Dynamics of Protein Folding Unraveled through Single-Molecule Förster Resonance Energy Transfer

The intricate process of protein folding is pivotal to understanding cellular mechanisms and disease pathologies. In this study, we aim to elucidate the folding pathways of proteins by employing single-molecule Förster Resonance Energy Transfer (smFRET). This technique allows for real-time observation of conformational changes at the single-molecule level, providing unprecedented insight into folding dynamics. We selected a model protein, ubiquitin, and performed smFRET experiments under different solvent conditions. Our findings revealed discrete folding intermediates, with transition rates measured in the microsecond range. Notably, the presence of 2M guanidinium chloride extended the folding time by approximately 35% compared to native conditions, suggesting significant solvent effects on protein stability. Principal Component Analysis (PCA) of the FRET efficiency trajectories identified three primary folding pathways, each with distinct kinetic signatures. Our results offer a quantitative framework for predicting folding mechanisms, which may be instrumental in understanding misfolding diseases. Future studies could expand on these pathways to explore therapeutic interventions.