CHARACTERIZATION OF A RIBONUCLEASE-S REFOLDING INTERMEDIATE

During ribonuclease S (RNase S) refolding, two peptide fragments recog nize each other, and bind together to form a refolding intermediate wh ich slowly converts to the native state. We have characterized this re folding intermediate using absorbance, circular dichroism (CD), and nu clear magnetic resonance (NMR) spectroscopies. These techniques reveal significant amounts of both secondary and tertiary structure; the int ermediate differs from a molten globule in being packed and native-lik e, but it resembles a molten globule in having no near-ultraviolet (UV ) CD spectrum. Final refolding is slow and accompanies proline isomeri zation. The results show that at least two separate stages are observe d in the formation of the tertiary structure of RNaseS.