Multiple Solvent Crystal Structures (MSCS) is a crystallographic technique to identify energetically favorable positions and orientations of small organic molecules on the surface of proteins. We determined the high-resolution crystal structures of thermolysin (TLN), generated from crystals soaked in 50--70% acetone, 50--80% acetonitrile and 50 mM phenol. The structures of the protein in the aqueous-organic mixtures are essentially the same as the native enzyme and a number of solvent interaction sites were identified. The distribution of probe molecules shows clusters in the main specificity pocket of the active site and a buried subsite. Within the active site, we compared the experimentally determined solvent positions with predictions from two computational functional group mapping techniques, GRID and Multiple Copy Simultaneous Search (MCSS). The experimentally determined small molecule positions are consistent with the structures of known protein--ligand complexes of TLN.

Multiple-solvent crystal structure determination (MSCS) allows the position and orientation of bound solvent fragments to be identified by determining the structure of protein crystals soaked in organic solvents. We have extended this technique by the determination of high-resolution crystal structures of thermolysin (TLN), generated from crystals soaked in 2% to 100% isopropanol. The procedure causes only minor changes to the conformation of the protein, and an increasing number of isopropanol interaction sites could be identified as the solvent concentration is increased. Isopropanol occupies all four of the main subsites in the active site, although this was only observed at very high concentrations of isopropanol for three of the four subsites. Analysis of the isopropanol positions shows little correlation with interaction energy computed using a molecular mechanics force field, but the experimentally determined positions of isopropanol are consistent with the structures of known protein-ligand complexes of TLN.

Multiple-solvent crystal structure determination (MSCS) allows the position and orientation of bound solvent fragments to be identified by determining the structure of protein crystals soaked in organic solvents. We have extended this technique by the determination of high-resolution crystal structures of thermolysin (TLN), generated from crystals soaked in 2% to 100% isopropanol. The procedure causes only minor changes to the conformation of the protein, and an increasing number of isopropanol interaction sites could be identified as the solvent concentration is increased. Isopropanol occupies all four of the main subsites in the active site, although this was only observed at very high concentrations of isopropanol for three of the four subsites. Analysis of the isopropanol positions shows little correlation with interaction energy computed using a molecular mechanics force field, but the experimentally determined positions of isopropanol are consistent with the structures of known protein-ligand complexes of TLN.

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