Anfinsen Redux: Ribonuclease Folding in the Single-Molecule Regime.

Ribonuclease A (RNase A) has long been known to spontaneously refold to its native conformation and recover catalytic activity following denaturation. Yet, the molecular heterogeneity of its folding pathway has remained unclear due to ensemble averaging in traditional bulk measurements. Here, we use single-molecule enzymology (SME) to monitor the catalytic recovery of urea-denatured RNase A at the single-molecule level. We directly observe that individual RNase A molecules exhibit either fast or slow refolding kinetics, indicating distinct renaturation pathways that implicate slow prolyl peptide bond isomerization. These findings establish the utility of SME in revealing hidden heterogeneity in protein folding pathways.