A thermodynamic characterization of the interaction of 8‐anilino‐1‐naphthalenesulfonic acid with native globular proteins: the effect of the ligand dimerization in the analysis of the binding isotherms

Andújar‐Sánchez, Montserrat; Jara‐Pérez, Vicente; Cobos, Eva S.; Cámara-Artigas, A.

doi:10.1002/jmr.1065

Cited by 6 publications

(5 citation statements)

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“…The estimated second-order rate constant is about 1.4 10 5 m À1 s À1 ,w hich is in agreement with weak bindingo fA NS to CHT in the first stage. Experimental artefacts due to the dimerisationo ft he probe ligand atl ower pH [39] in the observation of the differential pathways of molecular recognition can be ruled out for the followingr easons: Careful experiments [39] show that the ligand remains in its monomeric form at about % 100 mm at pH 3, 5 and 7. The molecular recognition pathway involving much weaker binding in the first stage and complexation through structural reorganisation is consistent with the induced-fit mechanism.…”

Section: Resultssupporting

confidence: 66%

“…Experimental artefacts due to the dimerisation of the probe ligand at lower pH39 in the observation of the differential pathways of molecular recognition can be ruled out for the following reasons: Careful experiments39 show that the ligand remains in its monomeric form at about ≈100 μ M at pH 3, 5 and 7. The concentration used in our experiment is around 10 μ M .…”

Section: Resultsmentioning

confidence: 99%

“…Experimental artefacts due to the dimerisationo ft he probe ligand atl ower pH [39] in the observation of the differential pathways of molecular recognition can be ruled out for the followingr easons: Careful experiments [39] show that the ligand remains in its monomeric form at about % 100 mm at pH 3, 5 and 7. The concentrationu sed in our experiment is around 10 mm.M oreover,i nacontrol experiment on the complexation of ANS (anionic) with cationic CTAB micelles (inset of Figure 2c)u nder similar experimental conditions, we obtained k 1 and k 2 values of 1486 and 19.8 s À1 ,r espectively,f or pH 6.3, whereas at pH 3.6 the corresponding k 1 and k 2 values were 1076 and 10.0 s À1 ,r espectively.T his observation clearlyr ules out the possibility of any interference of the host buffer (of different pH) with the differential CHT-ANS complexation kinetics at the two pH values discussed above.C onversion of the pseudo-first-order rate constant, k 1 ,t oi ts second-order counterpart reveals av alue of 10 4 m À1 s À1 ,w hichi sc onsistentw ith the reported value of molecular complexation of an organic ligand with am icelle.…”

Section: Resultsmentioning

confidence: 99%

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Direct Observation of Kinetic Pathways of Biomolecular Recognition

Choudhury

Batabyal

Mondal

et al. 2015

Chemistry A European J

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The pathways of molecular recognition, which is a central event in all biological processes, belong to the most important subjects of contemporary research in biomolecular science. By using fluorescence spectroscopy in a microfluidics channel, it can be determined that molecular recognition of α-chymotrypsin in hydrous surroundings at two different pH values (3.6 and 6.3) follows two distinctly different pathways. Whereas one corroborates an induced-fit model (pH 3.6), the other one (pH 6.3) is consistent with the selected-fit model of biomolecular recognition. The role of massive structural perturbations of differential recognition pathways could be ruled out by earlier XRD studies, rather was consistent with the femtosecond-resolved observation of dynamic flexibility of the protein at different pH values. At low concentrations of ligands, the selected-fit model dominates, whereas increasing the ligand concentration leads to the induced-fit model. From molecular modelling and experimental results, the timescale associated with the conformational flexibility of the protein plays a key role in the selection of a pathway in biomolecular recognition.

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Section: Resultssupporting

confidence: 66%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

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Direct Observation of Kinetic Pathways of Biomolecular Recognition

Choudhury

Batabyal

Mondal

et al. 2015

Chemistry A European J

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“…Protein interactions with other proteins Protein interactions with small ligands (including co‐factors and drugs) Protein/peptide interactions with metals and ions …”

Section: Introductionmentioning

confidence: 99%

Applications of isothermal titration calorimetry in pure and applied research—survey of the literature from 2010

Ghai

Falconer

Collins

2011

J of Molecular Recognition

167

108

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Isothermal titration calorimetry (ITC) is a biophysical technique for measuring the formation and dissociation of molecular complexes and has become an invaluable tool in many branches of science from cell biology to food chemistry. By measuring the heat absorbed or released during bond formation, ITC provides accurate, rapid, and label-free measurement of the thermodynamics of molecular interactions. In this review, we survey the recent literature reporting the use of ITC and have highlighted a number of interesting studies that provide a flavour of the diverse systems to which ITC can be applied. These include measurements of protein-protein and protein-membrane interactions required for macromolecular assembly, analysis of enzyme kinetics, experimental validation of molecular dynamics simulations, and even in manufacturing applications such as food science. Some highlights include studies of the biological complex formed by Staphylococcus aureus enterotoxin C3 and the murine T-cell receptor, the mechanism of membrane association of the Parkinson's disease-associated protein α-synuclein, and the role of non-specific tannin-protein interactions in the quality of different beverages. Recent developments in automation are overcoming limitations on throughput imposed by previous manual procedures and promise to greatly extend usefulness of ITC in the future. We also attempt to impart some practical advice for getting the most out of ITC data for those researchers less familiar with the method.

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“…Analytical solutions of cubic equations are available that describe ligand depletion (with and without NSB) of two binding sites and one ligand or of two sites with homologous competition [3,9,18,46,47]. Analytical solutions of a quartic polynomial describing ligand depletion and NSB of three binding sites and one ligand or of two binding sites with homologous competition can be derived from the general solution of a quartic polynomial [48].…”

Section: Methodsmentioning

confidence: 99%

Characterizing low affinity epibatidine binding to α4β2 nicotinic acetylcholine receptors with ligand depletion and nonspecific binding

Person

Wells

2011

BMC Biophys

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BackgroundAlong with high affinity binding of epibatidine (Kd1≈10 pM) to α4β2 nicotinic acetylcholine receptor (nAChR), low affinity binding of epibatidine (Kd2≈1-10 nM) to an independent binding site has been reported. Studying this low affinity binding is important because it might contribute understanding about the structure and synthesis of α4β2 nAChR. The binding behavior of epibatidine and α4β2 AChR raises a question about interpreting binding data from two independent sites with ligand depletion and nonspecific binding, both of which can affect equilibrium binding of [3H]epibatidine and α4β2 nAChR. If modeled incorrectly, ligand depletion and nonspecific binding lead to inaccurate estimates of binding constants. Fitting total equilibrium binding as a function of total ligand accurately characterizes a single site with ligand depletion and nonspecific binding. The goal of this study was to determine whether this approach is sufficient with two independent high and low affinity sites.ResultsComputer simulations of binding revealed complexities beyond fitting total binding for characterizing the second, low affinity site of α4β2 nAChR. First, distinguishing low-affinity specific binding from nonspecific binding was a potential problem with saturation data. Varying the maximum concentration of [3H]epibatidine, simultaneously fitting independently measured nonspecific binding, and varying α4β2 nAChR concentration were effective remedies. Second, ligand depletion helped identify the low affinity site when nonspecific binding was significant in saturation or competition data, contrary to a common belief that ligand depletion always is detrimental. Third, measuring nonspecific binding without α4β2 nAChR distinguished better between nonspecific binding and low-affinity specific binding under some circumstances of competitive binding than did presuming nonspecific binding to be residual [3H]epibatidine binding after adding a large concentration of cold competitor. Fourth, nonspecific binding of a heterologous competitor changed estimates of high and low inhibition constants but did not change the ratio of those estimates.ConclusionsInvestigating the low affinity site of α4β2 nAChR with equilibrium binding when ligand depletion and nonspecific binding are present likely needs special attention to experimental design and data interpretation beyond fitting total binding data. Manipulation of maximum ligand and receptor concentrations and intentionally increasing ligand depletion are potentially helpful approaches.

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A thermodynamic characterization of the interaction of 8‐anilino‐1‐naphthalenesulfonic acid with native globular proteins: the effect of the ligand dimerization in the analysis of the binding isotherms

Cited by 6 publications

References 35 publications

Direct Observation of Kinetic Pathways of Biomolecular Recognition

Direct Observation of Kinetic Pathways of Biomolecular Recognition

Applications of isothermal titration calorimetry in pure and applied research—survey of the literature from 2010

Characterizing low affinity epibatidine binding to α4β2 nicotinic acetylcholine receptors with ligand depletion and nonspecific binding

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