Elucidating the microscopic properties of a β-barrel protein and the solvent confined in and around it

Intracellular lipid binding proteins (iLBPs) possess different characteristics, including a rigid protein structure consisting of a β-barrel, an α-helix cap, and a substantial internalized water cluster. Despite X-ray crystallographic research providing insights into the three-dimensional structures of iLBPs, the protein conformations, and the function of the internal water molecules inside the protein remain uncertain. In this study, we conducted molecular dynamics (MD) simulations on free (apo) and oleate-bound (holo) rat liver fatty acid binding proteins (rLFABPs), which are common intracellular lipid binding proteins (iLBPs) found in the liver of rats. Efforts have been made to obtain a comprehensive microscopic understanding of the conformational motions of different segments of the protein, namely, the β-strands, the helix-turn-helix (HTH) motif, and the loop regions, along with the impact of ligand binding on the microscopic structure and ordering of water molecules confined within the core and at the exterior surface of the protein. The calculations revealed fluctuating nature of the HTH region, characterized by the development and disruption of distinct secondary structural components. Furthermore, the coexistence of spatially heterogeneous ordered and disordered water molecules within the core regions of the apo and holo forms has been observed. A high degree of ordering of core water molecules has been attributed to those that are doubly coordinated. In contrast, the randomly oriented ones are found to be surrounded by three neighboring water molecules in their first coordination shells. Such non-uniform ordering of core water molecules suggests their important role in the ligand binding process for this class of proteins.