Ligand entry into the calyx of β‐lactoglobulin

Although the thermodynamic principles that control the binding of drug molecules to their protein targets are well understood, the detailed process of how a ligand reaches a protein binding site has been an intriguing question over decades. The short time interval between the encounter between a ligand and its receptor to the formation of the stable complex has prevented experimental observations. Bovine β-lactoglobulin (βlg) is a lipocalin member that carries fatty acids (FAs) and other lipids in the cellular environment. Βlg accommodates a FA molecule in its highly hydrophobic cavity and exhibits the capability of recognizing a wide variety of hydrophobic ligands. To elucidate the ligand entry process on βlg, we report molecular dynamics simulations of the encounter between palmitate (PA) or laurate (LA) and βlg. Our results show that residues localized in loops at the cavity entrance play an important role in the ligand penetration process. Analysis of the short-term interaction energies show that the forces operating on the systems lead to average conformations very close to the crystallographic holo-forms. Whereas the binding free energy analysis using the molecular mechanics Generalized Born surface area method shows that these conformations were thermodynamically favorable. © 2013 Wiley Periodicals, Inc. Biopolymers 101: 744–757, 2014.