In depth,thermodynamic analysis of Ca2+ binding to human cardiac troponin C: Extracting buffer-independent binding parameters

BackgroundCharacterizing the thermodynamic parameters behind metal-biomolecule interactions is fundamental to understanding the roles metal ions play in biology. Isothermal Titration Calorimetry (ITC) is a “gold-standard” for obtaining these data. However, in addition to metal-protein binding, additional equilibria such as metal-buffer interactions must be taken into consideration prior to making meaningful comparisons between metal-binding systems.MethodsIn this study, the thermodynamics of Ca²⁺ binding to three buffers (Bis-Tris, MES, and MOPS) were obtained from Ca²⁺-EDTA titrations using ITC. These data were used to extract buffer-independent parameters for Ca²⁺ binding to human cardiac troponin C (hcTnC), an EF-hand containing protein required for heart muscle contraction.ResultsThe number of protons released upon Ca²⁺ binding to the C– and N-domain of hcTnC were found to be 1.1 and 1.2, respectively. These values permitted determination of buffer-independent thermodynamic parameters of Ca²⁺-hcTnC binding, and the extracted data agreed well among the buffers tested. Both buffer and pH-adjusted parameters were determined for Ca²⁺ binding to the N-domain of hcTnC and revealed that Ca²⁺ binding under aqueous conditions and physiological ionic strength is both thermodynamically favorable and driven by entropy.ConclusionsTaken together, the consistency of these data between buffer systems and the similarity between theoretical and experimental proton release is indicative of the reliability of the method used and the importance of extracting metal-buffer interactions in these studies.General significanceThe experimental approach described herein is clearly applicable to other metal ions and other EF-hand protein systems.     

The thermodynamics of protein interactions with essential first row transition metals

BackgroundThe binding of metal ions to proteins is a crucial process required for their catalytic activity, structural stability and/or functional regulation. Isothermal titration calorimetry provides a wealth of fundamental information which when combined with structural data allow for a much deeper understanding of the underlying molecular mechanism.Scope of reviewA rigorous understanding of any molecular interaction requires in part an in-depth quantification of its thermodynamic properties. Here, we provide an overview of recent studies that have used ITC to quantify the interaction of essential first row transition metals with relevant proteins and highlight major findings from these thermodynamic studies.General significanceThe thermodynamic characterization of metal ion–protein interactions is one important step to understanding the role that metal ions play in living systems. Such characterization has important implications not only to elucidating proteins' structure-function relationships and biological properties but also in the biotechnology sector, medicine and drug design particularly since a number of metal ions are involved in several neurodegenerative diseases.Major conclusionsIsothermal titration calorimetry measurements can provide complete thermodynamic profiles of any molecular interaction through the simultaneous determination of the reaction binding stoichiometry, binding affinity as well as the enthalpic and entropic contributions to the free energy change thus enabling a more in-depth understanding of the nature of these interactions. This article is part of a Special Issue entitled Microcalorimetry in the BioSciences — Principles and Applications, edited by Fadi Bou-Abdallah.     

Application of isothermal titration calorimetry in bioinorganic chemistry

The thermodynamics of metals ions binding to proteins and other biological molecules can be measured with isothermal titration calorimetry (ITC), which quantifies the binding enthalpy (ΔH°) and generates a binding isotherm. A fit of the isotherm provides the binding constant (K), thereby allowing the free energy (ΔG°) and ultimately the entropy (ΔS°) of binding to be determined. The temperature dependence of ΔH° can then provide the change in heat capacity (ΔC _p°) upon binding. However, ITC measurements of metal binding can be compromised by undesired reactions (e.g., precipitation, hydrolysis, and redox), and generally involve competing equilibria with the buffer and protons, which contribute to the experimental values (K _ITC, ΔH _ITC). Guidelines and factors that need to be considered for ITC measurements involving metal ions are outlined. A general analysis of the experimental ITC values that accounts for the contributions of metal–buffer speciation and proton competition and provides condition-independent thermodynamic values (K, ΔH°) for metal binding is developed and validated.     

Protonation linked equilibria and apparent affinity constants: the thermodynamic profile of the α-chymotrypsin–proflavin interaction

Protonation/deprotonation equilibria are frequently linked to binding processes involving proteins. The presence of these thermodynamically linked equilibria affects the observable thermodynamic parameters of the interaction (K _obs, ΔH _obs⁰). In order to try and elucidate the energetic factors that govern these binding processes, a complete thermodynamic characterisation of each intrinsic equilibrium linked to the complexation event is needed and should furthermore be correlated to structural information. We present here a detailed study, using NMR and ITC, of the interaction between α-chymotrypsin and one of its competitive inhibitors, proflavin. By performing proflavin titrations of the enzyme, at different pH values, we were able to highlight by NMR the effect of the complexation of the inhibitor on the ionisable residues of the catalytic triad of the enzyme. Using ITC we determined the intrinsic thermodynamic parameters of the different equilibria linked to the binding process. The possible driving forces of the interaction between α-chymotrypsin and proflavin are discussed in the light of the experimental data and on the basis of a model of the complex. This study emphasises the complementarities between ITC and NMR for the study of binding processes involving protonation/deprotonation equilibria. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Thermodynamics of substrate binding to the metal site in homoprotocatechuate 2,3-dioxygenase: Using ITC under anaerobic conditions to study enzyme–substrate interactions

BackgroundExtradiol dioxygenases are a family of nonheme iron (and sometimes manganese) enzymes that catalyze an O₂-dependent ring-opening reaction in a biodegradation pathway of aromatic compounds. Here we characterize the thermodynamics of two substrates binding in homoprotocatechuate 2,3-dioxygenase (HPCD) prior to the O₂ activation step.MethodsThis study uses microcalorimetry under an inert atmosphere to measure thermodynamic parameters associated with catechol binding to nonheme metal centers in HPCD. Several stopped-flow rapid mixing experiments were used to support the calorimetry experiments.ResultsThe equilibria constant for 4-nitrocatechol and homoprotocatechuate binding to the iron(II) and manganese(II) forms of HPCD range from 2 × 10⁴ to 1 × 10⁶, suggesting there are distinctive differences in how the enzyme–substrate complexes are stabilized. Further experiments in multiple buffers allowed us to correct the experimental ΔH for substrate ionization and to fully derive the pH and buffer independent thermodynamic parameters for substrate binding to HPCD. Fewer protons are released from the iron(II) dependent processes than their manganese(II) counterparts.ConclusionsCondition independent thermodynamic parameters for 4-nitrocatechol and homoprotocatechuate binding to HPCD are highly consistent with each other, suggesting these enzyme–substrate complexes are more similar than once thought, and the ionization state of metal coordinated waters may be playing a role in tuning redox potential and in governing reactivity.General significanceSubstrate binding to HPCD is a complex set of equilibria that includes ionization of substrate and water release, yet it is also the key step in O₂ activation. This article is part of a Special Issue entitled Microcalorimetry in the BioSciences — Principles and Applications, edited by Fadi Bou-Abdallah.     

Biomolecule–nanoparticle interactions: Elucidation of the thermodynamics by isothermal titration calorimetry

BackgroundNanomaterials (NMs) are often exposed to a broad range of biomolecules of different abundances. Biomolecule sorption driven by various interfacial forces determines the surface structure and composition of NMs, subsequently governs their functionality and the reactivity of the adsorbed biomolecules. Isothermal titration calorimetry (ITC) is a nondestructive technique that quantifies thermodynamic parameters through in-situ measurement of the heat absorption or release associated with an interaction.Scope of reviewThis review highlights the recent applications of ITC in understanding the thermodynamics of interactions between various nanoparticles (NPs) and biomolecules. Different aspects of a typical ITC experiment that are crucial for obtaining accurate and meaningful data, as well as the strengths, weaknesses, and challenges of ITC applications to NP research were discussed.Major conclusionsITC reveals the driving forces behind biomolecule–NP interactions and the effects of the physicochemical properties of both NPs and biomolecules by quantifying the crucial thermodynamics parameters (e.g., binding stoichiometry, ΔH, ΔS, and ΔG). Complimentary techniques would strengthen the interpretation of ITC results for a more holistic understanding of biomolecule–NP interactions.General significanceThe thermodynamic information revealed by ITC and its complimentary characterizations is important for understanding biomolecule–NP interactions that are fundamental to the biomedical and environmental applications of NMs and their toxicological effects. This article is part of a Special Issue entitled Microcalorimetry in the BioSciences — Principles and Applications, edited by Fadi Bou-Abdallah.     

An interfacial equilibria model for the electrokinetic properties of a fat emulsion. Part III: A predictive computer model

Abstract

The semi-empirical thermodynamic model for the binding of metal ions to an emulsion (intralipid 20%) reported previously (Hall et al., 1991 ; Gaskin et al., 1993) is incorporated into a thermodynamic computer model. This permits the zeta potential and emulsion stability together with precipitate formation to be estimated for any intravenous nutrition regimen. A regimen frequently used in the intravenous nutrition of patients is considered in this modeling study. The effect of solution pH and calcium on the zeta potential of the emulsion is predicted. Calcium and magnesium are the only metal cations which are predicted to be of importance when considering stability of this emulsion.     

Hot Biological Catalysis: Isothermal Titration Calorimetry to Characterize Enzymatic Reactions

Isothermal titration calorimetry (ITC) is a well-described technique that measures the heat released or absorbed during a chemical reaction, using it as an intrinsic probe to characterize virtually every chemical process. Nowadays, this technique is extensively applied to determine thermodynamic parameters of biomolecular binding equilibria. In addition, ITC has been demonstrated to be able of directly measuring kinetics and thermodynamic parameters (k_cat, K_M, ΔH) of enzymatic reactions, even though this application is still underexploited. As heat changes spontaneously occur during enzymatic catalysis, ITC does not require any modification or labeling of the system under analysis and can be performed in solution. Moreover, the method needs little amount of material. These properties make ITC an invaluable, powerful and unique tool to study enzyme kinetics in several applications, such as, for example, drug discovery.In this work an experimental ITC-based method to quantify kinetics and thermodynamics of enzymatic reactions is thoroughly described. This method is applied to determine k_cat and K_M of the enzymatic hydrolysis of urea by Canavalia ensiformis (jack bean) urease. Calculation of intrinsic molar enthalpy (ΔH_int) of the reaction is performed. The values thus obtained are consistent with previous data reported in literature, demonstrating the reliability of the methodology.     

Simultaneous determination of thermodynamic and kinetic data by isothermal titration calorimetry

BackgroundThermodynamic and binding kinetic data increasingly support and guide the drug optimization process.MethodsBecause ITC thermograms contain binding thermodynamic and kinetic information, an efficient protocol for the simultaneous extraction of thermodynamic and kinetic data for 1:1 protein ligand reactions from AFFINImeter kinITC in one single experiment are presented.ResultsThe effort to apply this protocol requires the same time as for the standard protocol but increases the precision of both thermodynamic and kinetic data.ConclusionsThe protocol enables reliable extraction of both thermodynamic and kinetic data for 1:1 protein-ligand binding reactions with improved precision compared to the ‘standard protocol’.General significanceThermodynamic and kinetic data are recorded under exactly the same conditions in solution without any labeling or immobilization from a protein sample that is not 100% active and would otherwise render the extraction of kinetic parameters impossible.     

What Are the Conformational Changes Induced by Hard and Soft Metal Ion Binding to 2′-Deoxyguanosine?

Abstract

The NMR study on the interactions of 2′-dG with Mg²⁺, Zn²⁺ and Hg²⁺ ions in D₂O solution has shown that binding of softer metal ions to N7 shifts N <!—graphic—> S pseudorotational equilibrium slightly towards N-type sugar conformations. There are no detectable changes for the conformational equilibria across C4′-C5′ bond, whereas the population of the syn conformers is slightly increased.     

Application of ITC in foods: A powerful tool for understanding the gastrointestinal fate of lipophilic compounds

BackgroundIsothermal titration calorimetry (ITC) is a biophysical technique widely used to study molecular interactions in biological and non-biological systems. It can provide important information about molecular interactions (such as binding constant, number of binding sites, free energy, enthalpy, and entropy) simply by measuring the heat absorbed or released during an interaction between two liquid solutions.Scope of the reviewIn this review, we present an overview of ITC applications in food science, with particular focus on understanding the fate of lipids within the human gastrointestinal tract. In this area, ITC can be used to study micellization of bile salts, inclusion complex formation, the interaction of surface-active molecules with proteins, carbohydrates and lipids, and the interactions of lipid droplets.Major conclusionsITC is an extremely powerful tool for measuring molecular interactions in food systems, and can provide valuable information about many types of interactions involving food components such as proteins, carbohydrates, lipids, surfactants, and minerals. For systems at equilibrium, ITC can provide fundamental thermodynamic parameters that can be used to establish the physiochemical origin of molecular interactions.General significanceIt is expected that ITC will continue to be utilized as a means of providing fundamental information about complex materials such as those found in foods. This knowledge may be used to create functional foods designed to behave in the gastrointestinal tract in a manner that will improve human health and well-being. This article is part of a Special Issue entitled Microcalorimetry in the BioSciences — Principles and Applications, edited by Fadi Bou-Abdallah.     

Simultaneous determination of thermodynamic and kinetic parameters of aminopolycarbonate complexes of cobalt(II) and nickel(II) based on isothermal titration calorimetry data

The influence of the different side chain residues on the thermodynamic and kinetic parameters for complexation reactions of the Co²⁺ and Ni²⁺ ions has been investigated by using the isothermal titration calorimetry (ITC) technique supported by potentiometric titration data. The study was concerned with the 2 common tripodal aminocarboxylate ligands, namely, nitrilotriacetic acid and N‐(2‐hydroxyethyl) iminodiacetic acid. Calorimetric measurements (ITC) were run in the 2‐(N‐morpholino)ethanesulfonic acid hydrate (2‐(N‐morpholino) ethanesulfonic acid), piperazine‐N ,N ′‐bis(2‐ethanesulfonic acid), and dimethylarsenic acid buffers (0.1 mol L⁻¹, pH 6) at 298.15 K. The quantification of the metal‐buffer interactions and their incorporation into the ITC data analysis enabled to obtain the pH‐independent and buffer‐independent thermodynamic parameters (K , ΔG , ΔH , and ΔS ) for the reactions under study. Furthermore, the kinITC method was applied to obtain kinetic information on complexation reactions from the ITC data. Correlations, based on kinetic and thermodynamic data, between the kinetics of formation of Co²⁺ and Ni²⁺ complexes and their thermodynamic stabilities are discussed.     

On the link between conformational changes,ligand binding and heat capacity

BackgroundConformational changes coupled to ligand binding constitute the structural and energetics basis underlying cooperativity, allostery and, in general, protein regulation. These conformational rearrangements are associated with heat capacity changes. ITC is a unique technique for studying binding interactions because of the simultaneous determination of the binding affinity and enthalpy, and for providing the best estimates of binding heat capacity changes.Scope of reviewStill controversial issues in ligand binding are the discrimination between the “conformational selection model” and the “induced fit model”, and whether or not conformational changes lead to temperature dependent apparent binding heat capacities. The assessment of conformational changes associated with ligand binding by ITC is discussed. In addition, the “conformational selection” and “induced fit” models are reconciled, and discussed within the context of intrinsically (partially) unstructured proteins.Major conclusionsConformational equilibrium is a major contribution to binding heat capacity changes. A simple model may explain both conformational selection and induced fit scenarios. A temperature-independent binding heat capacity does not necessarily indicate absence of conformational changes upon ligand binding. ITC provides information on the energetics of conformational changes associated with ligand binding (and other possible additional coupled equilibria).General significancePreferential ligand binding to certain protein states leads to an equilibrium shift that is reflected in the coupling between ligand binding and additional equilibria. This represents the structural/energetic basis of the widespread dependence of ligand binding parameters on temperature, as well as pH, ionic strength and the concentration of other chemical species. This article is part of a Special Issue entitled Microcalorimetry in the BioSciences — Principles and Applications, edited by Fadi Bou-Abdallah.     

Transition Metal Ligands as Novel DNA-Base Substitutes

Abstract

The base modified nucleoside dBP, carrying a non-hydrogen-bonding non-shape complementary base was incorporated into oligonucleotides (Brotschi, C.; Häberli, A.; Leumann C.J. Angew. Chem. Int. Ed. 2001, 40, 3012–3014). This base was designed to coordinate transition metal ions into well defined positions within a DNA double helix. Melting experiments revealed that the stability of a dBP: dBP base couple in a DNA duplex is similar to a dG: dC base pair even in the absence of transition metal ions. In the presence of transition metal ions, melting experiments revealed a decrease in duplex stability which is on a similar order for all metal ions (Mn²⁺, Cu²⁺, Zn²⁺, Ni²⁺) tested.     

Metal ions in sugar binding, sugar specificity and structural stability of Spatholobus parviflorus seed lectin

Spatholobus parviflorus seed lectin (SPL) is a heterotetrameric lectin, with two α and two β monomers. In the crystal structure of SPL α monomer, two residues at positions 240 and 241 are missing. This region was modeled based on the positional and sequence similarities. The role of metal ions in SPL structure was analyzed by 10 ns molecular dynamics simulation. MD simulations were performed in the presence and absence of metal ions to explain the loss of haemagglutinating property of the lectin due to demetallization. Demetallized structure was found to deviate drastically at the metal binding loop region. Affinity of different sugars like N-acetyl galactosamine (GalNAc), D-galactose and lactose towards the native and demetallized protein was calculated by molecular docking studies. It was found that the sugar binding site got severely distorted in demetallized lectin. Consequently, sugar binding ability of lectin might be decreasing in the demetallized condition. Isothermal titration calorimetric (ITC) analysis of the sugars in the presence of native and demetallized protein confirmed the in silico results. It was observed after molecular dynamics simulations, that significant structural deviations were not caused in the quaternary structure of demetallized lectin. It was confirmed that the structural changes modified the sugar binding ability, as well as sugar specificity of the present lectin. The role of metal ions in sugar binding is described based on the in silico studies and ITC analysis. A comprehensive analysis of the ITC data suggests that the sugar specificity of the metal bound lectin and the loss of sugar specificity due to metal chelation are not linear.

Selectivity of Ni(II) and Zn(II) binding to Sporosarcina pasteurii UreE, a metallochaperone in the urease assembly: a calorimetric and crystallographic study

Urease is a nickel-dependent enzyme that plays a critical role in the biogeochemical nitrogen cycle by catalyzing the hydrolysis of urea to ammonia and carbamate. This enzyme, initially synthesized in the apo form, needs to be activated by incorporation of two nickel ions into the active site, a process driven by the dimeric metallochaperone UreE. Previous studies reported that this protein can bind different metal ions in vitro, beside the cognate Ni(II). This study explores the metal selectivity and affinity of UreE from Sporosarcina pasteurii (Sp, formerly known as Bacillus pasteurii) for cognate [Ni(II)] and noncognate [Zn(II)] metal ions. In particular, the thermodynamic parameters of SpUreE Ni(II) and Zn(II) binding have been determined using isothermal titration calorimetry. These experiments show that two Ni(II) ions bind to the protein dimer with positive cooperativity. The high-affinity site involves the conserved solvent-exposed His¹⁰⁰ and the C-terminal His¹⁴⁵, whereas the low-affinity site comprises also the C-terminal His¹⁴⁷. Zn(II) binding to the protein, occurring in the same protein regions and with similar affinity as compared to Ni(II), causes metal-driven dimerization of the protein dimer. The crystal structure of the protein obtained in the presence of equimolar amounts of both metal ions indicates that the high-affinity metal binding site binds Ni(II) preferentially over Zn(II). The ability of the protein to select Ni(II) over Zn(II) was confirmed by competition experiments in solution as well as by analysis of X-ray anomalous dispersion data. Overall, the thermodynamics and structural parameters that modulate the metal ion specificity of the different binding sites on the protein surface of SpUreE have been established.     

Competitive binding of anticancer drugs 5-fluorouracil and cyclophosphamide with serum albumin: Calorimetric insights

BackgroundIsothermal titration calorimetry (ITC) has emerged as an excellent method to characterize drug–protein interactions. 5-Fluorouracil and cyclophosphamide have been used in combination for the treatment of breast carcinoma, though individually these drugs have also been useful in treating other types of cancer. A quantitative understanding of binding of these drugs with the transport protein under different conditions is essential for optimizing recognition by the protein and delivery at the target.MethodsThe values of binding constant, enthalpy, and entropy of binding have been determined by using ITC. Fluorescence and circular dichroism spectroscopies have been used to obtain further support to calorimetric observations, monitor conformational changes in the protein and establishing stoichiometry of association.ResultsThe thermodynamic parameters have enabled a quantitative understanding of the affinity of 5-fluorouracil and cyclophosphamide with bovine serum albumin. The nature of binding has been unraveled based on effect of ionic strength, tetrabutyl-ammonium bromide, and sucrose which interfere in ionic, hydrophobic, and hydrogen bonding interactions. The binding site has been identified by using site marker warfarin in combination with 5-fluorouracil and cyclophosphamide. Further, the experiments have been done to establish whether both the drugs share the same binding site, and the effect of antibiotic drug carbenecillin and anti-inflammatory drug naproxen on their association.General significanceTuning optimum association of drugs with the transport vehicles for effective drug delivery requires identification of the nature of interacting groups in terms of energetics of interactions. Such studies employing ITC have direct significance in rational drug design. This article is part of a Special Issue entitled Microcalorimetry in the BioSciences — Principles and Applications, edited by Fadi Bou-Abdallah.     

Cadmium(II) sorption from water samples by powdered marble wastes

Abstract

A series of batch adsorption experiments were carried out, with the aim of removing cadmium ions from aqueous solutions and water samples using powdered marble wastes (PMW) as an effective inorganic sorbent. PMW is inexpensive, widespread, and may be considered as environmental problem. The main parameters (i.e. solution pH, sorbent and cadmium concentrations, stirring time, and temperature) influencing the sorption process were investigated. The results obtained for sorption of cadmium ions onto PMW are well described by the Freundlich and Langmuir models. The Dubinin-Radushkevick (D–R) isotherm model was applied to describe the nature of the adsorption of the metal ion; it was found that the adsorption process was chemical in nature. The thermodynamic parameters were also calculated from the Gibbs free energy change (ΔG°), enthalpy (AH°) and entropy (ΔS°). These parameters indicated that the adsorption process of cadmium(II) ions on PMW was spontaneous and endothermic in nature. Under the optimum experimental conditions employed the removal of ca ~100% of Cd²⁺ ions was attained. The procedure was successfully applied to removal of the cadmium ions from aqueous and various natural water samples. The adsorption mechanism is discussed.