Studies of the intermediates in the folding of ribonuclease A

Abstract

Previous work from this laboratory has shown that proline isomerization can be used as a kinetic trap for intermediates in the folding of RNase A. There are two classes of unfolded RNase A: a fast-folding class, U/sub F/, and a major (80%) slow folding class, U/sub S/ (1), The U/sub F/ in equilibrium U/sub S/ interconversion reaction in unfolded RNase A has been shown to be acid-catalyzed and to have other specific properties of proline isomerization. Thus, it is probable that U/sub S/ contains one or more proline isomers in wrong cis or trans conformations. We have evidence that, in the folding of RNase A at low temperatures, structural intermediates accumulate before proline isomerization. An assay has been devised for wrong proline isomers which can be used during folding. These data point to the existence of a quasi-native intermediate, I/sub N/, formed late in the folding reaction. I/sub N/ appears to be folded, as measured both by tyrosine absorbance and by 2'CMP binding, but has one or more prolines in the wrong cis-trans conformation, as measured by the assay for wrong proline isomers. Proline isomerization provides a suitable trap for intermediates in folding since: (a) proline isomerization is the finalmore » and slowest step of the folding reaction; (b) folding does occur before proline isomerization takes place; and (c) the kinetic trap is applicable to the major form of the unfolded protein (U/sub S/).« less