CONFORMATIONAL STABILITY AND THERMODYNAMICS OF FOLDING OF RIBONUCLEASES SA, SA2 AND SA3

Ribonucleases Sa, Sa2, and Sa3 are three small, extracellular enzymes produced by different strains of Streptomyces nureofaciens with amino acid sequences that are 50% identical. We have studied the unfolding o f these enzymes by heat and urea to determine the conformational stabi lity and its dependence on temperature, pH, NaCl, and the disulfide bo nd. All three of the Sa ribonucleases unfold reversibly by a two-state mechanism with melting temperatures, T-m, at pH 7 of 48.4 degrees C ( Sa), 41.1 degrees C (Sa2), and 47.2 degrees C (Sa3). The T-m values ar e increased in the presence of 0.5 M NaCl by 4.0 deg. C (Sa), 0.1 deg. C (Sa2), and 7.2 deg. C (Sa3). The T-m values are decreased by 20.0 d eg. C (Sa), 31.5 deg. C (Sa2), and 27.0 deg. C (Sa3) when the single d isulfide bond in the molecules is reduced. We compare these results wi th similar studies on two Other members of the microbial ribonuclease family, RNase T-1 and RNase Ba (barnase), and with a member of the mam malian ribonuclease family, RNase A. At pH 7 and 25 degrees C, the con formational stabilities of the ribonucleases are (kcal/mol): 2.9 (Sa2) , 5.6 (Sa3), 6.1 (Sa), 6.6 (T-1), 8.7 (Ba), and 9.2 (A). Our analysis of the stabilizing forces suggests that the hydrophobic effect contrib utes from 90 to 110 kcal/mol and that hydrogen bonding contributes fro m 70 to 105 kcal/mol to the stability of these ribonucleases. Thus, we think that the hydrophobic effect and hydrogen bonding make large but comparable contributions to the conformational stability of these pro teins. (C) 1998 Academic Press Limited.