Work

Anfinsen Redux: Ribonuclease Folding in the Single-Molecule Regime

Year: 2025

Type: article

Abstract: 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.

Source: Journal of the American Chemical Society

DOI: 10.1021/jacs.5c13815

Authors: Arek V. Melkonian, Evans C. Wralstad, Ronald T. Raines, David R. Walt

Institutions: Brigham and Women's Hospital, Harvard University, Wyss Institute for Biologically Inspired Engineering, Massachusetts Institute of Technology

Language: English

Topic: Protein Structure and Dynamics

Subfield: Molecular Biology

Field: Biochemistry, Genetics and Molecular Biology

Domain: Life Sciences

Open Access status: green

Funders: National Institutes of Health, National Cancer Institute, National Institute of General Medical Sciences, National Science Foundation Graduate Research Fellowship Program

Awards: G1418158339, G1431139938, G9000048456, G9000059961, Protein Chemistry