The structure of a trimeric domain-swapped form of barnase (EC 3.1.27.3) was determined by x-ray crystallography at a resolution of 2.2 Å from crystals of space group R32. Residues 1-36 of one molecule associate with residues 41-110 from another molecule related through threefold symmetry. The resulting cyclic trimer contains three protein folds that are very similar to those in monomeric barnase. Both swapped domains contain a nucleation site for folding. The formation of a domain-swapped trimer is consistent with the description of the folding process of monomeric barnase as the formation and subsequent association of two foldons.

In the crystalline state, ribonuclease T 1 binds calcium ions at different lattice-dependent positions. In solution, its conformational stability is also remarkably increased in the presence of divalent metal ions. Combining urea unfolding studies and X-ray crystallography, we compared the presence of several metal ions at specific sites in the protein to their contribution to the overall stabilizing effect in solution. We constructed thermodynamic cycles involving particular metal ions and specific carboxylate functions. The resulting coupling energies indicate that some (but not all) metal ions found at lattice contacts in crystal structures may indeed significantly contribute to stability enhancement in the presence of metal ions in solution.Keywords: conformational stability; metal binding; RNase T 1 .Ribonuclease T 1 (RNase T 1 ) is a small, well-characterized enzyme excreted from Aspergillus oryzae. In an aqueous environment it cleaves the P-O5 H ester bond of singlestranded RNA specifically at the 3 H side of guanosine residues [1,2]. Over the years, a wealth of RNase T 1 crystal structures have been solved. Many of these structures contain calcium ions at different lattice contacts. The relevance of these particular calciums to the protein's properties in solution has never been investigated. Nevertheless, the enzyme is remarkably stable at high salt concentrations. The first studies on the stabilizing effect of salts on RNase T 1 were done by Oobatake and coworkers [3,4]. These authors measured an increase in melting temperature with 20 8C in the presence of 2 m NaCl. Moreover, the unfolding of the native reduced conformation can be prevented by the addition of salts [5]. Pace and Grimsley [6] suggested that the stabilizing character of ions on RNase T 1 is mainly due to weak but specific binding of cations and anions by the native, folded conformation, rather than to an ionic-strength effect. In the present work, we set out to test this hypothesis. We have also investigated whether metal ions found at crystal lattice contacts can contribute to protein stability in solution.To analyze the contribution of particular ion-binding sites (identified by crystallography) to stability, we disrupted these sites using site-directed mutagenesis and compared the conformational stability of the wild-type and the mutant enzymes in the presence and absence of metal ions. The coupling between the disrupting mutation and a particular ion was calculated using thermodynamic cycles [7,8]. We also determined the structure of RNase T 1 in crystals soaked with different metal ions. By comparing the thermodynamic and the structural data, we found that particular metal ion-binding sites contribute to the conformational stability of T 1 . Other sites are not relevant to the conformational stability in solution and may be artefacts of the crystallization procedure. E X P E R I M E N T A L P R O C E D U R E S Site-directed mutagenesis and enzyme purificationThe expression of recombinant RNase T 1 has been described previously [9]....