Fluctuation-Dominated Ligand Binding in Molten Globule Protein

A molten globule (MG) state is an intermediate state of protein observed during the unfolding of the native structure. In MG states, milk protein α-lactalbumin (aLA) binds to oleic acid (OLA). This MG-aLA-OLA complex, popularly known as XAMLET, performs cytotoxic activities against cancer cell lines. However, the microscopic understanding of ligand recognition ability in the MG state of the protein has not yet been explored. Motivated by this, we explore the binding of bovine aLA with OLA using all-atom molecular dynamics (MD) simulations. We find the binding mode between MG-aLA and OLA using the conformational thermodynamics method. We also estimate the binding free energy using the umbrella sampling (US) method for both the MG state and the neutral state. We find that the binding free energy obtained from US is comparable with earlier experimental results. We characterize the dihedral fluctuations as the ligand is liberated from the active site of the protein using steered MD. The low energy fluctuations occur near the ligand binding site, which eventually transfer toward the Ca²⁺-binding site as the ligand is taken away from the protein.