Nature of the early folding intermediate of ribonuclease A

Abstract

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. Sco. U.S.A. 87, 8197-8201] showed that there is a major early folding intermediate (I₁) 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 I₁ has a well-developed teriary structure even though its tyroside side chains are not buried. First, optical stopped-flow experimentes suggest that I₁ binds 2'CMP. Second, the protection against hydrogen exchange is similar in I₁ and I_N for almost all protected amide protons studied. Third, analysis of the mechanism of hydrogen exchange in I₁ confirms the large protection factors reported earlier for probes in the β-sheet of ribonuclease A and indicated that β-sheet is formed in I₁. Other experiments are also reported that test the interpretation of pulsed hydrogen exchange studies of the folding pathway of ribonuclease A.