Locating interaction sites on proteins: The crystal structure of thermolysin soaked in 2% to 100% isopropanol

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 exte nded this technique by the determination of high-resolution crystal structu res of thermolysin (TLN), generated from crystals soaked in 2% to 100% isop ropanol. The procedure causes only minor changes to the conformation of the protein, and an increasing number of isopropanol interaction sites could b e identified as the solvent concentration is increased. Isopropanol occupie s 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 s ubsites. Analysis of the isopropanol positions shows little correlation wit h 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, Proteins 1999;37:6 28-640, (C) 1999 Wiley-Liss, Inc.