Understanding the binding mechanisms of hydroxy-chalcone-based 24-membered macrocyclic bis-epoxide with beta-lactoglobulin

The lipocalin carrier protein, (3- lactoglobulin ((3-lg), stands out as a crucial protein in the food industry, known for its ability to effectively bind with hydrophobic small molecules. However, it was unclear how (3-lg interacts with macrocyclic molecules. In this research, we focused on two key aspects. First, we synthesized a 24-membered macrocycle 4d by modifying a natural product chalcone to create a macrocycle by connecting two orthohydroxyl groups of each phenyl ring of two chalcone units with alkyl chains. To enhance solubility, we converted the chalcone C--C bonds to epoxide rings. Second, we investigated the binding ability and mechanism of binding of the compound with the (3- lg. The (3-lg and 4d interaction shows an isoemissive point at 382 nm with Kb = 4.64 +/- 0.02 x 105 at 298 K, indicating the excellent protein binding ability of 4d . Remarkably, despite its size, 4d binds to the protein without altering its conformation, suggesting the availability of a spacious binding site on the protein where the molecule fits well. Molecular docking analysis confirmed the presence of such a site at the mouth of the calyx. Additionally, our 200 ns molecular dynamics simulation demonstrated that 4d adopts a conformation to interact with the hydrophobic amino acids of the binding site, ultimately stabilizing the protein.