Interaction of multimicrobial synthetic inhibitor 1,2-bis(2-benzimidazolyl)-1,2-ethanediol with serum albumin: spectroscopic and computational studies.

The molecule, 1,2-Bis(2-benzimidazolyl)-1,2-ethanediol (BBE) is known to act as a selective inhibitor of poliovirus, rhinovirus, Candida albicans, several bacterial species, and is easily synthesized by Phillips reaction. The interaction of BBE with BSA and the effects of its binding on the conformation and unfolding/refolding pathways of the protein were investigated using multispectroscopic techniques and molecular modeling. The binding studies indicate that BSA has one high affinity BBE binding site with association constant 6.02±0.05×10(4) M(-1) at 298 K. By measuring binding at different temperatures, we determined the changes in enthalpy (ΔH = -15.13±2.15 kJ mol(-1)), entropy (ΔS = 40.87±7.25 J mol(-1) K(-1)) and free energy (ΔG( = )26.78±1.02) of interaction, which indicate that the binding was spontaneous and both enthalpically and entropically driven. Based on molecular modeling and thermodynamic parameters, we proposed that the complex formation involved mainly hydrophilic interaction such as hydrogen bonding between hydroxyl groups of ethane-1,2-diol fragment with Tyr410 and benzimidazole sp(2) nitrogen atom with Ser488 and hydrophobic interaction between phenyl ring of one benzimidazole of the ligand and hydrophobic residues namely, Ile387, Cys391, Phe402, Val432 and Cys437. The sequential unfolding mechanism of BSA, site-specific marker displacement experiments and molecular modeling showed that the molecule preferably binds in subdomain IIIA. The BBE binding to BSA was found to cause both secondary and tertiary structural alterations in the protein as studied by intrinsic fluorescence, near-UV and far-UV circular dichroism results. The unfolding/refolding study showed that BBE stabilized native to intermediate states (N⇌I) transition of the protein by ∼2 kJ mol(-1) without affecting the intermediate to unfolded states (I⇌U) transition and general mechanism of unfolding of BSA

1,2<i>-</i>Bis(2-Benzimidazolyl) 1,2-Ethanediol induced conformational alteration in BSA.

<p>1,2<i>-</i>Bis(2-Benzimidazolyl) 1,2-Ethanediol induced conformational alteration in BSA.</p

The binding and thermodynamic parameters of BBE-BSA complex.

<p>Log[{(F0-F)}/F ] versus log[Q] plots of the BBE-BSA binding data at three different temperatures for the determination of the association constant (K_a) and number of binding sites (n). The inset shows the van’t Hoff plot for BBE-BSA interaction for the determination of thermodynamic parameters.</p

1,2<i>-</i>Bis(2-Benzimidazolyl) 1,2-Ethanediol interaction and thermodynamic parameters.

<p>1,2<i>-</i>Bis(2-Benzimidazolyl) 1,2-Ethanediol interaction and thermodynamic parameters.</p

The equilibrium unfolding process and stability of BBE-BSA complex.

<p>The urea-induced unfolding profile of BSA and BBE-BSA complex at BBE/BSA molar ratios of 0∶1 and 2∶1 monitored by intrinsic fluorescence of the protein (A). The fraction denatured (f_d) versus [urea] plots for NI and IU transitions of the protein and complex. Lines represent the nonlinear regression fitting of the data according to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053499#pone.0053499.e008" target="_blank">equation 7</a> (B). The dependence of free energy change as a function of urea concentrations for the transitions shown in Figure B (C).</p

Preparation and structure of 1,2-Bis(2-Benzimidazolyl)-1,2-Ethanediol (BBE).

<p>Preparation and structure of 1,2-Bis(2-Benzimidazolyl)-1,2-Ethanediol (BBE).</p

1,2-Bis(2-Benzimidazolyl)-1,2-Ethanediol (BBE) induced fluorescence quenching mechanism of bovine serum albumin.

<p>The BBE induced intrinsic fluorescence quenching (A) and Stern-Volmer plots for fluorescence quenching data (B) of BSA at different temperatures. The inset of Figure A shows fluorescence spectra of BSA in the absence and presence of increasing BBE concentrations at 298 K. The concentration of BSA was 5 µM and the intrinsic fluorescence of the protein was measured in 60 mM sodium phosphate buffer of pH 7.4 upon excitation at 280 nm.</p

The BBE binding to BSA is specific.

<p>Particle formation by BBE in the absence and presence of 0.01% Triton X-100 as monitored by Rayleigh light scattering at 350 nm (A). Stern-Volmer plots of BBE-induced fluorescence quenching of BSA with increasing concentrations of the protein (B).</p

The effect of 1,2-<i>Bis</i>(2-Benzimidazolyl) 1,2-Ethanediol binding on unfolding transition of BSA.

<p>The effect of 1,2-<i>Bis</i>(2-Benzimidazolyl) 1,2-Ethanediol binding on unfolding transition of BSA.</p