0 1998 The Protein Society - FOR THE RECORD Structure and stability of the P 93 G variant of ribonuclease A

The peptide bonds preceding Pro 93 and Pro I14 of bovine pancreatic ribonuclease A (RNase A) are in the cis conformation. The trans-to-cis isomerization of these bonds had been indicted as the slow step during protein folding. Here, site-directed mutagenesis was used to replace Pro 93 or Pro 114 with a glycine residue, and the crystalline structure of the P93G variant was determined by X-ray diffraction analysis to a resolution of 1.7 w. This structure is essentially identical to that of the wild-type protein, except for the 9 1-94 p-turn containing the substitution. In the wild-type protein, the p-turn is of type Vla. In the P93G variant, this turn is of type I1 with the peptide bond preceding Gly 93 being trans. The thermal stabilities of the P93G and P114G variants were assessed by differential scanning calorimetry and thermal denaturation experiments monitored by ultraviolet spectroscopy. The value of AAGm, which reports on the stability lost in the variants, is 1 S-fold greater for the PI 14G variant than for the P93G variant. The greater stability of the P93G variant is likely due to the relatively facile accommodation of residues 91-94 in a type I1 turn, which has a preference for a glycine residue in its i + 2 position.