A Native‐Like Intermediate on the Ribonuclease A Folding Pathway

Abstract

Unfolded ribonuclease A consists of a mixture of fast‐folding (UF) and slow‐folding species (US); US folds slowly because it contains non‐native proline isomers. The folding kinetics of US have been determined by measuring the changes in tyrosine fluorescence upon refolding. Under conditions where the energy of stabilization of the native protein is small (in marginally native conditions) the fluorescence‐detected kinetics of folding coincide with the ones measured by other probes. However, when refolding is carried out under conditions where the native protein is highly stabilized (in strongly native conditions), the fluorescence changes become progressively slower than folding monitored by tyrosine absorbance or by 2′CMP binding. Under these conditions the fluorescence‐detected kinetics of folding show properties of proline isomerization reactions. In addition they match proline isomerization measured during refolding of Us by a two‐step assay for non‐native proline isomers.The following conclusions are drawn. (1) There is a kinetic intermediate between US and the native enzyme in the folding pathway of RNase A. This has been shown by the divergent progress curves monitored by different probes. Although the intermediate contains non‐native proline isomers, it is folded and hence sensitive to agents that affect the stability of native proteins. (2) Fluorescence‐detected folding closely follows proline isomerization during folding. Refolding of US is not blocked by incorrect prolinc isomers, but US is able to fold to a structural intermediate before proline isomerization occurs. (3) The quantum yield of the fluorescence of native RNase A depends on the precise spatial location of the tyrosine side chains with respect to their hydrogen bond acceptors and to the disulfide bonds. Therefore the native fluorescence is not regained until all essential proline peptide bonds are in the right configuration.