Structural intermediates trapped during the folding of ribonuclease A by amide proton exchange.

In the folding reaction of the slow-folding species (US) of ribonuclease A (RNase A), the slow isomerization of wrong proline isomers provides a suitable trap for kinetic folding intermediates at low temperatures (0--10 degrees C). Partly folded intermediates are known to accumulate before proline isomerization takes place, after which native RNase A is formed. We have been able to measure the protection from amide proton exchange which is provided by structure in the intermediates at different times along the folding pathway. Previous work has shown that, by labeling the amide protons of the unfolded protein before initiating refolding, an early folding intermediate can be detected. The new pulse-labeling method presented here can be used to label later folding intermediates. Our results indicate that, in conditions which strongly favor the native protein, intermediates are formed which provide protection against exchange. However, when folding is initiated in 2.5 M Gdn . HCl, 10 degrees C, pH 7.5, conditions in which folding goes to completion but there are no spectroscopically detectable intermediates, then no intermediates are detected by our method. Alternate minimal mechanisms for the folding of US are presented.