THE ROLE OF A TRANS-PROLINE IN THE FOLDING MECHANISM OF RIBONUCLEASE-T1

Protein folding is often retarded by the cis reversible arrow trans is omerizations of prolyl peptide bonds both in vitro and in vivo. An imp ortant role for the folding mechanism is well established for the prol yl peptide bonds that are cis in the native protein, but not for those that are trans. Here we investigated the role of trans-Pro73 for the folding of ribonuclease T1 (which additionally contains two cis-prolin es) by comparing the wild-type protein with the Pro73-->Val variant. T he Pro73-->Val substitution led to a destabilization of the folded pro tein by 8.5 kJ/mol, which is explained by the strong, 25-fold increase in the rate of unfolding. In contrast, the rates and amplitudes of th e fast and slow refolding reactions were virtually unchanged. trans-Pr oline residues remain largely trans after unfolding, and therefore the ir contributions to the observed folding kinetics should indeed be ins ignificant for proteins which also contain one or more cis prolines. T he cis-proline residues dominate the kinetics of refolding, because al most all slow-folding molecules contain the respective incorrect (tran s) isomers, and because trans-->cis isomerizations are slower than cis -->trans isomerizations. The inability to detect contributions from a trans-proline to the kinetics of folding does not imply that this prol ine is non-essential for folding in the sense that its cis reversible arrow trans isomerization is energetically uncoupled from conformation al folding.