Resolving the nanoscale structure of β-sheet assemblies using single-molecule orientation-localization microscopy

Abstract

Synthetic peptides that self-assemble into cross-β fibrils have remarkable utility as engineered biomaterials due to their modularity and biocompatibility, but their structural and morphological similarity to amyloid species has been a long-standing concern for their translation. Further, their polymorphs are difficult to characterize using spectroscopic and imaging techniques that rely on ensemble averaging to achieve high resolution. Here, we utilize single-molecule orientation-localization microscopy (SMOLM) to characterize fibrils formed by the designed amphipathic enantiomers, KFE8Land KFE8D, and the pathological amyloid-beta peptide Aβ42. SMOLM reveals that the orientations of Nile red, as it transiently binds to both KFE8 and Aβ42, are consistent with a helical (bilayer) ribbon structure and convey the precise tilt of the fibrils' inner and outer backbones. SMOLM also finds polymorphic branched and curved morphologies of KFE8 whose backbones exhibit much more heterogeneity than those of more typical straight fibrils. Thus, SMOLM is a powerful tool to interrogate the structural differences and polymorphism between engineered and pathological cross β-rich fibrils.

Single-Molecule Orientation Imaging Reveals the Nano-Architecture of Amyloid Fibrils Undergoing Growth and Decay

“…Through careful analysis of the SMOLM data, structural polymorphisms in the β-sheet assemblies may be resolvable. 38 Beyond our proof-of-principle experiments, different Aβ42 seedings can be utilized to prepare and analyze a selection of pathologically relevant Aβ42 structures, thereby enabling the study of how different preparations affect structural dynamics. More broadly, future studies can also target other amyloid peptides implicated in neurodegeneration, such as Aβ40.…”