http://www.loc.gov/mods/v3 http://www.loc.gov/standards/mods/v3/mods-3-2.xsd
Mutational effects on the conformational stability and activity of Ribonuclease T1
Ribonuclease T1 (RNase T1) and fourteen mutants were prepared from a chemically synthesized gene, cloned and expressed in Escherichia coli. The wild-type RNase T1 prepared from E. coli was identical in every functional and physical property examined to RNase T1 prepared from Aspergillus oryzae. Urea and thermal unfolding experiments show that the amino acid substitution Gln25 [to] Lys is 0.9 kcal/mol more stable and Glu58 [to] Ala is 0.8 kcal/mol less stable than wild-type RNase T1. In the mutant Glu58 [to] Ala (K25) (i.e. the double mutant), these contributions cancel and the stability does not differ significantly from that of wild-type RNase T1. The activity of Gln25 [to] Lys is identical with that of wild-type RNase T1. However, the activities of Glu58 [to] Ala and Glu58 [to] Ala (K25) are 7% of wild-type when GpC hydrolysis is measured (due to a 35-fold decrease in k[cat]), and 37% of wild-type when RNA hydrolysis is measured. Thus, Glu58 is important, but not essential to the activity of RNase T1. Urea and thermal unfolding curves were determined for eleven mutants that remove specific hydrogen bonds from the native state structure of RNase T1. These studies show that the deletion of a partner from a neutral-neutral hydrogen bond pair reduces stability by 0.6 to 1.5 kcal/mol, and the deletion of a partner from a neutral-charged pair reduces stability by as much as 2 kcal/mol. The most surprising finding was that for two of these mutants, the removal of a hydrogen bonding partner resulted in an increase in the conformational stability of RNase T1. These results suggest that hydrogen bonds make a significant contribution to the conformational stability of globular proteins.