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

Area of Science:

• Biochemistry
• Protein dynamics
• Enzymology

Background:

• Ribonuclease A (RNase A) is known for its ability to refold and regain catalytic function after denaturation.
• Traditional bulk measurements average protein behavior, obscuring heterogeneity in folding pathways.

Purpose of the Study:

• To investigate the molecular heterogeneity of RNase A refolding at the single-molecule level.
• To identify distinct protein folding pathways and their underlying mechanisms.

Main Methods:

• Utilized single-molecule enzymology (SME) to monitor urea-denatured RNase A.
• Observed the catalytic recovery of individual RNase A molecules over time.

Main Results:

• Direct observation of distinct fast and slow refolding kinetics in individual RNase A molecules.
• Identified distinct renaturation pathways, suggesting slow prolyl peptide bond isomerization as a key factor.

Conclusions:

• Single-molecule enzymology is effective in uncovering hidden heterogeneity in protein folding.
• RNase A exhibits molecular heterogeneity in its refolding pathways, challenging previous ensemble-averaged views.