Intermediates in the refolding of ribonuclease at subzero temperatures. 3. Multiple folding pathways.

The kinetics of refolding of ribonuclease A have been measured at -15 degrees C by monitoring the intrinsic fluorescence and absorbance signals from the six tyrosine residues. For each probe multiphasic kinetics were observed. The burial of tyrosine residues, as determined by the change in absorbance at 286 nm, revealed four phases, whereas the kinetics of refolding monitored by fluorescence revealed only two phases. The rates of the transients detected by fluorescence were independent of pH. One of the faster transients detected by delta A286 involved a decrease in absorbance, which is consistent with solvent exposure, rather than burial, and suggests the possibility of an abortive partially folded intermediate in the earlier stages of folding. Double-jump unfolding assays were used to follow the buildup and decay of an intermediate in the refolding reaction at -15 degrees C. At both pH* 3.0 and pH* 6.0 the maximum concentration of the intermediate was 25-30% of the total protein. The existence of a second pathway of slow folding was inferred from the difference in rate of formation of native enzyme and breakdown of the observed intermediate, and by computer simulations. In addition, the unfolding assay demonstrated that 20% of the unfolded protein was converted to native at a much faster rate, consistent with observations in aqueous solution that 80% of unfolded ribonuclease A consists of slow-folding species. Kinetics and amplitude data from these and other refolding experiments with different probes were used to develop possible models for the pathway of refolding. The simplest system consistent with the results for the slow-refolding species involves two parallel pathways with multiple intermediates on each of them. Several independent lines of evidence indicate that about 30% of the unfolded state refolds by the minor pathway, in which the slowest observed phase is attributed to the isomerization of Pro-93. The major pathway involves 50% of the unfolded state; the reason why it refolds slowly is not apparent. A native-like intermediate is formed considerably more rapidly in the major slow-refolding pathway, compared to the minor pathway.