Mapping Surface Hydrophobicity of α-Synuclein Oligomers at the Nanoscale

Abstract

Proteins fold into a single structural ensemble but can also misfold into many diverse structures including small aggregates and fibrils, which differ in their toxicity. The aggregate surface properties play an important role in how they interact with the plasma membrane and cellular organelles, potentially inducing cellular toxicity, however, these properties have not been measured to date due to the lack of suitable methods. Here, we used a spectrally resolved, super-resolution imaging method combined with an environmentally sensitive fluorescent dye to measure the surface hydrophobicity of individual aggregates formed by the protein α-synuclein (αS), whose aggregation is associated with Parkinson’s disease. We show that the surface of soluble oligomers is more hydrophobic than fibrils and populates a diverse range of coexisting states. Overall, our data show that the conversion of oligomers to fibril-like aggregates and ultimately to fibrils results in a reduction in both hydrophobicity and the variation in hydrophobicity. This funneling characteristic of the energy landscape explains many of the observed properties of αS aggregates and may be a common feature of aggregating proteins.

Identification of on- and off-pathway oligomers in amyloid fibril formation

“…Oligomers have variously been dened in the literature as species having smaller size, lower growth rate, less ordered structure, distinct surface properties or higher toxicity compared to brils 8,11,12,[14][15][16][17][18][19] (Fig. 2).…”

“…16). (d) Differences in surface properties, for example hydrophobicity, may be detected using fluorescence probes such as 8-anilino-1-naphthalenesulfonic acid (ANS), Nile red, 17 or by cryo-EM, in which case oligomers may have a higher tendency than fibrils to adsorb to the grid surface (adapted from ref. 14).…”

Long-term, super-resolution imaging of amyloid structures using transient amyloid binding microscopy

“…The binding affinity of Nile red to amyloid structures has been used to map the hydrophobicity of amyloid structures. 21 The image sequence (Fig. 1E) shows the blinking of single ThT molecules on an amyloid fibril.…”

PAINT using proteins: A new brush for super‐resolution artists

“…Since its initial implementation in 2006, PAINT-based SMLM has seen many innovative applications. 1,2,5,[7][8][9][10][11][12][13][14][15][16][17][18][19][20][21][22]36 The development of DNA-PAINT in 2010 represented a sea change, by providing a means to incorporate high specificity into the transient interaction. DNA-PAINT has since been optimized and extended in many ways.…”