Investigation of α-synuclein fibril structure by site-directed spin labeling

The misfolding and fibril formation of alpha- synuclein plays an important role in neurodegenerative diseases such as Parkinson disease. Here we used electron paramagnetic resonance spectroscopy, together with site-directed spin labeling, to investigate the structural features of alpha- synuclein fibrils. We generated fibrils from a total of 83 different spin- labeled derivatives and observed single-line, exchange-narrowed EPR spectra for the majority of all sites located within the core region of alpha- synuclein fibrils. Such exchange narrowing requires the orbital overlap between multiple spin labels in close contact. The core region of alpha- synuclein fibrils must therefore be arranged in a parallel, inregister structure wherein same residues from different molecules are stacked on top of each other. This parallel, in-register core region extends from residue 36 to residue 98 and is tightly packed. Only a few sites within the core region, such as residues 62-67 located at the beginning of the NAC region, as well as the N- and C- terminal regions outside the core region, are significantly less ordered. Together with the accessibility measurements that suggest the location of potential alpha- sheet regions within the fibril, the data provide significant structural constraints for generating three-dimensional models. Furthermore, the data support the emerging view that parallel, in-register structure is a common feature shared by a number of naturally occurring amyloid fibrils.