NATURE OF THE EARLY FOLDING INTERMEDIATE OF RIBONUCLEASE-A

A previous study of the folding pathway of the major unfolded species of ribonuclease A by pulsed hydrogen exchange [Udgaonkar, J. B., and Baldwin, R. L. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 8197-8201] showed that there is a major early folding intermediate (I-1) that resembles a molten globule species in having stable secondary structure while lacking buried tyrosine side chains. Earlier work showed that there is also a late native-like folding intermediate (I-N) that can bind the specific inhibitor 2'CMP and that has buried tyrosine side chains. Results are reported here indicating that I-1 has a well-developed tertiary structure even though its tyrosine side chains are not buried. First, optical stopped-flow experiments suggest that I-1 binds 2'CMP. Second, the protection against hydrogen exchange is similar in I-1 and I-N for almost all protected amide protons studied. Third, analysis of the mechanism of hydrogen exchange in I-1 confirms the large protection factors reported earlier for probes in the beta-sheet of ribonuclease A and indicates that the beta-sheet is formed in I-1. Other experiments are also reported that test the interpretation of pulsed hydrogen exchange studies of the folding pathway of ribonuclease A.