Locating missing water molecules in protein cavities by the three-dimensional reference interaction site model theory of molecular solvation

Water molecules confined in protein cavities are of great importance in understanding the protein structure and functions. However, it is a nontrivial task to locate such water molecules in protein by the ordinary molecular simulation and modeling techniques as well as experimental methods. The present study proves that the three-dimensional reference interaction site model (3D-RISM) theory, a recently developed statistical-mechanical theory of molecular solvation, has an outstanding advantage in locating such water molecules. In this paper, we demonstrate that the 3D-RISM theory is able to reproduce the structure and the number of water molecules in cavities of hen egg-white lysozyme observed commonly in the Xray structures of different resolutions and conditions. Furthermore, we show that the theory successfully identified a water molecule in a cavity, the existence of which has been ambiguous even from the X-ray results. In contrast, we confirmed that molecular dynamics simulation is helpless at present to find such water molecules because the results substantially depend on the initial coordinates of water molecules. Possible applications of the theory to problems in the fields of biochemistry and biophysics are also discussed.