Elucidation of molecular interactions of theaflavin monogallate with camel milk lactoferrin: detailed spectroscopic and dynamic simulation studies

Abstract

Lactoferrin is a heme-binding multifunctional glycoprotein known for iron transportation in the blood and also contributes to innate immunity. In this study, the interaction of theaflavin monogallate, a polyphenolic component of black tea, with camel milk lactoferrin was studied using various biophysical and computational techniques. Fluorescence quenching at different temperatures suggests that theaflavin monogallate interacted with lactoferrin by forming a non-fluorescent complex, i.e., static quenching. Theaflavin monogallate shows a significant affinity towards lactoferrin with a binding constant of ∼10⁴-10⁵ M^-1 at different temperatures. ANS binding shows that the binding of polyphenol resulted in the burial of hydrophobic domains of lactoferrin. Moreover, thermodynamic parameters (ΔH, ΔS and ΔG) suggested that the interaction between protein and polyphenol was entropically favored and spontaneous. Circular dichroism confirmed there was no alteration in the secondary structure of lactoferrin. The energy transfer efficiency (FRET) from lactoferrin to theaflavin was found to be approximately 50%, with a distance between protein and polyphenol of 2.44 nm. Molecular docking shows that the binding energy of lactoferrin-theaflavin monogallate interaction was -9.7 kcal mol^-1. Theaflavin monogallate was bound at the central cavity of lactoferrin and formed hydrogen bonds with Gln89, Tyr192, Lys301, Ser303, Gln87, and Val250 of lactoferrin. Other residues, such as Tyr82, Tyr92, and Tyr192, were involved in hydrophobic interactions. The calculation of various molecular dynamics simulations parameters indicated the formation of a stable complex between protein and polyphenol. This study delineates the binding mechanism of polyphenol with milk protein and could be helpful in milk formulations and play a key role in the food industry.

Fig. 1. Interaction between lactoferrin and theaflavin monogallate. (A) Quenching in fluorescence intensity of lactoferrin (5 μM) in the absence and presence of varying theaflavin monogallate concentration (0–15 μM) at 298 K, (B) Stern–Volmer plot at different temperatures, (C) modified Stern–Volmer plot at different temperatures, and (D) van't Hoff thermodynamics plot at three different temperatures.

Fig. 2. Conformational changes in lactoferrin due to the binding of theaflavin monogallate. (A) Far-UV CD spectra of lactoferrin (5 μM) in the absence and presence of theaflavin monogallate at 1 : 5 and 1 : 10 molar ratios, and (B) ANS fluorescence spectra of lactoferrin (5 μM) in the absence and presence of varying theaflavin monogallate concentration (0–15 μM).

Fig. 3. Forster resonance energy transfer (FRET) plot depicting the overlap between the fluorescence intensity of lactoferrin (5 μM) and the absorption spectrum of theaflavin monogallate (10 μM).

Fig. 4. Molecular docking of theaflavin monogallate with lactoferrin. (A) Cartoon representation of theaflavin monogallate (balls and stick model) binding with lactoferrin, (B) binding of theaflavin monogallate at the central cavity of lactoferrin, and (C) 2D depiction of lactoferrin interacting with theaflavin monogallate and the nature of forces involved in stabilizing lactoferrin–theaflavin monogallate complex.

Fig. 5. Molecular dynamics (MD) simulation of lactoferrin and theaflavin monogallate interaction. (A) Variation in RMSD (root mean square deviation) of lactoferrin alone and lactoferrin–theaflavin monogallate complex as a function of simulation, (B) RMSF (root mean square fluctuation) in lactoferrin in the absence and presence of theaflavin monogallate, (C) dependency of R_g (radius of gyration) of lactoferrin alone and lactoferrin–theaflavin monogallate complex as a function of simulation, and (D) variation in SASA (solvent accessible surface area) of lactoferrin alone and lactoferrin–theaflavin monogallate complex as a function of simulation.