Proline Isomerization in Unfolded Ribonuclease A

Unfolded ribonuclease A (RNase A) consists of a mixture of fast refolding (UF) and slow-refolding (US) species. The slow UF⇌US equilibration reaction is rate-limited by proline peptide bond isomerization. Investigations of the dependence on temperature of the UF⇌US equilibrium have led to conflicting results and different molecular interpretations. Here the dependence on temperature of the UF: US ratio was reinvestigated by using a new assay for the fast-folding molecules UF. Between 0°C and 60°C the proportion of UF present in unfolded RNase A at 6M guanidine · HCl was found to be independent of temperature. Consequently, no conclusions can be drawn regarding the role and importance of particular prolines in the UF⇌US transition solely from these results.