Rapid Scan Electron Paramagnetic Resonance Spectroscopy Is a Suitable Tool to Study Intermolecular Interactions of Intrinsically Disordered Protein

SIMPLE SUMMARY: To understand complex cellular processes, the investigation of intrinsically disordered proteins that are involved in the most crucial mechanisms is of utmost importance. However, intrinsically disordered proteins lack a well-defined structure and exhibit vast flexibility. A prominent example is α-synuclein, that under pathological conditions, self-interacts and forms highly-ordered filamentous aggregates characteristic of neurodegenerative disorders, e.g., Parkinson’s disease. Due to its broad conformational ensemble, its investigation is hampered, and suitable biophysical methods to elucidate underlying interactions and mechanisms on a molecular level are scarce. Investigating the aggregation process and prevailing kinetics is essential to gain insight into the causes of the pathological processes and eventually invent effective treatments. Here, we set out to investigate the accelerated aggregation process of α-synuclein and its disease mutants in the presence of ethanol. For the first time, we made use of rapid scan electron paramagnetic resonance spectroscopy to study the kinetics of α-synuclein aggregation. We were able to highlight differences between different protein variants and demonstrated that this approach outperforms conventional techniques in terms of sensitivity and rapidity of data acquisition and successfully demonstrated that this technique is suitable for studying intermolecular interactions with fast kinetics. ABSTRACT: Intrinsically disordered proteins (IDPs) are involved in most crucial cellular processes. However, they lack a well-defined fold hampering the investigation of their structural ensemble and interactions. Suitable biophysical methods able to manage their inherent flexibility and broad conformational ensemble are scarce. Here, we used rapid scan (RS) electron paramagnetic resonance (EPR) spectroscopy to study the intermolecular interactions of the IDP α-synuclein (aS). aS aggregation and fibril deposition is the hallmark of Parkinson’s disease, and specific point mutations, among them A30P and A53T, were linked to the early onset of the disease. To understand the pathological processes, research intensively investigates aS aggregation kinetics, which was reported to be accelerated in the presence of ethanol. Conventional techniques fail to capture these fast processes due to their limited time resolution and, thus, lose kinetic information. We have demonstrated that RS EPR spectroscopy is suitable for studying aS aggregation by resolving underlying kinetics and highlighting differences in fibrillization behavior. RS EPR spectroscopy outperforms traditional EPR methods in terms of sensitivity by a factor of 5 in our case while significantly reducing data acquisition time. Thus, we were able to sample short time intervals capturing single events taking place during the aggregation process. Further studies will therefore be able to shed light on biological processes proceeding on fast time scales.