The aggregation and amyloid formation of α-synuclein is associated with Parkinson’s
disease and other synucleinopathies. In its native, monomeric form α-synuclein is
an intrinsically disordered protein represented by highly dynamic conformational ensembles.
Inhibition of α-synuclein aggregation using small molecules, peptides, or proteins
has been at the center of interest in recent years. Our aim was to explore the effects
of cross-linking on the structure and aggregation/amyloid formation properties of
α-synuclein. Comparative analysis of available high-resolution amyloid structures
and representative structural models and MD trajectory of monomeric α-synuclein revealed
that potential cross-links in the monomeric protein are mostly incompatible with the
amyloid forms and thus might inhibit fibrillation. Monomeric α-synuclein has been
intramolecularly chemically cross-linked under various conditions using different
cross-linkers. We determined the location of cross-links and their frequency using
mass spectrometry and found that most of them cannot be realized in the amyloid structures.
The inhibitory potential of cross-linked proteins has been experimentally investigated
using various methods, including thioflavin-T fluorescence and transmission electron
microscopy. We found that conformational constraints applied by cross-linking fully
blocked α-synuclein amyloid formation. Moreover, DTSSP-cross-linked molecules exhibited
an inhibitory effect on the aggregation of unmodified α-synuclein as well.
