Conformational stability and thermodynamics of folding of ribonucleases Sa, Sa2 and Sa3

C N Pace, E J Hebert, K L Shaw, D Schell, V Both, D Krajcikova, J Sevcik, K S Wilson, Z Dauter, R W Hartley, G R Grimsley

Research output: Contribution to journalLiterature reviewpeer-review


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 of these enzymes by heat and urea to determine the conformational stability and its dependence on temperature, pH, NaCl, and the disulfide bond. 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 are 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 deg. C (Sa), 31.5 deg. C (Sa2), and 27.0 deg. C (Sa3) when the single disulfide bond in the molecules is reduced. We compare these results with similar studies on two Other members of the microbial ribonuclease family, RNase T-1 and RNase Ba (barnase), and with a member of the mammalian ribonuclease family, RNase A. At pH 7 and 25 degrees C, the conformational 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 contributes from 90 to 110 kcal/mol and that hydrogen bonding contributes from 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 proteins. (C) 1998 Academic Press Limited.

Original languageEnglish
Pages (from-to)271-286
Number of pages16
JournalJournal of Molecular Biology
Issue number1
Publication statusPublished - 29 May 1998

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