Optical Structural Analysis of Individual α‐Synuclein Oligomers

Abstract

Supportinginformation and the ORCID identification number(s) for the author(s) of this article can be found under https://doi.org/10.

Single-Molecule Localization Microscopy of 3D Orientation and Anisotropic Wobble using a Polarized Vortex Point Spread Function

“…S1 By carefully analyzing images from the polarized vortex microscope, we can further measure the 3D orientation and rotational diffusion of each NR molecule. As observed previously, 10,65 azimuthal orientations φ show that individual NR molecules bind mostly parallel to the long axis of the fibrils (Figure 4B, std. dev.…”

“…This anisotropy gradually disappears as NR orientations become more varied (large σ φ ), suggesting that β-sheet stacking is largely disordered within these small aggregates. We note that SMOLM data clearly distinguish between more ordered, "fibril-like" oligomeric structures 65 (Figures 4C and S14D) vs. disordered, amorphous oligomers (Figures 4D,E and S14E), even for amyloid structures aggregated under identical conditions within the same field of view. Differences between these particles cannot be resolved in standard SMLM reconstructions (Figures 4C-E and S14D,E insets).…”

“…61,62 In particular, the cross-β architecture of amyloid fibrils exhibit β-sheet strands that run perpendicular to the long axis of the fibril with grooves formed by its amino-acid side chains. 63 These grooves serve as binding sites of amyloid-sensitive fluorescent molecules, such as NR and Thioflavin T. 64,65 In addition to sensing hydrophobicity, 66 we hypothesize that the rotational diffusion of NR molecules, while bound transiently to surfaces of amyloid aggregates, may be used to quantify the organization of the stacked β sheets. Here, we incubated recombinant amyloid-β (1-42) peptides (Aβ42) on ozone-cleaned coverslips to form amyloid fibrils (see Methods for further details).…”