BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 248, 273–277 (1998) ARTICLE NO. RC988937 Organic Solvent Binding to Crystalline Subtilisin 1 in Mostly Aqueous Media and in the Neat Solvents Jennifer L. Schmitke, Lawrence J. Stern, and Alexander M. Klibanov 2 Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139 Received May 18, 1998 proach is that it is very difficult to determine crystal The X-ray crystal structures of the protease subtil- structures of proteins in neat organic solvents due to isin Carlsberg in 40% acetonitrile and in 20% dioxane crystal lattice distortions (7,8). It should be far easier have been determined to at least 2.3 A ˚ resolution, and to solve the structure of a lightly cross-linked enzyme their solvent binding patterns have been compared to crystal placed in a predominantly aqueous solution con- those observed in the neat organic solvents. The struc- taining organic solvent. In addition, since the ultimate tures of the protein in the two aqueous-organic mix- goal of rational drug design is to identify molecules tures are essentially the same as in pure water, aceto- that bind to proteins in aqueous solution, determining nitrile, and dioxane. Interestingly, the enzyme-bound the binding sites of organic solvent molecules in the organic solvent molecules tend to congregate in the neat solvent may yield positions involving weak associ- active site. Three of the five bound acetonitrile mole- ations not observed in aqueous solution. cules observed in the structure of subtilisin in 40% ace- The preceding considerations pose the following tonitrile are situated in the enzyme active site, as is questions: (i) Do solvent molecules bind to crystalline the single enzyme-bound dioxane molecule observed enzymes in aqueous-organic, principally aqueous mix- in 20% dioxane (whose location is distinct from that tures?, If so, (ii) Where are these bound organic solvent of any bound acetonitrile molecule). Furthermore, the organic solvent molecules detected in the enzyme ac- molecules located in the enzyme? and (iii) Are these tive site in the aqueous-organic mixtures are in the solvent molecules bound in the same locations as in same locations as in the structures in the correspond- the neat organic solvent? ing neat organic solvents.  1998 Academic Press To answer these questions, herein we determined the X-ray crystal structures of subtilisin in 40% acetoni- trile and in 20% dioxane and compared them to those previously determined in the corresponding neat sol- vents (refs. 1 and 8, respectively). There is much interest in obtaining functional group binding maps of protein surfaces. The objective of this mapping is to construct larger, tighter-binding mole- MATERIALS AND METHODS cules, ‘drug candidates’, which incorporate these func- tional groups. The determination of protein X-ray crys- Crystal preparation. Subtilisin Carlsberg (serine protease from Bacillus licheniformis, EC 3.4.21.14) was purchased from Sigma tal structures in organic solvents has been suggested Chemical Co. Crystals were grown from an aqueous 330 mM cacodyl- as a direct experimental technique for such mapping ate buffer, pH 5.6, saturated with (Ç13%) Na 2 SO 4 (9). Single crystals (1-4) analogous to the original computational methods (Ç1.0 1 0.1 1 0.08 mm) were placed in 1 mL of a 10% glutaraldehyde (5,6). In the crystallographic strategy, structures of the cross-linking solution (aged for three days at room temperature be- protein in several neat organic solvents are deter- fore reaction (8)) containing 30 mM cacodylate buffer, pH 7.5, and 10% Na 2 SO 4 . We presume a relatively low extent of cross-linking: mined, and the locations of the protein-bound organic there are only 9 lysine residues in subtilisin of which only 5 which solvent molecules, each representing a given functional could reasonably be involved in an intermolecular cross-linking event group, are ascertained (1-4). in this crystal form (distances below 20 A ˚ ). No glutaraldehyde or A major obstacle faced by the crystallographic ap- modified Lys electron density was detected in the electron density maps, but the cross-linking rendered the crystals insoluble in water and more robust against distortions caused by organic solvents (7,8). The crystals were incubated in the cross-linking solution for 30 1 Coordinates and data from the structures reported in this paper have been deposited in the Protein Data Bank (PDB; Brookhaven min and washed five times with 2 mL of distilled water. The water was then removed and replaced with either 40% (v/v) acetonitrile or National Laboratory, codes pending). 2 To whom correspondence should be addressed. Fax: (617) 252- 20% (v/v) dioxane by washing five times with 2 mL each. The crystals were finally mounted in a 0.7-mm glass capillary. 1609; E-mail: klibanov@mit.edu. 0006-291X/98 $25.00 Copyright  1998 by Academic Press All rights of reproduction in any form reserved. 273