Binding of the Iminium and Alkanolamine Forms of Sanguinarine to Lysozyme: Spectroscopic Analysis, Thermodynamics, and Molecular Modeling Studies

Abstract

Sanguinarine (SGR) exists in charged iminium (SGRI) and neutral alkanolamine (SGRA) forms. The binding of these two forms to the protein lysozyme (Lyz) was investigated by fluorescence, UV-vis absorbance and circular dichroism spectroscopy, and in silico molecular docking approaches. Binding thermodynamics were studied by microcalorimetry. Both forms of sanguinarine quenched the intrinsic fluorescence of Lyz, but the quenching efficiencies varied on the basis of binding that was derived after correction for an inner-filter effect. The equilibrium binding constants at 25 ± 1.0 °C for the iminium and alkanolamine forms were 1.17 × 10(5) and 3.32 × 10(5) M(-1), respectively, with approximately one binding site for both forms of the protein. Conformational changes of the protein in the presence of SGR were confirmed by absorbance, circular dichroism, three-dimensional fluorescence, and synchronous fluorescence spectroscopy. Microcalorimetry data revealed that SGRI binding is endothermic and predominantly involves electrostatic and hydrophobic interactions, whereas SGRA binding is exothermic and dominated by hydrogen-bonding interactions. The molecular distances (r) of 3.27 and 3.04 nm between the donor (Lyz) and the SGRI and SGRA acceptors, respectively, were calculated according to Förster's theory. These data suggested that both forms were bound near the Trp-62/63 residues of Lyz. Stronger binding of SGRA than SGRI was apparent from the results of both structural and thermodynamic experiments. Molecular docking studies revealed that the putative binding site for the SGR analogues resides at the catalytic site. The docking results are in accordance with the spectroscopic and thermodynamic data, further validating the stronger binding of SGRA over SGRI to Lyz. The binding site is situated near a deep crevice on the protein surface and is close to several crucial amino acid residues, including Asp-52, Glu-35, Trp-62, and Trp-63. This study advances our knowledge of the structural nature and thermodynamic aspects of binding between the putative anticancer alkaloid sanguinarine and lysozyme.

Chelerythrine–lysozyme interaction: spectroscopic studies, thermodynamics and molecular modeling exploration

“…It has promising role as an antineoplastic growth inhibitor against various tumor cell lines, such as human breast cancer (MCF-7), human uveal melanoma (OCM-1), human neuroblastoma, colon carcinoma (HCT116) cell lines, and neonatal rat cardiac myocytes. 15 Although there are only small structural differences between sanguinarine and chelerythrine, large functional differences have been observed. 8,9 This might include binding to duplex and quadruplex nucleic acid structures.…”

“…It is known for its unique ability to damage bacterial cell wall by cleaving the b-linkages between the N-acetyl-muramic acid and N-acetylglucosamine of the peptidoglycan thereby protecting against bacterial infections. 15 The binding of sanguinarine to lysozyme has identified the buckled alkanolamine to bind stronger than the planar iminium. [19][20][21] Other activities include anti-inflammatory, antiviral, antiseptic, antihistamine, and antineoplastic effects.…”

“…Lysozyme is also used widely as a food preservative and as an antimicrobial agent. 15 Recently, the binding studies of a number of small molecules, alkaloids, nanoparticles and model membranes to lysozyme were reported. Owing to its small size, high stability, natural abundance, propensity to undergo amyloid aggregation, and ability to carry drugs, Lyz has been preferred as a model protein in many studies to understand protein folding, structurefunction, dynamics, and ligand interactions.…”

Interaction of Lysozyme with Rhodamine B: A combined analysis of spectroscopic & molecular docking

“…Then the α-helical content was calculated from the MRE value at 208 nm using the following equation. [68,69] − ℎ (%) = − 208 − 4000 33000 − 4000 × 100 (11) where MRE 208 is the observed MRE value at 208 nm, 4000 is the MRE value of the β-form and random coil conformation at 208 nm, and 33,000 is the MRE value of a pure α-helix at 208 nm.…”

“…It is also used in the pharmaceutical industries such as in bacterial and viral diseases as well as in inflammatory diseases. [9][10][11][12] Due to its higher natural abundance, stability, small size and drug binding ability, it has been used as a model protein for studying the interaction with variety of small molecules (metal ions, dyes, several pharmaceuticals drugs and non-metal ions etc. ), in understanding the relation between protein folding and dynamics, structure-function relationship etc.…”