The contribution of metal ions to the conformational stability of ribonuclease T1: crystal versus solution.

In the crystalline state, ribonuclease T1 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.

Analysis of a water mediated protein-protein interactions within RNase T1.

Buried and well-ordered solvent molecules are an integral part of each folded protein. For a few individual water molecules, the exchange kinetics with solvent have been described in great detail. So far, little is known about the energetics of this exchange process.

Hydrogen-exchange stabilities of RNase T1 and variants with buried and solvent-exposed Ala --> Gly mutations in the helix.

Hydrogen-exchange rates were measured for RNase T1 and three variants with Ala --> Gly substitutions at a solvent-exposed (residue 21) and a buried (residue 23) position in the helix: A21G, G23A, and A21G + G23A. These results were used to measure the stabilities of the proteins. The hydrogen-exchange stabilities (DeltaG(HX)) for the most stable residues in each variant agree with the equilibrium conformational stability measured by urea denaturation (DeltaG(U)), if the effects of D(2)O and proline isomerization are included [Huyghues-Despointes, B.

Conserved water molecules in a large family of microbial ribonucleases.

We systematically analyzed the crystallographically determined water molecules of all known structures of RNase T1 and compared them to the ordered solvent in a large number of related microbial nucleases. To assess the crystallographers' impact on the interpretation of the solvent structure, we independently refined five validation structures from diffraction data derived from five isomorphous crystals of RNase T1. We also compared the positions of water molecules found in 11 published isomorphous RNase T1 inhibitor complexes.

Dissection of the structural and functional role of a conserved hydration site in RNase T1.

The reoccurrence of water molecules in crystal structures of RNase T1 was investigated. Five waters were found to be invariant in RNase T1 as well as in six other related fungal RNases. The structural, dynamical, and functional characteristics of one of these conserved hydration sites (WAT1) were analyzed by protein engineering, X-ray crystallography, and (17)O and 2H nuclear magnetic relaxation dispersion (NMRD).

Dissecting histidine interactions of ribonuclease T1 with asparagine and glutamine replacements: analysis of double mutant cycles at one position.

His92 of Ribonuclease T1 combines functional and structural features involving both imidazole nitrogens. To evaluate the use of Asn and Gln substitutions in dissecting the properties of histidines, we analysed the consequences of the His92Gln and His92Asn substitutions on the enzyme's structure, function, and conformational stability by protein engineering and X-ray crystallographic methods. In the X-ray structures of wild-type and His92Gln RNase T1 in complex with 2'-GMP the His92-N epsilon 2 and Gln92-N epsilon 2 atoms are isosterically equivalent.