Higher throughput calorimetry: opportunities, approaches and challenges

Higher throughput thermodynamic measurements can provide value in structure-based drug discovery during fragment screening, hit validation, and lead optimization. Enthalpy can be used to detect and characterize ligand binding, and changes that affect the interaction of protein and ligand can sometimes be detected more readily from changes in the enthalpy of binding than from the corresponding free-energy changes or from protein-ligand structures. Newer, higher throughput calorimeters are being incorporated into the drug discovery process. Improvements in titration calorimeters come from extensions of a mature technology and face limitations in scaling. Conversely, array calorimetry, an emerging technology, shows promise for substantial improvements in throughput and material utilization, but improved sensitivity is needed.     

Thermodynamic studies on the interaction between sodium n-dodecyl sulphate and histone H2B

The thermodynamic parameters for the interaction of the anionic detergent sodium n-dodecyl sulphate (SDS) with H2B at pH 3.2, 6.4 and 10 have been measured at 27 degrees C and 37 degrees C by equilibrium dialysis to determine the Gibbs energies of detergent binding. The data have been used to obtain the enthalpy of interaction from the temperature dependence of the equilibrium constants from the Van't Hoff relation. The enthalpy of interaction between H2B and SDS is endothermic at pH 3.2, 6.4 and 10. The shapes of the enthalpy curves at pH 3.2 and 10 show some small exothermic contribution which probably indicates folding of H2B. The interactions of H2B-SDS are dominated by the increase in entropy on detergent binding. The larger negative free energy, enthalpy and entropy changes at pH 6.4 are consistent with greater denaturation relative to pH 3.2 and 10.     

Interaction between ricin hemagglutinin and its ligands, galactose and lactose. Microcalorimetry and equilibrium dialysis]

The interaction of Ricinus communis hemagglutinin with galactose and lactose has been studied by means of microcalorimetry, equilibrium dialysis and analytical ultracentrifugation. A first class of beta-galactoside-binding sites involves two similar and independent sites of which affinity constants are 2600 M-1 for galactose and 26700 M-1 for lactose at 25 degrees C. The binding of one galactose or one lactose molecule leads to enthalpy changes of--12.3 Kcal and--11 Kcal, respectively. Considering the negative entropy changes of the association, and as for ricin, the binding of galactosides with hemagglutinin is driven by favorable enthalpic contributions. In presence of high lactose concentrations, a second endothermic step of the calorimetric titration curve was observed. This result and the biphasic nature of Scatchard plots of equilibrium dialysis suggest the existence of a second class of binding sites on the lectin molecule. As for ricin, the interaction between these secondary sites and lactose would be entropically driven.     

Fatty acid interactions with native and mutant fatty acid binding proteins

The interactions of long chain fatty acids (FA) with wild type (WT) fatty acid binding proteins (FABP) and engineered FABP mutants have been monitored to determine the equilibrium binding constants as well as the rate constants for binding and dissociation. These measurements have been done using the fluorescent probes, ADIFAB and ADIFAB2, that allow the determination of the free fatty acid (FFA) concentration in the reaction of FA with proteins and membranes. The results of these studies indicate that for WT proteins from adipocyte, heart, intestine, and liver, Kd values are in the nM range and affinities decrease with increasing aqueous solubility of the FA. Binding affinities for heart and liver are generally greater than those for adipocyte and intestine. Moreover, measurements of the rate constants indicate that binding equilibrium at 37øC is achieved within seconds for all FA and FABPs. These results, together with the level of serum (unbound) FFA, suggests a buffering action of FABPs that helps to maintain the intracellular concentration of FFA so that the flux of FFA between serum and cells occurs down a concentration gradient. Measurements of the temperature dependence of binding reveal that the free energy is predominately enthalpic and that the enthalpy of the reaction results from FA-FABP interactions within the binding cavity. The nature of these interactions were investigated by determining the thermodynamics of binding to engineered point mutants of the intestinal FABP. These measurements showed that binding affinities did not report accurately the changes in protein-FA interactions because changes in the binding entropy and enthalpy tend to compensate. For example, an alanine substitution for arginine 106 yields a 30 fold increase in binding affinity, because the loss in enthalpy due to the elimination of the favorable interaction between the FA carboxylate and Arg106, is more than compensated for by an increase in entropy. Thus understanding the effects of amino acid replacements on FA-FABP interactions requires measurements of enthalpy and entropy, in addition to affinity.     

Interaction between yeast eukaryotic initiation factor eIF4E and mRNA 5' cap analogues differs from that for murine eIF4E

Measurements of interaction of 7-methyl-GTP eIF4E from S. cerevisiae were performed by means of two methods: Isothermal Titration Calorimetry (ITC) and fluorescence titration. The equilibrium association constants (Kas) derived from the two methods show significantly different affinity of yeast eIF4E for the mRNA 5' cap than those of the murine and human proteins. The observed differences in the Kas values and the enthalpy changes of the association (deltaH(o)) suggest some dissimilarity in the mode of binding and stabilization of cap in the complexes with eIF4E from various sources.     

Direct comparison of binding equilibrium, thermodynamic, and rate constants determined by surface- and solution-based biophysical methods

The binding interactions of small molecules with carbonic anhydrase II were used as model systems to compare the reaction constants determined from surface- and solution-based biophysical methods. Interaction data were collected for two arylsulfonamide compounds, 4-carboxybenzenesulfonamide (CBS) and 5-dimethyl-amino-1-naphthalene-sulfonamide (DNSA), binding to the enzyme using surface plasmon resonance, isothermal titration calorimetry, and stopped-flow fluorescence. We demonstrate that when the surface plasmon resonance biosensor experiments are performed with care, the equilibrium, thermodynamic, and kinetic constants determined from this surface-based technique match those acquired in solution. These results validate the use of biosensor technology to collect reliable data on small molecules binding to immobilized macromolecular targets. Binding kinetics were shown to provide more detailed information about complex formation than equilibrium constants alone. For example, although carbonic anhydrase II bound DNSA with twofold higher affinity than CBS, kinetic analysis revealed that CBS had a fourfold slower dissociation rate. Analysis of the binding and transition state thermodynamics also revealed significant differences in the enthalpy and entropy of complex formation. The lack of labeling requirements, high information content, and high throughput of surface plasmon resonance biosensors will make this technology an important tool for characterizing the interactions of small molecules with enzymes and receptors.     

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.     

Interactions in solution of cobalt(II) and nickel(II) with nicotinamide adenine dinucleotide: a potentiometric and calorimetric study

The equilibrium constants and the thermodynamic parameters enthalpy and entropy of the interaction between Ni(II) and Co(II) with NAD(+) in aqueous solution were determined by calorimetry and potentiometry methods (ionic strength adjusted to 0.1 with sodium nitrate at 25 degrees C). The macrochelation of the systems was also studied. All the data, including the protonation enthalpy data of NAD(+) (very similar to the protonation enthalpy of 5'-AMP) suggest a less restrictive model for the NAD(+) "folded" conformation without intramolecular stacking between the bases, in agreement both with recent theoretical calculations and with the X-ray structure of trimethylene-bisadeninium or the free acid form of NAD(+).     

Quantitative analysis of drug-receptor interactions: I. Determination of kinetic and equilibrium properties

It has recently become possible to characterize a variety of different receptors by studying the binding of appropriate drugs labelled with ³H or ¹²⁵I. The goal of this review is to describe the basic mathematical analyses that should be used to characterize a particular receptor in terms of its interactions with ligands. Methods for direct determination of kinetic and equilibrium constants of simple bimolecular drug-receptor interactions are described as is the use of these measurements to verify the existence of a simple second order reaction. Some of the causes of deviations from second order behavior which imply more complex interactions are also discussed. $\text{In vitro}$ studies of radioligand binding provide a means of indirectly determining equilibrium dissociation constants of unlabelled drugs. The appropriate equations for these determinations are presented and the assumptions underlying these calculations are identified. Analysis of the temperature dependence of kinetic and equilibrium constants allows determination of the energetics of binding and methods are presented for calculation of the changes in Gibbs free energy, enthalpy and entropy that are associated with the binding of ligands to receptors. Studies of the interactions of agonists and antagonists with β-adrenergic receptors are presented as examples of the various types of calculations.     

Mapping heat exchange in an allosteric protein

Nicotinic acetylcholine receptors (AChRs) are synaptic ion channels that spontaneously isomerize (i.e., gate) between resting and active conformations. We used single-molecule electrophysiology to measure the temperature dependencies of mouse neuromuscular AChR gating rate and equilibrium constants. From these we estimated free energy, enthalpy, and entropy changes caused by mutations of amino acids located between the transmitter binding sites and the middle of the membrane domain. The range of equilibrium enthalpy change (13.4 kcal/mol) was larger than for free energy change (5.5 kcal/mol at 25°C). For two residues, the slope of the rate-equilibrium free energy relationship (Φ) was approximately constant with temperature. Mutant cycle analysis showed that both free energies and enthalpies are additive for energetically independent mutations. We hypothesize that changes in energy associated with changes in structure mainly occur close to the site of the mutation, and, hence, that it is possible to make a residue-by-residue map of heat exchange in the AChR gating isomerization. The structural correlates of enthalpy changes are discussed for 12 different mutations in the protein.     

Magnesium ion dependent equilibria, kinetics, and thermodynamic parameters of Artemia ribosome dissociation and subunit association

The influence of magnesium ion concentration on the equilibrium and kinetics of Artemia ribosome dissociation and subunit association has been studied by laser light scattering. Ribosomal aggregation was found to be reduced by addition of 0.1-0.05 mM spermidine and KCl concentrations of 100 mM. The ribosomes were found to be stable at low [Mg2+], and the curves obtained for ribosome-subunit equilibrium were independent of the direction and origin of the magnesium ion titration. Thermodynamic parameters were obtained from the temperature-dependent equilibria and have been compared to those of wheat germ and Escherichia coli type A ribosomes. The entropy term calculated for the association of 40S and 60S subunits is small, and the reaction is exothermic. The entropy term is negative, favoring subunit dissociation, and contributes less to the free energy than the enthalpy term. Rate constants for ribosome dissociation and subunit association have been determined. The reaction curves gave no evidence for sequential processes and were homogeneous.     

Thermodynamic analysis of protein interactions with biosensor technology

A methodology using biosensor technology for combined kinetic and thermodynamic analysis of biomolecular interactions is described. Rate and affinity constants are determined with BIAcore. Thermodynamics parameters, changes in free energy, enthalpy and entropy, are evaluated from equilibrium data and by using rate constants and transition state theory. The methodology using van't Hoff theory gives complementary information to microcalorimetry, since only the direct binding is measured with BIAcore whereas microcalorimetry measures all components, including e.g. hydration effects. Furthermore, BIAcore gives possibilities to gain new information by thermodynamic analysis of the rate constants.     

Interpretation of the temperature dependence of equilibrium and rate constants

The objective of this review is to draw attention to potential pitfalls in attempts to glean mechanistic information from the magnitudes of standard enthalpies and entropies derived from the temperature dependence of equilibrium and rate constants for protein interactions. Problems arise because the minimalist model that suffices to describe the energy differences between initial and final states usually comprises a set of linked equilibria, each of which is characterized by its own energetics. For example, because the overall standard enthalpy is a composite of those individual values, a positive magnitude for DeltaH(o) can still arise despite all reactions within the subset being characterized by negative enthalpy changes: designation of the reaction as being entropy driven is thus equivocal. An experimenter must always bear in mind the fact that any mechanistic interpretation of the magnitudes of thermodynamic parameters refers to the reaction model rather than the experimental system. For the same reason there is little point in subjecting the temperature dependence of rate constants for protein interactions to transition-state analysis. If comparisons with reported values of standard enthalpy and entropy of activation are needed, they are readily calculated from the empirical Arrhenius parameters.     

Interaction Between Yeast Eukaryotic Initiation Factor eIF4E and mRNA 5′ Cap Analogues Differs from That for Murine eIF4E

Abstract

Measurements of interaction of 7-methyl-GTP eIF4E from S. cerevisiae were performed by means of two methods: Isothermal Titration Calorimetry (ITC) and fluorescence titration. The equilibrium association constants (K_as) derived from the two methods show significantly different affinity of yeast eIF4E for the mRNA 5′ cap than those of the murine and human proteins. The observed differences in the K_as values and the enthalpy changes of the association (ΔH°) suggest some dissimilarity in the mode of binding and stabilization of cap in the complexes with eIF4E from various sources.     

Cooperative and thermodynamic parameters for oligoinosinate-polycytidylate complexes

The cooperative nature of the binding between polycytidylate and the oligoinosinates I(pI)_5–10 has been determined. Using the data of Tazawa, Tazawa, and Ts'o, it is shown that knowledge of the slope of the adsorption isothern allows one to determine the oligomer-polymer binidng constant, the oligomer–oligomer interaction constant, and the average degree of association (cooperative clustering) of the oligomers on the polymer. Knowledge of the above equilibrium constants as a function of temperature yields the respective thermodynamic parameters; no assumptions need to be made about the nature of the equilibrium constants or the thermodynamic parameters. For very long chains of polycytidylate, simple, explicit relations are given for the determination of the equilibrium constants involved. For finite chains of polycytidylate, the calculation of a single graph for each oligomer and polymer size allows the equilibrium constants to be determined for all experimental conditions of temperature and concentration. We find that the enthalpy and entropy of binding an oligomer n, bases to be δH_n = ±13.7 ? n(6.65) and δS_n = +32.5 ? n(18.8) given, respectively, in kcal/mole and e.u.; these parameters predict a melting temperature of 81°C for the poly(I)·poly(C) complex compared with the experimental value of 75°C. If the enthalpy is interpreted as arising from a sum of hydrogen bonding and stacking interactions, then the enthalpy of stacking is ?13.7 kcal/mole while the enthalpy of hydrogen bonding is +7 ± 4 kcal/mole; the positive enthalpy of hydrogen bonding presumably is a result of the fact that in the inosine-cytosine base pair, only two of the three sites on cytosine can hydrogen bond, the third being blocked from hydrogen bonding with water. The enthalpy of interaction between neighboring bound oligomers is found to be ?10.4 kcal/mole while the corresponding entropy is ?26.1 e.u. The binding is bound to be cooperative, though the extent of clustering varies markedly with temperature; the average number of oligomers in a cluster on the polymer is found to about five at a melting temperature of 25°C. The approach and equations given have generally applicability to oligomer-polymer associations.     

Structural and physicochemical analysis of the reaction between the anti-lysozyme antibody D1.3 and the anti-idiotopic antibodies E225 and E5.2

The reaction between the mouse (BALB/c) anti-idiotiopic monoclonal antibodies E225 and E5.2 and idiotopes on the (BALB/c) anti-lysozyme monoclonal antibody D1.3 has been characterized by titration calorimetry, by equilibrium sedimentation and by the determination of binding association and dissociation rates. The reaction between E5.2 and D1.3 is driven by a large negative enthalpy and its rate and equilibrium association constants are comparable to those observed in other antigen–antibody reactions. In contrast, the reaction between E225 and D1.3 is entropically driven and characterized by slow association kinetic (1 × 10³ M^?1 sec^?1) and a resulting low equilibrium constant (K_a = 2 × 10⁵M ^?1). A correlation of these properties with the three-dimensional structure of the Fab225-FabD1.3 complex, previously determined by X-ray diffraction methods to 2.5 Å resolution, indicates that conformational changes of several D1.3 contacting residues, located in its complementarity determining regions, may explain these features of the reaction.     

Standard thermodynamic formation properties for the adenosine 5'-triphosphate series.

The criterion for chemical equilibrium at specified temperature, pressure, pH, concentration of free magnesium ion, and ionic strength is the transformed Gibbs energy, which can be calculated from the Gibbs energy. The apparent equilibrium constant (written in terms of the total concentrations of reactants like adenosine 5'-triphosphate, rather than in terms of species) yields the standard transformed Gibbs energy of reaction, and the effect of temperature on the apparent equilibrium constant at specified pressure, pH, concentration of free magnesium ion, and ionic strength yields the standard transformed enthalpy of reaction. From the apparent equilibrium constants and standard transformed enthalpies of reaction that have been measured in the adenosine 5'-triphosphate series and the dissociation constants of the weak acids and magnesium complexes involved, it is possible to calculate standard Gibbs energies of formation and standard enthalpies of formation of the species involved at zero ionic strength. This requires the convention that the standard Gibbs energy of formation and standard enthalpy of formation for adenosine in dilute aqueous solutions be set equal to zero. On the basis of this convention, standard transformed Gibbs energies of formation and standard transformed enthalpies of formation of adenosine 5'-trisphosphate, adenosine 5'-diphosphate, adenosine 5'-monophosphate, and adenosine at 298.15 K, 1 bar, pH = 7, a concentration of free magnesium ions of 10(-3) M, and an ionic strength of 0.25 M have been calculated.     

Kinetic and thermodynamic study of the tetramerization equilibrium of phosphorylase b

The association equilibrium of phosphorylase b, induced by AMP and in the presence of Mg2+, has been shown to be a reversible process that follows second order and first order reversible rate laws in the direction of tetramerization and dimerization respectively, this fact being independent of temperature and of enzyme and AMP concentrations. Moreover, rate and equilibrium constants have been evaluated and their dependence on temperature and AMP concentration studied in this work. An important role that the existence of two classes of AMP binding sites per enzymatic subunit plays in the aggregation properties of the enzyme has also been emphasized. In the presence of 0.1 and 1 mM AMP (binding to the high affinity site), the values of the change in enthalpy, activation energy of dimerization and activation energy of tetramerization are: -36 kcal/mol, +36 kcal/mol, and 0 kcal/mol respectively. Binding of AMP to the low affinity site (10 mM AMP) yields significant changes in the self-association equilibrium, since the preceding parameters reach the following values: -18, +32, and +14 kcal/mol.     

Temperature-induced switching of the bacterial flagellar motor.

Chemotaxis signaling proteins normally control the direction of rotation of the flagellar motor of Escherichia coli. In their absence, a wild-type motor spins exclusively counterclockwise. Although the signaling pathway is well defined, relatively little is known about switching, the mechanism that enables the motor to change direction. We found that switching occurs in the absence of signaling proteins when cells are cooled to temperatures below about 10 degrees C. The forward rate constant (for counterclockwise to clockwise, CCW to CW, switching) increases and the reverse rate constant (for CW to CCW switching) decreases as the temperature is lowered. At about -2 degrees C, most motors spin exclusively CW. At temperatures for which reversals are frequent enough to generate a sizable data set, both CCW and CW interval distributions appear to be exponential. From the rate constants we computed equilibrium constants and standard free energy changes, and from the temperature dependence of the standard free energy changes we determined standard enthalpy and entropy changes. Using transition-state theory, we also calculated the activation free energy, enthalpy, and entropy. We conclude that the CW state is preferred at very low temperatures and that it is relatively more highly bonded and restricted than the CCW state.