Cis proline mutants of ribonuclease A. II. Elimination of the slow‐folding forms by mutation

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Abstract
Ribonuclease A is known to form an equilibrium mixture of fast‐folding (UF) and slow‐folding (US) species. Rapid unfolding to UF is then followed by a reaction in the unfolded state, which produces a mixture of UF, USII, USI, and possibly also minor populations of other US species. The two cis proline residues, P93 and P114, are logical candidates for producing the major US species after unfolding, by slow cis → trans isomerization. Much work has been done in the past on testing this proposal, but the results have been controversial. Site‐directed mutagenesis is used here. Four single mutants, P93A, P93S, P114A, and P114G, and also the double mutant P93A, P114G have been made and tested for the formation of US species after unfolding. The single mutants P114G and P114A still show slow isomerization reactions after unfolding that produce US species; thus, Pro 114 is not required for the formation of at least one of the major US species of ribonuclease A. Both the refolding kinetics and the isomerization kinetics after unfolding of the Pro 93 single mutants are unexpectedly complex, possibly because the substituted amino acid forms a cis peptide bond, which should undergo cis → trans isomerization after unfolding. The kinetics of peptide bond isomerization are not understood at present and the Pro 93 single mutants cannot be used yet to investigate the role of Pro 93 in forming the US species of ribonuclease A. The double mutant P93A, P114G shows single exponential kinetics measured by CD, and it shows no evidence of isomerization after unfolding. Thus, the unfolding and refolding kinetics of the double mutant indicate that replacement of Pro 93 and Pro 114 has removed the residues responsible for forming the major US species. This result, taken together with data for the Pro 114 single mutants, indicates that Pro 93 and Pro 114 are collectively responsible for forming the major US species.