Crystallographic Characterization of Wild-Type and Mutant Ribonuclease T₁ Complexes with Several Ribonucleotides

Abstract

Ribonuclease T₁ and the mutant enzymes were cocrystallized with several ribonucleotides, including non-hydrolyzable substrate analogs of di- and triribonucleotides, which have a novel guanylate in which the 2'-hydroxyl group of the ribose is replaced by a fluorine atom. One of the mutant enzymes has a tryptophan residue, instead of Tyr45 of the wild-type enzyme, to enhance the binding of ribonucleotides to the enzyme and the other mutant enzyme has histidine and aspartate residues, instead of Asn43 and Asn44, respectively, to reproduce the natural substitutions found in ribonuclease M_s. Polymorphism of the crystals was observed for wild-type and mutant enzymes. However, orthorhombic crystals, which are virtually all isomorphous to each other, were successfully obtained from wild-type and mutant (Y45W) enzymes by the macroscopic seeding technique using mother crystals of the wild-type ribonuclease T₁ complexed with 2'GMP or 3'GMP. The diffraction patterns of these crystals extend beyond 2.5 Å resolution and the diffraction data were collected from some of the crystals on a diffractometer up to a range of 2.5 to 1.8 Å resolution.