Abstract
The binding interactions of bovine serum albumin (BSA) with tetraphenylborate ions ([B(Ph)4]−) have been investigated by a set of experimental methods (isothermal titration calorimetry, steady-state fluorescence spectroscopy, differential scanning calorimetry and circular dichroism spectroscopy) and molecular dynamics-based computational approaches. Two sets of structurally distinctive binding sites in BSA were found under the experimental conditions (10 mM cacodylate buffer, pH 7, 298.15 K). The obtained results, supported by the competitive interactions experiments of SDS with [B(Ph)4]− for BSA, enabled us to find the potential binding sites in BSA. The first site is located in the subdomain I A of the protein and binds two [B(Ph)4]− ions (logK(ITC)1 = 7.09 ± 0.10; ΔG(ITC)1 = −9.67 ± 0.14 kcal mol−1; ΔH(ITC)1 = −3.14 ± 0.12 kcal mol−1; TΔS(ITC)1 = −6.53 kcal mol−1), whereas the second site is localized in the subdomain III A and binds five ions (logK(ITC)2 = 5.39 ± 0.06; ΔG(ITC)2 = −7.35 ± 0.09 kcal mol−1; ΔH(ITC)2 = 4.00 ± 0.14 kcal mol−1; TΔS(ITC)2 = 11.3 kcal mol−1). The formation of the {[B(Ph)4]−}–BSA complex results in an increase in the thermal stability of the alfa-helical content, correlating with the saturation of the particular BSA binding sites, thus hindering its thermal unfolding.
Highlights
In aqueous media, proteins undergo many physicochemical changes such as conformational alterations, denaturation, folding/unfolding processes, and ligand exchange [1,2,3,4,5,6]
We have focused our attention on bovine serum albumin (BSA), as it represents one of the most common model molecular systems for binding studies [18]
We have proven that the isothermal titration calorimetry technique supported by experimental and in silico methods can be used as an alternative to fluorescence spectroscopy for studying a stoichiometry of the resulting albumin–ligand complexes, the type of the binding interactions, and the number of the binding sites [20]
Summary
Proteins undergo many physicochemical changes such as conformational alterations, denaturation, folding/unfolding processes, and ligand exchange [1,2,3,4,5,6]. These phenomena can be invoked by variations in the temperature and/or the pH of a solution. The environmental conditions may affect the binding properties of a biomolecule, such as the affinity of low-molecular weight compounds to a protein, the number of potential binding sites, and the stoichiometry of the resulting protein–ligand complexes [7].
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