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.

Conformational fluctuations in the molten globule state of α-lactalbumin.

A molten globule (MG) state is an intermediate state of a protein observed during the unfolding of the native structure. The MG state of the protein is induced by various denaturing agents (like urea), extreme pH, pressure, and heat. Experiments suggest that the MG state of some proteins is functionally relevant even if there is no well-defined tertiary structure. Earlier experimental and theoretical studies show that the MG state of a protein is dynamic in nature, where conformational states are interconverted on nanosecond time scales. These observations lead us to study and compare the conformational fluctuations of the MG state to those of intrinsic disordered proteins (IDPs). We consider a milk protein, α-lactalbumin (aLA), which shows an MG state at low pH upon removal of the calcium (Ca2+) ion. We use the constant pH molecular dynamics (CpHMD) simulation to maintain the protonation state of titratable residues at a low pH during the simulation. We use the dihedral principal component analysis, the density based clustering method, and the machine learning technique to identify the conformational fluctuations. We observe metastable states in the MG state. The residues containing the essential coordinates responsible for metastability belong to a stable helix in the crystal structure, but most of them prefer unstructured or bent conformation in the MG state. These residues control the exposure of the putative binding residues for fatty acids. Thus, the MG state of a protein behaves as an intrinsic disorder protein, although the disorder here is induced by external conditions.