Hydrophobicity-directed structural alteration in cytochrome C induced by bile salts: physicochemical, spectroscopic, and atomic force microscopic studies with molecular docking analysis

Abstract

Despite the biological relevance of bile salts in digestion and cellular signaling, their interaction with biological macromolecules, particularly with proteins, remains poorly understood. The current study shows a comprehensive and comparative analysis of the interaction between the bile salts, sodium cholate (NaC) and sodium deoxycholate (NaDC), and a small heme protein, cytochrome C (Cyt C), in aqueous phosphate buffer medium. Adsorption isotherm analysis suggests that the aggregation behavior and the surface activity of bile salts are significantly improved in the presence of Cyt C. Based on UV-vis spectroscopy measurements, the binding isotherms show that three different zones describe the interaction between bile salts and Cyt C, which are also replicated successfully from the steady-state fluorescence measurements. The calorimetric profile (ITC) of Cyt C's interaction with the two bile salts exhibited a distinct pattern, indicating a different mode of interaction involved for the two bile salts. An 8–10% alpha helicity change was observed for Cyt C by exposure to NaC/NaDC, as indicated by CD spectroscopy measurements. DLS results indicate that swelling in protein structure and protein oligomer formation occur in the presence of bile salts. Electrochemical results reveal that the electron transfer process at the electroactive center of Cyt C is greatly modified in the presence of bile salts. The calculated binding constant value indicates that NaDC binds more strongly with Cyt C than NaC. Finally, we performed molecular docking studies, and the results were in agreement with the experimental results. The results of the entire experimental analysis imply that hydrophobicity of bile salts is a significant factor in protein structure modification in addition to electrostatic interaction. As bile salts have an indirect effect on the process of cell apoptosis, and cytochrome C plays a direct function in it, their interaction is of great importance in relation to cell apoptosis. Also, the modulation of the electron transfer process of Cyt C in the presence of NaC/NaDC makes it very important in biological and biomedical fields.