Folding of ribonuclease A from a partially disordered conformation. Kinetic study under transition conditions

Bovine pancreative ribonuclease A (RNase A), denatured by 3.5 M LiClO4 (pH 3.0), has some ordered conformation as indicated by a high retention of alpha-helix and compact structure. This effect of LiClO4 was confirmed by the observation that the alpha-helix of isolated S-peptide is stabilized in the presence of 3.5 M LiClO4 (pH 3.0), as measured by circular dichroism and nuclear magnetic resonance. The effect of the retained alpha-helices and compact structure on the folding kinetics of RNase A was studied by comparison with the kinetic folding from urea-denatured RNase A, which has no ordered structure. In contrast to our previous study under folding conditions [Denton, J.B., Konishi, Y., & Scheraga, H. A. (1982) Biochemistry 21, 5155], the kinetic folding/unfolding experiments were carried out here in the transition regions between native and LiClO4-denatured RNase A and between native and urea-denatured RNase A. The measured relaxation times were extrapolated to the triple point, where native RNase A, LiClO4-denatured RNase A, and urea-denatured RNase A have the same thermodynamic stability and are at the same concentration, in order to compare the rates of these two processes under the same solvent conditions. Under these conditions, both folding and unfolding pathways are studied simultaneously without any accumulated intermediates. No significant acceleration of folding was observed from LiClO4-denatured RNase A as compared to that from urea-denatured RNase A. This indicates that all ordered structures in RNase A are not equivalent in their influence on the folding pathway; some may play an essential role and some may not.(ABSTRACT TRUNCATED AT 250 WORDS)