MECHANISTIC INSIGHTS INTO PROTEIN MISFOLDING IN NEURODEGENERATIVE DISEASES USING SINGLE-MOLECULE TECHNIQUES

Abstract

Neurodegenerative diseases such as Alzheimer's, Parkinson's, and amyotrophic lateral sclerosis (ALS) are strongly associated with the misfolding and aggregation of key proteins, often driven by oxidative stress. In this study, we employed single-molecule techniques—including fluorescence resonance energy transfer (smFRET), atomic force microscopy, and optical tweezers—to investigate how oxidative stress modulates the folding dynamics of disease-associated proteins including amyloid-β, tau, α-synuclein, TDP-43, and FUS. Experimental conditions simulated increasing oxidative stress through graded hydrogen peroxide (H₂O₂) exposure. Our results revealed a clear dose-dependent relationship between oxidative stress and both the rate of protein misfolding and extent of aggregation. Quantitative analysis across eight datasets demonstrated that higher ROS levels significantly elevated misfolding rates (up to a 12-fold increase) and aggregation percentages (reaching 70% in high-stress conditions). Single-molecule tracking enabled detection of transient misfolding intermediates otherwise missed by ensemble techniques. Fitting data of ten kinetic sets to thermodynamic models showed that whenever oxygen was removed, proteins kept their native shapes. The findings point to oxidative stress being an important early reason why proteins accumulate dangerously in brain disorders. Moreover, what we do helps prove the importance of single-molecule studies in understanding events that steer cells away from functioning correctly. Thanks to these findings, scientists are designing ways to help manage redox balance and protein folding which could slow or stop age-related decline in the brain.