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Protein Science, Vol 1, Issue 7 910-916, Copyright © 1992 by Cold Spring Harbor Laboratory Press

ARTICLE

Cis proline mutants of ribonuclease A. I. Thermal stability

D. A. SCHULTZ and R. L. BALDWIN
Present address: Department of Molecular Biophysics and Biochemistry, Yale University, P.O. Box 6666, New Haven, Connecticut 06511.

A chemically synthesized gene for ribonuclease A has been expressed in Escherichia coli using a T7 expression system (Studier, F.W., Rosenberg, A.H., Dunn, J.J., & Dubendorff, J.W., 1990, Methods Enzymol. 185, 60-89). The expressed protein, which contains an additional N-terminal methionine residue, has physical and catalytic properties close to those of bovine ribonuclease A. The expressed protein accumulates in inclusion bodies and has scrambled disulfide bonds; the native disulfide bonds are regenerated during purification. Site-directed mutations have been made at each of the two cis proline residues, 93 and 114, and a double mutant has been made. In contrast to results reported for replacement of trans proline residues, replacement of either cis proline is strongly destabilizing. Thermal unfolding experiments on four single mutants give {delta}T(m) {complex} 10{deg}C and {delta}{delta}G(0) (apparent) = 2-3 kcal/mol. The reason is that either the substituted amino acid goes in cis, and cis {complex} trans isomerization after unfolding pulls the unfolding equilibrium toward the unfolded state, or else there is a conformational change, which by itself is destabilizing relative to the wild-type conformation, that allows the substituted amino acid to from a trans peptide bond.
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