Ligand Entry into Fatty Acid Binding Protein via Local Unfolding Instead of Gap Widening

Fatty acid binding proteins play an important role in the transportation of fatty acids. Despite intensive studies, how fatty acids enter the protein cavity for binding is still controversial. Here, a gap-closed variant of human intestinal fatty acid binding protein was generated by mutagenesis, in which the gap is locked by a disulfide bridge. According to its structure determined here by NMR, this variant has no obvious openings as the ligand entrance and the gap cannot be widened by internal dynamics. Nevertheless, it still takes up fatty acids and other ligands. NMR relaxation dispersion, chemical exchange saturation transfer, and hydrogen-deuterium exchange experiments show that the variant exists in a major native state, two minor native-like states, and two locally unfolded states in aqueous solution. Local unfolding of either βB-βD or helix 2 can generate an opening large enough for ligands to enter the protein cavity, but only the fast local unfolding of helix 2 is relevant to the ligand entry process.