A Novel Tool to Investigate the Early and Late Stages of α‑Synuclein Aggregation

The accumulation of an inherently disordered protein α-synuclein (α-syn) aggregates in brain tissue play a pivotal role in the pathology and etiology of Parkinson’s disease. Aggregation of α-syn has been found to be complex and heterogeneous, occurring through multitudes of early- and late-stage intermediates. Because of the inherent complexity and large dynamic range (between a few microseconds to several days under in vitro measurement conditions), it is difficult for the conventional biophysical and biochemical techniques to sample the entire time window of α-syn aggregation. Here, for the first time, we introduced the Z-scan technique as a novel tool to investigate different conformations formed in the early and late stage of temperature and mechanical stress-induced α-syn aggregation, in which different species showed its characteristic nonlinear characteristics. A power-dependent study was also performed to observe the changes in the protein nonlinearity. The perceived nonlinearity was accredited to the thermal-lensing effect. A switch in the sign of the refractive nonlinearity was observed for the first time as a signature of the late oligomeric conformation, a prime suspect that triggers cell death associated with neurodegeneration. We validate Z-scan results using a combination of different techniques, like thioflavin-T fluorescence assay, fluorescence correlation spectroscopy, Fourier-transform infrared spectroscopy, and atomic force microscopy. We believe that this simple, inexpensive, and sensitive method can have potential future applications in detecting/monitoring conformations in other essential peptides/proteins related to different neurodegenerative and other human diseases.