Resolving the Nanoscale Structure of β-Sheet Peptide Self-Assemblies Using Single-Molecule Orientation–Localization Microscopy

Abstract

Synthetic peptides that self-assemble into cross-β fibrils are versatile building blocks for engineered biomaterials due to their modularity and biocompatibility, but their structural and morphological similarities to amyloid species have been a long-standing concern for their translation. Further, their polymorphs are difficult to characterize by using spectroscopic and imaging techniques that rely on ensemble averaging to achieve high resolution. Here, we utilize Nile red (NR), an amyloidophilic fluorogenic probe, and single-molecule orientation−localization microscopy (SMOLM) to characterize fibrils formed by the designed amphipathic enantiomers KFE8 L and KFE8 D and the pathological amyloid-beta peptide Aβ42. Importantly, NR SMOLM reveals the helical (bilayer) ribbon structure of both KFE8 and Aβ42 and quantifies the precise tilt of the fibrils' inner and outer backbones in relevant buffer conditions without the need for covalent labeling or sequence mutations. SMOLM also distinguishes polymorphic branched and curved morphologies of KFE8, whose backbones exhibit much more heterogeneity than those of 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…”

“…[35][36][37] By combining the TAB labeling technique with SMOLM, it is possible to visualize how the nanoscale organization of amyloid assemblies affects the orientations of dye molecules bound to their surfaces. In this way, SMOLM has resolved nanoscale structural heterogeneities in amyloid fibers 38,39,40 and distinct dye binding configurations dependent on amyloid fibril orientation. 17 Despite these developments, the precise links between the dye orientations sensed by SMOLM, which we call the amyloid fiber's orientation signature, and their structural dynamics remains unknown.…”

“…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.…”

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

“…To address these weaknesses, one may augment SMLM with spectroscopic capabilities − or measure fluorophores’ positions and orientations via single-molecule orientation localization microscopy (SMOLM). − SMOLM has been successfully applied to study actin filaments, − molecular motors, − DNA conformations, , and membrane composition and fluidity. − By combining the TAB labeling technique with SMOLM, it is possible to visualize how the nanoscale organization of amyloid assemblies affects the orientations of dye molecules bound to their surfaces. In this way, SMOLM has resolved nanoscale structural heterogeneities in amyloid fibers − and distinct dye binding configurations dependent on amyloid fibril orientation . Despite these developments, the precise links between the dye orientations sensed by SMOLM, which we call the amyloid fiber’s orientation signature, and their structural dynamics remains unknown.…”

“…Correlated multidye studies, , where each fluorophore has unique binding behaviors and/or environmental sensitivity, could elucidate new nanoscale insights into amyloid structure when combined with SMOLM. Through careful analysis of the SMOLM data, structural polymorphisms in the β-sheet assemblies may be resolvable …”