Binding Heat Capacity Change Research Articles

Calculation of Heat Capacity Changes in Enzyme Catalysis and Ligand Binding.

It has been suggested that heat capacity changes in enzyme catalysis may be the underlying reason for temperature optima that are not related to unfolding of the enzyme. If this were to be a common phenomenon, it would have major implications for our interpretation of enzyme kinetics. In most cases, the support for the possible existence of a nonzero (negative) activation heat capacity, however, only relies on fitting such a kinetic model to experimental data. It is therefore of fundamental interest to try to use computer simulations to address this issue. One way is simply to calculate the temperature dependence of the activation free energy and determine whether the relationship is linear or not. An alternative approach is to calculate the absolute heat capacities of the reactant and transition states from plain molecular dynamics simulations using either the temperature derivative or fluctuation formula for the enthalpy. Here, we examine these different approaches for a designer enzyme with a temperature optimum that is not caused by unfolding. Benchmark calculations for the heat capacity of liquid water are first carried out using different thermostats. It is shown that the derivative formula for the heat capacity is generally the most robust and insensitive to the thermostat used and its parameters. The enzyme calculations using this method give results in agreement with direct calculations of activation free energies and show no sign of a negative activation heat capacity. We also provide a simple scheme for the calculation of binding heat capacity changes, which is of clear interest in ligand design, and demonstrate it for substrate binding to the designer enzyme. Neither in that case do the simulations predict any negative heat capacity change.

The interplay of protein-ligand and water-mediated interactions shape affinity and selectivity in the LAO binding protein.

The study of binding thermodynamics is essential to understand how affinity and selectivity are acquired in molecular complexes. Periplasmic binding proteins (PBPs) are macromolecules of biotechnological interest that bind a broad number of ligands and have been used to design biosensors. The lysine-arginine-ornithine binding protein (LAO) is a PBP of 238 residues that binds the basic amino acids l-arginine and l-histidine with nm and μm affinity, respectively. It has been shown that the affinity difference for arginine and histidine binding is caused by enthalpy, this correlates with the higher number of protein-ligand contacts formed with arginine. In order to elucidate the structural bases that determine binding affinity and selectivity in LAO, the contribution of protein-ligand contacts to binding energetics was assessed. To this end, an alanine scanning of the LAO-binding site residues was performed and arginine and histidine binding were characterized by isothermal titration calorimetry and X-ray crystallography. Although unexpected enthalpy and entropy changes were observed in some mutants, thermodynamic data correlated with structural information, especially, the binding heat capacity change. We found that selectivity is conferred by several residues rather than exclusive arginine-protein interactions. Furthermore, crystallographic structures revealed that protein-ligand contributions to binding thermodynamics are highly influenced by the solvent. Finally, we found a similar backbone conformation in all the closed structures obtained, but different structures in the open state, suggesting that the binding site residues of LAO play an important role in stabilizing not only the holo conformation, but also the apo state. DATABASE: Structural data are available in the Protein Data Bank database under the accession numbers 6MLE, 6MLN, 6MLG, 6MKX, 6MLI, 6MLA, 6MKU, 6MKW, 6ML0, 6MLD, 6MLV, 6MLO, 6MLP, 6ML9, 6MLJ.

Kinetic and Thermodynamic Analyses of Interaction between a High-Affinity RNA Aptamer and Its Target Protein.

AML1 (RUNX1) protein is an essential transcription factor involved in the development of hematopoietic cells. Several genetic aberrations that disrupt the function of AML1 have been frequently observed in human leukemia. AML1 contains a DNA-binding domain known as the Runt domain (RD), which recognizes the RD-binding double-stranded DNA element of target genes. In this study, we identified high-affinity RNA aptamers that bind to RD by systematic evolution of ligands by exponential enrichment. The binding assay using surface plasmon resonance indicated that a shortened aptamer retained the ability to bind to RD when 1 M potassium acetate was used. A thermodynamic study using isothermal titration calorimetry (ITC) showed that the aptamer-RD interaction is driven by a large enthalpy change, and its unfavorable entropy change is compensated by a favorable enthalpy change. Furthermore, the binding heat capacity change was identified from the ITC data at various temperatures. The aptamer binding showed a large negative heat capacity change, which suggests that a large apolar surface is buried upon such binding. Thus, we proposed that the aptamer binds to RD with long-range electrostatic force in the early stage of the association and then changes its conformation and recognizes a large surface area of RD. These findings about the biophysics of aptamer binding should be useful for understanding the mechanism of RNA-protein interaction and optimizing and modifying RNA aptamers.

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.

The Role of Cys-298 in Aldose Reductase Function

Diabetic tissues are enriched in an "activated" form of human aldose reductase (hAR), a NADPH-dependent oxidoreductase involved in sugar metabolism. Activated hAR has reduced sensitivity to potential anti-diabetes drugs. The C298S mutant of hAR reproduces many characteristics of activated hAR, although it differs from wild-type hAR only by the replacement of a single sulfur atom with oxygen. Isothermal titration calorimetry measurements revealed that the binding constant of NADPH to the C298S mutant is decreased by a factor of two, whereas that of NADP(+) remains the same. Similarly, the heat capacity change for the binding of NADPH to the C298S mutant is twice increased; however, there is almost no difference in the heat capacity change for binding of the NADP(+) to the C298S. X-ray crystal structures of wild-type and C298S hAR reveal that the side chain of residue 298 forms a gate to the nicotinamide pocket and is more flexible for cysteine compared with serine. Unlike Cys-298, Ser-298 forms a hydrogen bond with Tyr-209 across the nicotinamide ring, which inhibits movements of the nicotinamide. We hypothesize that the increased polarity of the oxidized nicotinamide weakens the hydrogen bond potentially formed by Ser-298, thus, accounting for the relatively smaller effect of the mutation on NADP(+) binding. The effects of the mutant on catalytic rate constants and binding constants for various substrates are the same as for activated hAR. It is, thus, further substantiated that activated hAR arises from oxidative modification of Cys-298, a residue near the nicotinamide binding pocket.

Binding dynamics and energetic insight into the molecular forces driving nucleotide binding by guanylate kinase

Plasmodium deoxyguanylate pathways are an attractive area of investigation for future metabolic and drug discovery studies due to their unique substrate specificities. We investigated the energetic contribution to guanylate kinase substrate binding and the forces underlying ligand recognition. In the range from 20 to 35°C, the thermodynamic profiles displayed marked decrease in binding enthalpy, while the free energy of binding showed little changes. GMP produced a large binding heat capacity change of -356 cal mol(-1) K(-1), indicating considerable conformational changes upon ligand binding. Interestingly, the calculated ΔCp was -32 cal mol(-1) K(-1), indicating that the accessible surface area is not the central change in substrate binding, and that other entropic forces, including conformational changes, are more predominant. The thermodynamic signature for GMP is inconsistent with rigid-body association, while dGMP showed more or less rigid-body association. These binding profiles explain the poor catalytic efficiency and low affinity for dGMP compared with GMP. At low temperature, the ligands bind to the receptor site under the effect of hydrophobic forces. Interestingly, by increasing the temperature, the entropic forces gradually vanish and proceed to a nonfavorable contribution, and the interaction occurs mainly through bonding, electrostatic forces, and van der Waals interactions.

Structural Insight into the Kinetics and ΔCp of Interactions between TEM-1 β-Lactamase and β-Lactamase Inhibitory Protein (BLIP)

In a previous study, we examined thermodynamic parameters for 20 alanine mutants in beta-lactamase inhibitory protein (BLIP) for binding to TEM-1 beta-lactamase. Here we have determined the structures of two thermodynamically distinctive complexes of BLIP mutants with TEM-1 beta-lactamase. The complex BLIP Y51A-TEM-1 is a tight binding complex with the most negative binding heat capacity change (DeltaG = approximately -13 kcal mol(-1) and DeltaCp = approximately -0.8 kcal mol(-1) K(-1)) among all of the mutants, whereas BLIP W150A-TEM-1 is a weak complex with one of the least negative binding heat capacity changes (DeltaG = approximately -8.5 kcal mol(-1) and DeltaCp = approximately -0.27 kcal mol(-1) K(-1)). We previously determined that BLIP Tyr51 is a canonical and Trp150 an anti-canonical TEM-1-contact residue, where canonical refers to the alanine substitution resulting in a matched change in the hydrophobicity of binding free energy. Structure determination indicates a rearrangement of the interactions between Asp49 of the W150A BLIP mutant and the catalytic pocket of TEM-1. The Asp49 of W150A moves more than 4 angstroms to form two new hydrogen bonds while losing four original hydrogen bonds. This explains the anti-canonical nature of the Trp150 to alanine substitution, and also reveals a strong long distance coupling between Trp150 and Asp49 of BLIP, because these two residues are more than 25 angstroms apart. Kinetic measurements indicate that the mutations influence the dissociation rate but not the association rate. Further analysis of the structures indicates that an increased number of interface-trapped water molecules correlate with poor interface packing in a mutant. It appears that the increase of interface-trapped water molecules is inversely correlated with negative binding heat capacity changes.

Energetic contributions to the initiation of transcription in E. coli

The thermodynamics of RNA polymerase (RNAP) binding to a 108 base pair (bp) synthetic promoter with consensus sequences at the − 35 and − 10 bp binding regions upstream from the transcription start point were determined using isothermal titration calorimetry (ITC). The binding constant at 25 °C is 2.37 ± 0.18 × 10 7 M − 1 , which is reduced to 0.17 ± 0.06 × 10 7 M − 1 with mutations in the − 10 bp region but remained the same with mutations in the − 35 binding region. The binding reactions were enthalpically-driven with exothermic binding enthalpies ranging from − 57 ± 6 kJ mol − 1 at 15 °C to − 271 ± 20 kJ mol − 1 at 35 °C yielding a large binding heat capacity change of − 10.7 ± 1.9 kJ mol − 1 K − 1 , indicating a conformational change upon binding to the RNAP. Differential scanning calorimetry (DSC) scans of the thermal unfolding of RNAP and the promoter–RNAP complex exhibited an unfolding transition at 55.5 ± 0.6 °C and at 58.9 ± 0.5 °C for the RNAP but only one transition at 60.5 ± 1.1 °C for the complex with van't Hoff enthalpy to transition enthalpy ratios of, resp., 3.2 ± 0.3 and 4.3 ± 0.5. The single transition of the complex results from a shift to 60.5 °C of the low temperature transition upon promoter binding to the structural unit unfolding at the lower temperature in RNAP. The large transition enthalpy ratios indicate that the σ, α, α, β, and β′ subunits unfold as almost independent entities. The dissociation thermodynamics of short transcription “bubble” duplexes of 7 promoters sequenced from − 1 to − 12 bp were determined from ITC and DSC measurements. The free energy change of the promoter binding to the RNAP and the free energy requirement for formation of the transcription bubble at the low promoter concentrations in the cell are sufficient to drive the initiation of transcription through the isomerization of the closed to the open form step of the RNAP–promoter complex.

Thermodynamics of T‐cell receptor–peptide/MHC interactions: progress and opportunities

alphabeta T-cell receptors (TCRs) recognize peptide antigens presented by class I or class II major histocompatibility complex molecules (pMHC). Here we review the use of thermodynamic measurements in the study of TCR-pMHC interactions, with attention to the diversity in binding thermodynamics and how this is related to the variation in TCR-pMHC interfaces. We show that there is no enthalpic or entropic signature for TCR binding; rather, enthalpy and entropy changes vary in a compensatory manner that reflects a narrow free energy window for the interactions that have been characterized. Binding enthalpy and entropy changes do not correlate with structural features such as buried surface area or the number of hydrogen bonds within TCR-pMHC interfaces, possibly reflecting the myriad of contributors to binding thermodynamics, but likely also reflecting a reliance on van't Hoff over calorimetric measurements and the unaccounted influence of equilibria linked to binding. TCR-pMHC binding heat capacity changes likewise vary considerably. In some cases, the heat capacity changes are consistent with conformational differences between bound and free receptors, but there is little data indicating these conformational differences represent the need to organize disordered CDR loops. In this regard, we discuss how thermodynamics may provide additional insight into conformational changes occurring upon TCR binding. Finally, we highlight opportunities for the further use of thermodynamic measurements in the study of TCR-pMHC interactions, not only for understanding TCR binding in general, but also for understanding specifics of individual interactions and the engineering of TCRs with desired molecular recognition properties.

Thermodynamic Investigation of the Role of Contact Residues of β-Lactamase-inhibitory Protein for Binding to TEM-1 β-Lactamase

We have determined the thermodynamics of binding for the interaction between TEM-1 beta-lactamase and a set of alanine substituted contact residue mutants ofbeta-lactamase-inhibitory protein (BLIP) using isothermal titration calorimetry. The binding enthalpies for these interactions are highly temperature dependent, with negative binding heat capacity changes ranging from -800 to -271 cal mol(-1) K(-1). The isoenthalpic temperatures (at which the binding enthalpy is zero) of these interactions range from 5 to 38 degrees C. The changes in isoenthalpic temperature were used as an indicator of the changes in enthalpy and entropy driving forces, which in turn are related to hydrophobic and hydrophilic interactions. A contact residue of BLIP is categorized as a canonical residue if its alanine substitution mutant exhibits a change of isoenthalpic temperature matching the change of hydrophobicity because of the mutation. A contact position exhibiting a change in isoenthalpic temperature that does not match the change in hydrophobicity is categorized as an anti-canonical residue. Our experimental results reveal that the majority of residues where alanine substitution results in a loss of affinity are canonical (7 of 10), and about half of the residues where alanine substitutions have a minor effect are canonical. The interactions between TEM-1beta-lactamase and BLIP canonical contact residues contribute directly to binding free energy, suggesting potential anchoring sites for binding partners. The anti-canonical behavior of certain residues may be the result of mutation-induced modifications such as structural rearrangements affecting contact residue configurations. Structural inspection of BLIP suggests that the Lys(74) side chain electrostatically holds BLIP loop 2 in position to bind to TEM-1 beta-lactamase, explaining a large loss of entropy-driven binding energy of the K74A mutant and the resulting anti-canonical behavior. The anti-canonical behavior of the W150A mutant may also be due to structural rearrangements. Finally, the affinity enhancing effect of the contact residue mutant Y50A may be due to energetic coupling interactions between Asp(49) and His(41).

Structural Reorganization and the Cooperative Binding of Single-stranded Telomere DNA in Sterkiella nova

In Sterkiella nova, alpha and beta telomere proteins bind cooperatively with single-stranded DNA to form a ternary alpha.beta.DNA complex. Association of telomere protein subunits is DNA-dependent, and alpha-beta association enhances DNA affinity. To further understand the molecular basis for binding cooperativity, we characterized several possible stepwise assembly pathways using isothermal titration calorimetry. In one path, alpha and DNA first form a stable alpha.DNA complex followed by the addition of beta in a second step. Binding energy accumulates with nearly equal free energy of association for each of these steps. Heat capacity is nonetheless dramatically different, with DeltaCp = -305 +/- 3 cal mol(-1) K(-1) for alpha binding with DNA and DeltaCp = -2010 +/- 20 cal mol(-1) K(-1) for the addition of beta to complete the alpha.beta.DNA complex. By examining alternate routes including titration of single-stranded DNA with a preformed alpha.beta complex, a significant portion of binding energy and heat capacity could be assigned to structural reorganization involving protein-protein interactions and repositioning of the DNA. Structural reorganization probably affords a mechanism to regulate high affinity binding of telomere single-stranded DNA with important implications for telomere biology. Regulation of telomere complex dissociation is thought to involve post-translational modifications in the lysine-rich C-terminal portion of beta. We observed no difference in binding energetics or crystal structure when comparing complexes prepared with full-length beta or a C-terminally truncated form, supporting interesting parallels between the intrinsically disordered regions of histones and this portion of beta.

Energetics of Interaction between the G-protein Chaperone, MeaB, and B12-dependent Methylmalonyl-CoA Mutase

MeaB is an auxiliary protein that supports the function of the radical B(12)-dependent enzyme, methylmalonyl-CoA mutase, although its precise role is not understood. Mutations in the human homolog of MeaB, MMAA, lead to methylmalonic aciduria, an inborn error of metabolism that can be fatal. To obtain insights into the function of this recently discovered protein, we have characterized the entropic and enthalpic contributions to DeltaGdegree (assoc) for complexation of MeaB (in the presence and absence of nucleotides) with methylmalonyl-CoA mutase (in the presence and absence of cofactor). The dissociation constant for binding of methylmalonyl-CoA mutase and MeaB ranges from 34 +/- 4 to 524 +/- 66 nm, depending on the combination of nucleotide and mutase form. Holomutase binds MeaB 15-fold more tightly when the nonhydrolyzable GTP analog, GMPPNP, is bound versus GDP. In contrast, the apomutase binds MeaB with similar affinity in the presence of either nucleotide. Our studies reveal that a large structural rearrangement accompanies interaction between these proteins and buries between approximately 4000 and 8600A(2) of surface area, depending on the combination of ligands in the active sites of the two proteins. Furthermore, we demonstrate that MeaB binds GTP and GDP with similar affinity (K(d) of 7.3 +/- 1.9 and 6.2 +/- 0.7 microm, respectively at 20 degrees C) and has low intrinsic GTPase activity (approximately 0.04 min(-1) at 37 degrees C), which is stimulated approximately 100-fold by methylmalonyl-CoA mutase. These studies provide insights into the energetics of interaction between the radical enzyme methylmalonyl-CoA mutase and MeaB, which are discussed.

Thermodynamics of Interaction of the Fusion-Inhibiting Peptide Z-D-Phe-L-Phe-Gly with Dioleoylphosphatidylcholine Vesicles: Direct Calorimetric Determination

The binding of the fusion-inhibiting peptide Z-D-Phe-L-Phe-Gly to unilamellar lipid vesicles of dioleoylphosphatidylcholine (DOPC) was investigated by isothermal titration calorimetry (ITC). The peptide Z-D-Phe-L-Phe-Gly is known to inhibit fusion of myxo- and paramyxoviruses with cells as well as cell-cell and vesicle-vesicle fusion in model systems. Calorimetric titrations conducted over a range of temperatures permitted characterization of the thermodynamics of the interaction of Z-D-Phe-L-Phe-Gly with model DOPC lipid membranes. Simultaneous global analysis of 15 ITC binding curves acquired at four different temperatures allowed determination of the equilibrium site association constant (K), stoichiometry of binding (n), binding enthalpy change (delta H), and heat capacity change of binding (delta Cp) in a single set of experiments. The binding affinity and enthalpy change per mole of DOPC bound at 25 degrees C was log K = 2.463 +/- 0.075 and delta H = -1.07 +/- 0.12 kcal/mol DOPC while the binding heat capacity change per mole of DOPC bound was delta Cp = -20.3 +/- 2.8 cal/(K.mol DOPC) with a temperature dependence (from 10-45 degrees C) of d(delta Cp)/dT = 0.37 +/- 0.18 cal/(K2.mol DOPC). A temperature-independent binding stoichiometry was determined to be n = 5.56 +/- 0.33 DOPC molecules per Z-D-Phe-L-Phe-Gly. A comparison of these results with previous peptide-lipid binding studies is discussed as is their relevance to a current model of the interaction of fusion-inhibiting peptides with phospholipid membranes.