions in the PA-IIL/saccharide binding.