Conformational stability of Ribonuclease T₁ and a derivative with the 2 to 10 disulfide bond broken and the cysteine residues carboxymethylated

Abstract

We have determined the conformational stability of Ribonuclease T1, (RNase T1), and a derivative of RNase T1 with the 2 to 10 disulfide bond broken and the cysteine residues carboxymethylated, at our standard conditions of pH 7.0, 25° C, and 30 mM MOPS buffer. In addition, we have also determined the conformational stability of RNase T1 as a function of salt concentration, type of salt, and pH. We have also presented preliminary data for the thermodynamic parameters for unfolding of RNase T1. Our results from the analysis of urea denaturation curves indicate that the folded conformation of RNase T1 is about 5.7 kcal/mole more stable than the unfolded conformations at our standard conditions. Breaking the 2 to 10 disulfide bond and blocking the cysteine residues by carboxymethylation decreases the conformational stability of RNase T1 by about 3.7 kcal/mole. The stability of the folded conformation of RNase T1 is increased by about 0.8, 1.8, and 3.3 kcal/mole in the presence of 0.1 M NaCl, MgCl2, and Na2HPO4, respectively. This remarkable increase in the conformational stability results primarily from the preferential binding to the native protein of one Mg[+2] or tow Na[+] ions at cation binding sites, and by the binding of one HPO4[-2] ion at an anion binding site. Only modest binding constants, 6.2 M^-1 (Na[+]), 155 M^-1 (Mg[2+]) and 282 M^-1 (HPO4[2-]) are required to account for the enhanced stability. An analysis of urea denaturation curves determined for RNase T1 at pHs 3.6, 5.0, 7.0, and 8.5 indicates that the protein is the most stable at pH 5.0. When compared to the pH of our standard conditions, pH 7.0, the protein is about 3.3 kcal/mole more stable at pH 5.0, and about 1.8 kcal/mole less stable at pH 8.5. We observe an increase in the stability of RNase T1 at all pHs in the presence of 0.5 M NaCl. The largest increases are observed at pHs 7.0 and 8.5...