In Situ Continuous Wave Electron Paramagnetic Resonance Investigation of the Amyloid Aggregation of Parkinson’s Protein Alpha-Synuclein—the Second Spin-Label Position

Abstract

Self-aggregation of amyloid proteins is a crucial step in neurodegenerative disease. The protein alpha-synuclein (αS) is implicated in Parkinson’s disease. In an extension of the demonstration of in situ observation of intermediates in αS-aggregation by continuous wave (cw) EPR at room temperature (Zurlo et al. PLoS One 16: e0245548, 2021) by spin-label EPR, here the spin label is attached to position 90 (R1αS90), rather than at position 56. The aim is to determine, if the spin-label position affects the kinetics of aggregation and if local information on the intermediates is accessible. Probed by the MTSL ((1-Oxyl-2,2,5,5-tetramethylpyrroline-3-methyl) methanethiosulfonate) spin label at position 90, using diamagnetic dilution of 9:1 wild type αS to R1αS90, similar aggregation kinetics are found. Rotation correlation times for the spin label in the oligomer cannot be determined with sufficient accuracy to obtain local information on the oligomer under the conditions used. At the present stage, higher resolution EPR approaches, such as high-field EPR are more promising.

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

“…Thus, we can sample only one single time point every 30 min. Obviously, we are able to probe smaller intervals in real-time compared to the reported studies [33,34]. However, the CD spectra clearly show global changes taking place even within the first half hour, underlining that we lack sufficient time resolution when performing conventional CW EPR experiments.…”

“…Conventional continuous wave (CW) EPR experiments probe local side chain dynamics of nitroxide spin labels reflected in the spectral shape and report on aggregation progression. Using this approach, Zurlo et al were able to reveal the aggregation kinetics of singly spin-labeled aS [33,34]. However, spectra acquisition time was considerably long (3 to 8 h depending on the sample), necessitating the intermediate storage of sample aliquots and waiting time.…”