IONIC INTERACTIONS IN CRYSTALLINE BOVINE PANCREATIC RIBONUCLEASE-A

Isomorphous crystals (space group P3(2)21) of bovine pancreatic ribonu clease A (RNase A) were prepared at a pH of 5.5 in a series of high sa lt conditions. where both the nature of the ions and the ionic strengt h varied: 80% ammonium sulfate (mu = 12.5); 8 M sodium formate (mu = 8 .0); 3 M NaCl, 30% ammonium sulfate (mu = 7.0); 3 M CsCl, 30% ammonium sulfate (mu = 7.0); and 3.5 M NaCl, 3.3 M sodium formate (mu = 5.8). These structures were independently refined to a resolution of 2.0 Ang strom or better with R-factors that range from 16.1% to 17.5%. A compa rison of these six structures and the monoclinic crystal form of RNase A grown from alcohol shows that changes in ionic strength do not alte r the secondary or tertiary sturcture and that there are no significan t changes in intramolecular salt bridges. These findings support the n otion that structures determined from crystals grown in high salt are representative of the overall structural and electrostatic features pr esent under physiological conditions. While little effect was observed on the main chain conformation, several residues adapted different si de chain conformations and altered hydrogen-bonding patterns, either a s result of direct anion binding or more subtle indirect effects, Chan ges in the ionic composition of the mother liquor allowed for the occu pancy of the active site with different anions, The direct observation of active site-bound chloride and formate anions supports the proposa l that these species act as true competitive inhibitors of RNase A and not through nonspecific electrostatic effects, The identification of bound formate anions allowed for an experimental validation of computa tional-based functional group mapping techniques and suggests a useful modification to these approaches. Electrostatic surface potential cal culations identify a nearly continuous band of positive potential, con sistent with an extended binding site for polynucleotide ligands and s ubstrates. The majority of these residues are not involved in salt bri dges, which may facilitate binding to extended polynucleotide substrat es. Selection of the appropriate solvent conditions results in an unoc cupied active site, which will allow this crystal form to be used for the crystallographic study of productive ligand-binding modes.