Analysis of the binding surfaces of proteins

We have developed an experimental approach to map the complete binding surf ace of any crystalline macromolecule that is fast and flexible. Crystals of the target protein are transferred into organic solvents and the crystal s tructures are determined at high (about 2 Angstrom) resolution. The sites w here the solvent molecules bind to the protein are thus identified directly . Different solvents serve as probes for different organic functional group s; thus, benzene is a probe for where aromatic groups like to bind, dimethy l formamide is a probe for peptide binding sites, and so forth. A series of about six such experiments suffices to locate the major binding regions on the protein surface unambiguously, These different sites can then be targe ted with "Hydra-headed" inhibitors that interact simultaneously with more t hat one site, thereby providing specificity for the desired target. We have used this method to map the complete binding surface of elastase, and find that three regions, including the active site cleft, are generally "sticky " and can make interactions with almost any functional group. Analyses of t hese binding sites on elastase and other proteins suggests that what makes a binding site is amphipathicity and the ease with which water can be displ aced. (C) 1999 John Wiley & Sons, Inc.