Binding Thermodynamic Parameters Research Articles

β-Lactoglobulin (BLG) binding to highly charged cationic polymer-grafted magnetic nanoparticles: Effect of ionic strength

Poly(2-(methacryloyloxy)ethyltrimethyl ammonium chloride) (PMATAC) modified magnetic nanoparticles (NPs) with a high zeta potential of ca. 50mV were synthesized by atom transfer radical polymerization (ATRP). The prepared NPs consist of a magnetic core around 13nm and a PMATAC shell around 20nm attached on the surface of magnetic nanoparticles. Thermodynamic binding parameters between β-lactoglobulin and these polycationic NPs were investigated at different ionic strengths by high-resolution turbidimetry, dynamic light scattering (DLS), and isothermal titration calorimetry (ITC). Both turbidity and ITC show that binding affinities for BLG display a non-monotonic ionic strength dependence trend and a maximum appears at ionic strength of 50mM. Such observation should arise from the coeffects of protein charge anisotropy visualized by DelPhi electrostatic modeling and the strong electrostatic repulsion among highly charged NPs at a variety of ionic strengths.

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.

Structural plasticity in Mycobacterium tuberculosis uracil-DNA glycosylase (MtUng) and its functional implications.

17 independent crystal structures of family I uracil-DNA glycosylase from Mycobacterium tuberculosis (MtUng) and its complexes with uracil and its derivatives, distributed among five distinct crystal forms, have been determined. Thermodynamic parameters of binding in the complexes have been measured using isothermal titration calorimetry. The two-domain protein exhibits open and closed conformations, suggesting that the closure of the domain on DNA binding involves conformational selection. Segmental mobility in the enzyme molecule is confined to a 32-residue stretch which plays a major role in DNA binding. Uracil and its derivatives can bind to the protein in two possible orientations. Only one of them is possible when there is a bulky substituent at the 5' position. The crystal structures of the complexes provide a reasonable rationale for the observed thermodynamic parameters. In addition to providing fresh insights into the structure, plasticity and interactions of the protein molecule, the results of the present investigation provide a platform for structure-based inhibitor design.

A Universal Stress Protein (USP) in Mycobacteria Binds cAMP

Mycobacteria are endowed with rich and diverse machinery for the synthesis, utilization, and degradation of cAMP. The actions of cyclic nucleotides are generally mediated by binding of cAMP to conserved and well characterized cyclic nucleotide binding domains or structurally distinct cGMP-specific and -regulated cyclic nucleotide phosphodiesterase, adenylyl cyclase, and E. coli transcription factor FhlA (GAF) domain-containing proteins. Proteins with cyclic nucleotide binding and GAF domains can be identified in the genome of mycobacterial species, and some of them have been characterized. Here, we show that a significant fraction of intracellular cAMP is bound to protein in mycobacterial species, and by using affinity chromatography techniques, we identify specific universal stress proteins (USP) as abundantly expressed cAMP-binding proteins in slow growing as well as fast growing mycobacteria. We have characterized the biochemical and thermodynamic parameters for binding of cAMP, and we show that these USPs bind cAMP with a higher affinity than ATP, an established ligand for other USPs. We determined the structure of the USP MSMEG_3811 bound to cAMP, and we confirmed through structure-guided mutagenesis, the residues important for cAMP binding. This family of USPs is conserved in all mycobacteria, and we suggest that they serve as "sinks" for cAMP, making this second messenger available for downstream effectors as and when ATP levels are altered in the cell.

Real-time monitoring of binding events on a thermostabilized human A2A receptor embedded in a lipid bilayer by surface plasmon resonance

Membrane proteins (MPs) are prevalent drug discovery targets involved in many cell processes. Despite their high potential as drug targets, the study of MPs has been hindered by limitations in expression, purification and stabilization in order to acquire thermodynamic and kinetic parameters of small molecules binding. These bottlenecks are grounded on the mandatory use of detergents to isolate and extract MPs from the cell plasma membrane and the coexistence of multiple conformations, which reflects biochemical versatility and intrinsic instability of MPs. In this work ,we set out to define a new strategy to enable surface plasmon resonance (SPR) measurements on a thermostabilized and truncated version of the human adenosine (A2A) G-protein-coupled receptor (GPCR) inserted in a lipid bilayer nanodisc in a label- and detergent-free manner by using a combination of affinity tags and GFP-based fluorescence techniques. We were able to detect and characterize small molecules binding kinetics on a GPCR fully embedded in a lipid environment. By providing a comparison between different binding assays in membranes, nanodiscs and detergent micelles, we show that nanodiscs can be used for small molecule binding studies by SPR to enhance the MP stability and to trigger a more native-like behaviour when compared to kinetics on A2A receptors isolated in detergent. This work provides thus a new methodology in drug discovery to characterize the binding kinetics of small molecule ligands for MPs targets in a lipid environment.

Isothermal titration calorimetry of ion-coupled membrane transporters

Binding of ligands, ranging from proteins to ions, to membrane proteins is associated with absorption or release of heat that can be detected by isothermal titration calorimetry (ITC). Such measurements not only provide binding affinities but also afford direct access to thermodynamic parameters of binding – enthalpy, entropy and heat capacity. These parameters can be interpreted in a structural context, allow discrimination between different binding mechanisms and guide drug design. In this review, we introduce advantages and limitations of ITC as a methodology to study molecular interactions of membrane proteins. We further describe case studies where ITC was used to analyze thermodynamic linkage between ions and substrates in ion-coupled transporters. Similar type of linkage analysis will likely be applicable to a wide range of transporters, channels, and receptors.

Intrinsic thermodynamics of trifluoromethanesulfonamide and ethoxzolamide binding to human carbonic anhydrase VII.

Human carbonic anhydrase (CA) isozyme VII is a cytosolic protein that is highly expressed in the cortex, hippocampus, and thalamus regions within mammalian brain, and expression disorders can cause epilepsy and several cases of malignant brain tumors. Therefore, CA VII is a potential antiepileptic and anticancer drug target. There are numerous sulfonamides that target CAs nonspecifically. It is important to understand the thermodynamics of inhibitor binding and the structural features of the protein-inhibitor complex in order to design specific inhibitors against CA VII. Isothermal titration calorimetry and fluorescent thermal shift assay were used to characterize the intrinsic thermodynamic parameters of trifluoromethanesulfonamide and ethoxzolamide binding to CA VII. Binding experiments were carried out at various pH in different buffers in order to dissect linked protonation of the water molecule bound to the CA VII active site, deprotonation of the sulfonamide group of the inhibitor, and protonation-deprotonation of buffer. Dissection of all those contributions yielded the intrinsic thermodynamic parameters of binding, such as Gibbs free energy, binding enthalpy, entropy, and protein pKa value. Thermal shift assay was also used to determine CA VII stability at various pH.

Intrinsic binding of 4-substituted-2,3,5,6-tetrafluorobenezenesulfonamides to native and recombinant human carbonic anhydrase VI.

Carbonic anhydrase (CA) VI is a potential drug target for cariogenesis and cancer of the salivary gland. It is the only secreted human CA isozyme which is found in saliva and milk. Here, CA VI was expressed in bacterial and mammalian cell cultures and directly affinity-purified from human saliva. The binding of 4-substituted-2,3,5,6-tetrafluorobenezenesulfonamides to the native and recombinant CA VI from these three sources was compared. Interaction between the enzyme and inhibitors was determined by fluorescent thermal shift assay and isothermal titration calorimetry. The observed dissociation constants were the same within the error margin for all three CA VI preparations. The intrinsic binding parameters for the compounds were obtained by determining and dissecting the binding-linked protonation reactions. Intrinsic thermodynamic parameters of binding arrange the compounds in a buffer- and pH-independent manner. Intrinsic binding constants of nonfluorinated compounds were significantly stronger than those of fluorinated benzenesulfonamides. An opposite result was determined for the observed binding constants. The increase in observed affinity of the fluorinated compounds was due to the fluorine effect on diminishing the pKa of the compounds but not due to direct recognition of the protein. The temperature-stability profiles for recombinant and native CA VI were compared and showed that CA VI is more stable in slightly acidic than neutral conditions.

Molecular Determinants and Kinetic Parameters of Ligand Binding to G Protein-Coupled Receptors using Markov State Model Analysis

G protein-coupled receptors (GPCRs) continue to hold leading positions as drug targets. However, many GPCR drug candidates fail in clinical trials because of limited in vivo efficacy. While binding affinity, i.e. the strength of association of a drug to its receptor, has traditionally been viewed as an appropriate surrogate for in vivo efficacy, retrospective analyses of marketed drugs suggest that kinetic parameters, such as the rates at which the drug associates with or dissociates from the target, may play a role that is as important as, or even more important than, binding affinity in determining in vivo efficacy. Thus, predictions of both kinetic and thermodynamic parameters of ligand binding to GPCRs are highly desirable because they may inform the rational discovery of improved therapeutics.The main challenge in studying molecular recognition by a GPCR using molecular dynamics (MD) is that ligand binding and unbinding are rare events on microscopic time-scales, and as such, they are difficult to observe using unbiased simulations. In this work, we present a general strategy that employs biased MD simulations to build Markov State Models (MSMs) of the binding of small molecules to a prototypic family A GPCR. Using Perron cluster analysis and transition path theory we were able to identify the kinetic basins of the binding process, and to characterize both the metastable and the transition states between bound and unbound conformations. By investigating the role of hydrophobic interactions, dewetting, and conformational changes in the binding pocket, we were able to characterize the microscopic determinants that influence association and dissociation rates of the ligand. This information has a direct applicability in rational drug design approaches.

Thermodynamic parameters for binding of some halogenated inhibitors of human protein kinase CK2

The interaction of human CK2α with a series of tetrabromobenzotriazole (TBBt) and tetrabromobenzimidazole (TBBz) analogs, in which one of the bromine atoms proximal to the triazole/imidazole ring is replaced by a methyl group, was studied by biochemical (IC50) and biophysical methods (thermal stability of protein–ligand complex monitored by DSC and fluorescence). Two newly synthesized tri-bromo derivatives display inhibitory activity comparable to that of the reference compounds, TBBt and TBBz, respectively. DSC analysis of the stability of protein–ligand complexes shows that the heat of ligand binding (Hbind) is driven by intermolecular electrostatic interactions involving the triazole/imidazole ring, as indicated by a strong correlation between Hbind and ligand pKa. Screening, based on fluorescence-monitored thermal unfolding of protein–ligand complexes, gave comparable results, clearly identifying ligands that most strongly bind to the protein. Overall results, additionally supported by molecular modeling, confirm that a balance of hydrophobic and electrostatic interactions contribute predominantly, relative to possible intermolecular halogen bonding, in binding of the ligands to the CK2α ATP-binding site.

Probing the binding of thioTEPA to human serum albumin using spectroscopic and molecular simulation approaches

The present work is devoted to probing the molecular interaction of N,N′,N″-triethylenethiophosphoramide (thioTEPA) with human serum albumin (HSA) using UV-visible and fluorescence spectroscopies. Further, molecular dynamics and molecular docking simulations were used to investigate the binding site of thioTEPA. The outcomes of the spectroscopic observations and also the Stern−Volmer and van’t Hoff equations were employed to determine the binding thermodynamic parameters. It was found out that the interaction of thioTEPA with HSA is enthalpy driven through a quenching mechanism that is both static and dynamic at domain I of HSA. No significant changes in the local and overall secondary structure, polarity, and hydrophobicity of HSA were observed. The low values of binding free energies and binding constants are evidence for necessary high dosage of thioTEPA in chemotherapies. These results may be attributed to the spherical geometry and steric hindrance of the free vibrating aziridinyl groups of thioTEPA.

Thermodynamic analysis of F1-ATPase rotary catalysis using high-speed imaging.

F1-ATPase (F1) is a rotary motor protein fueled by ATP hydrolysis. Although the mechanism for coupling rotation and catalysis has been well studied, the molecular details of individual reaction steps remain elusive. In this study, we performed high-speed imaging of F1 rotation at various temperatures using the total internal reflection dark-field (TIRDF) illumination system, which allows resolution of the F1 catalytic reaction into elementary reaction steps with a high temporal resolution of 72 µs. At a high concentration of ATP, F1 rotation comprised distinct 80° and 40° substeps. The 80° substep, which exhibited significant temperature dependence, is triggered by the temperature-sensitive reaction, whereas the 40° substep is triggered by ATP hydrolysis and the release of inorganic phosphate (Pi). Then, we conducted Arrhenius analysis of the reaction rates to obtain the thermodynamic parameters for individual reaction steps, that is, ATP binding, ATP hydrolysis, Pi release, and TS reaction. Although all reaction steps exhibited similar activation free energy values, ΔG(‡) = 53-56 kJ mol(-1), the contributions of the enthalpy (ΔH(‡)), and entropy (ΔS(‡)) terms were significantly different; the reaction steps that induce tight subunit packing, for example, ATP binding and TS reaction, showed high positive values of both ΔH(‡) and ΔS(‡). The results may reflect modulation of the excluded volume as a function of subunit packing tightness at individual reaction steps, leading to a gain or loss in water entropy.

Effect of copper oxide nanoparticles on the conformation and activity of β-galactosidase

The primary objective of this study is to explore the interaction of β-galactosidase with copper oxide nanoparticles (CuO NPs). Steady-state absorption, fluorescence and circular dichroism (CD) spectroscopic techniques have been employed to unveil the conformational changes of β-galactosidase induced by the binding of CuO NPs. Temperature dependent fluorescence quenching results indicates a static quenching mechanism in the present case. The binding thermodynamic parameters delineate the predominant role of H-bonding and van der Waals forces between β-galactosidase and CuO NPs binding process. The binding was studied by isothermal titration calorimetry (ITC) and the result revealed that the complexation is enthalpy driven, the ΔH°<0, ΔS°<0 indicates the formation of hydrogen bonds between β-galactosidase and CuO NPs occurs. Disruption of the native conformation of the protein upon binding with CuO NPs is reflected through a reduced functionality (in terms of hydrolase activity) of the protein CuO NPs conjugate system in comparison to the native protein and CuO NPs exhibited a competitive mode of inhibition. This also supports the general belief that H-bond formation occurs with NPs is associated with a lesser extent of modification in the native structure. Morphological features and size distributions were investigated using transmission electron microscopy (TEM) and dynamic light scattering (DLS). Additionally the considerable increase in the Rh following the addition of CuO NPs accounts for the unfolding of β-galactosidase. Chemical and thermal unfolding of β-galactosidase, when carried out in the presence of CuO NPs, also indicated a small perturbation in the protein structure. These alterations in functional activity of nanoparticle bound β-galactosidase which will have important consequences should be taken into consideration while using nanoparticles for diagnostic and therapeutic purposes.

Probing lectin-mucin interactions by isothermal titration microcalorimetry.

Isothermal titration microcalorimetry (ITC) can directly determine the thermodynamic binding parameters of biological molecules including affinity constant, binding stoichiometry, and heat of binding (enthalpy) and indirectly the entropy and free energy of binding. ITC has been extensively used to study the binding of lectins to mono- and oligosaccharides, but limited applications to lectin-glycoprotein interactions. Inherent experimental challenges to ITC include sample precipitation during the experiment and relative high amount of sample required, but careful design of experiments can minimize these problems and allow valuable information to be obtained. For example, the thermodynamics of binding of lectins to multivalent globular and linear glycoproteins (mucins) have been described. The results are consistent with a dynamic binding mechanism in which lectins bind and jump from carbohydrate to carbohydrate epitope in these molecules leading to increased affinity. Importantly, the mechanism of binding of lectins to mucins appears similar to that for a variety of protein ligands binding to DNA. Recent results also show that high affinity lectin-mucin cross-linking interactions are driven by favorable entropy of binding that is associated with the bind and jump mechanism. The results suggest that the binding of ligands to biopolymers, in general, may involve a common mechanism that involves enhanced entropic effects that facilitate binding interactions.

Kinetic and thermodynamic evaluation of phosphate ions binding onto sevelamer hydrochloride

Sevelamer hydrochloride is the first non-aluminium, non-calcium-based phosphate binder developed for the management of hyperphosphatemia in end stage renal diseases. It is a synthetic ion-exchange polymer which binds and removes phosphate ions due to the high content of cationic charge associated with protonated amine groups on the polymer matrix. This is the first in-depth study investigating phosphate removal in vitro from aqueous solutions using commercially available sevelamer hydrochloride at physiological conditions of phosphate level, pH and temperature. The kinetic and thermodynamic parameters of phosphate binding onto the sevelamer hydrochloride particles were evaluated in order to define the binding process. A series of kinetic studies were carried out in order to delineate the effect of initial phosphate concentration, absorbent dose and temperature on the rate of binding. The results were analysed using three kinetic models with the best-fit of the experimental data obtained using a pseudo-second order model. Thermodynamic parameters provide in-depth information on inherent energetic changes that are associated with binding. Free energy ΔG°, enthalpy ΔH°, and entropy ΔS° changes were calculated in this study in order to assess the relationship of these parameters to polymer morphology. The binding reaction was found to be a spontaneous endothermic process with increasing entropy at the solid–liquid interface.

Micellization of anionic gemini surfactants and their interaction with polyacrylamide

A series of anionic gemini surfactants have been synthesized. The surface properties and micellization process of as-prepared sulfonate gemini surfactants (SGS) and carboxylate gemini surfactant (CGS) have been studied by surface tension measurement and isothermal titration microcalorimetry. Meanwhile, the interaction of these five surfactants with polyacrylamide (PAM) was investigated using surface tension, steady-state fluorescence measurement, and isothermal titration microcalorimetry. The results show that the critical micelle concentrations (CMCs) of above-mentioned surfactants are more than 1 order of magnitude lower than those of corresponding single chain surfactants. Moreover, the enthalpy of micelle formation (ΔH mic) for the investigated gemini surfactants is negative. In the surfactant–PAM systems, the thermodynamic parameters of binding have also been determined. The conclusion may be drawn that the binding strength of SGS onto PAM is stronger than that of CGS, resulting from more compact structure of SGS aggregates. With increasing surfactant hydrophobicity, the values of ΔH agg become more exothermic and a ΔS agg decrease was observed. Therefore, the interaction between SGS and PAM is enthalpy-driven.

Intrinsic thermodynamics of sulfonamide inhibitor binding to human carbonic anhydrases I and II

Human carbonic anhydrase (CA) I and II are cytosolic proteins, where their expression disorders can cause diseases such as glaucoma, edema, epilepsy or cancer. There are numerous inhibitors that target these isozymes, but it is difficult to design compounds that could bind to one of these proteins specifically. The binding of sulfonamide inhibitor to a CA is linked to several protonation reactions, namely, deprotonation of the sulfonamide group, protonation of the active site zinc hydroxide and the compensating protonation–deprotonation of buffer. By performing binding experiments at various pHs and buffers, all those contributions were dissected and the “intrinsic” binding parameters were calculated. Intrinsic thermodynamic binding parameters to CA I and II were determined for such widely studied drugs as acetazolamide, ethoxzolamide, methazolamide, trifluoromethanesulfonamide and dichlorophenamide. The assignment of all contributions should enhance our understanding of the underlying energetics and increase our capability to design more potent and specific CA inhibitors.

Cyclic Voltammetric Investigation of Interactions between Bisnitroaromatic Compounds and ds.DNA

Herein, the cyclic voltammetric (CV) investigations of structurally similar bisnitrocompounds (N3, N4, N5, N6, having differentCH2� spacer length) is presented. CV study offered interesting interactional possibilities of bisnitrocom- pounds with chicken blood ds.DNA at physiological pH 4.7 and human body temperature, 310 K. The results indicated strong interaction by these symmetric molecules with ds.DNA and strength of binding is found to depend on length of CH2 spacer group in their molecular structure. Thermodynamics derived from electrochemical binding parameters also favored the irre- versible interactions. Moreover, threading intercalation mode of binding is suggested based on thermodynamic and kinetic binding parameters extracted from CV studies.

Structure–Activity Relationship of the Antimicrobial Peptide Gomesin: The Role of Peptide Hydrophobicity in Its Interaction with Model Membranes

Antimicrobial peptides are part of the innate immune system of animals and plants. Their lytic activity against microorganisms generally depends on their ability to disrupt and permeabilize membranes. Here we study the structure-activity relationship of the antimicrobial peptide gomesin (Gm), from the spider Acanthoscurria gomesiana, with large unilamellar vesicles (LUVs) composed of 3:7 palmitoyloleoyl phosphatidylglycerol: palmitoyloleoyl phosphatidylcholine. Several synthetic analogues of Gm were designed to alter the hydrophobicity/charge of the molecule, whereby selected amino acid residues were replaced by alanine. Isothermal titration calorimetry (ITC) was used to assess the thermodynamic parameters of peptide binding to LUVs and light scattering measurements were made to evaluated peptide-induced vesicle aggregation. The ability of the peptides to permeabilize vesicles was quantified through the leakage of an entrapped fluorescent probe. The activity of peptides could be quantified in terms of the leakage extent induced and their affinity to the membrane, which was largely dictated by the exothermic enthalpy change. The results show that analogues more hydrophobic than Gm display higher activity, whereas peptides more hydrophilic than Gm have their activity almost abolished. Vesicle aggregation, on the other hand, largely increases with peptide charge. We conclude that interaction of Gm with membranes depends on an interplay between surface electrostatic interactions, which drive anchoring to the membrane surface and vesicle aggregation, and insertion of the hydrophobic portion into the membrane core, responsible for causing membrane rupture/permeabilization.

The specification of the production of three different complexes via thermodynamic investigations between vanadyl (IV) bis(acetylacetonate) and D-penicillamine at three various pHs

d-H2Pen-binding behavior to [VO(acac)2] at pH=7.00, 7.50 and 10.0 has been studied in thermodynamic viewpoint using UV/VIS spectroscopy. The optical absorption spectra of [VO(acac)2] were analyzed in order to obtain binding constants and stoichiometries using SQUAD software. The estimation of binding constant at various temperatures enabled us to calculate all of the thermodynamic parameters of binding using the van't Hoff equation. Studies of described reactions at pH=7.00, 7.50 and 10.0 show exothermic, endothermic and exothermic modality, respectively. d-penicillamine is a potentially tridentate ligand, but the pKa for the ‒ COOH, NH3 and S‒ H groups are 1.99–2.00, 8.0 and 10.6, respectively, as a result S‒H can not release proton at pH=7.00 and pH=7.50, so the coordination of S is not possible except at strong alkali mediums. At pH=7.00, d-H2Pen converts [VO(acac)2] to a vanadyl Schiff base complex (coordination mode is N2O2). At pH=7.50, d-H2Pen is converted to HPen− and the exchange of acac− with HP...