Single-Molecule Orientation Imaging Reveals Two Distinct Binding Configurations on Amyloid Fibrils

Abstract

Fluorescence readouts for an amyloid fibril sensor critically depend on its molecular interaction and local environment offered by the available structural motifs. Here we employ polarized points accumulation for imaging in nanoscale topography with intramolecular charge transfer probes transiently bound to amyloid fibrils to investigate the organization of fibril nanostructures and probe binding configurations. Besides the in-plane (θ ≈ 90°) mode for binding on the fibril surface parallel to the long fibril axis, we also observed a sizable population of over 60% out-of-plane (θ < 60°) dipoles for rotor probes experiencing a varying degree of orientational mobility. Highly confined dipoles exhibiting an out-of-plane configuration probably reflect tightly bound dipoles in the inner channel grooves, while the weakly bound ones on amyloid enjoy rotational flexibility. Our observation of an out-of-plane binding mode emphasizes the pivotal role played by the electron donor amino group toward fluorescence detection and hence the emergence of anchored probes alongside conventional groove binders.

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

“…As such, it is important to expand the library of TAB-compatible fluorophores , both to probe dye–structure interactions and to broaden the applicability of TAB SMOLM to various classes of peptide assemblies. Even using existing probes, our imaging strategy should be readily adaptable to helices of different radii, periodicity, peptide sequences, and chirality.…”

“…In the past decade, single-molecule localization microscopy (SMLM) has become a powerful super-resolution fluorescence modality for quantitatively investigating the structural features of amyloids. − By repeatedly localizing “flashes” from individual fluorophores over time, SMLM has visualized the static and dynamic behavior of self-assembling fibrillar systems and their intermolecular interactions. , Beyond covalent labeling, binding-activated blinking of environment-sensitive amyloidophilic dyes can also be used for SMLM. Termed transient amyloid binding (TAB), a variety of dyes, including p-TFAA, Nile red (NR), − thioflavin T, thioflavin X, SYPRO orange, and LDS772, can visualize self-assembling fibrillar systems under physiological conditions for hours to days. However, with localization precisions of ∼10 nm, ,− , TAB SMLM cannot interrogate nanometer-scale details.…”

“…To overcome this limitation, combined imaging of fluorophore positions and orientations, termed single-molecule orientation–localization microscopy (SMOLM), has been explored to characterize fibrillar nanostructures with exquisite detail. ,,,, Orientation precisions of 4–8° are possible when imaging dim emitters (∼500 photons), and when using bright fluorophores (∼5000 photons), 2.0° orientation precision in 3D can be achieved . Analogous to structural tools like X-ray and electron diffraction, high-precision imaging of fluorophore orientations (angles) yields exquisite nanoscale details of the fibril architecture without the need for nanometer-scale spatial resolution.…”

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

“…We anticipate that recent advances in utilizing fluorogenic dyes for SMOLM will enable improved understanding of amyloid aggregation. Correlated multi-dye studies, 17,42 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.…”

“…In addition, super-resolved single-molecule localization microscopy (SMLM) has become popular for amyloid imaging. Both binding activated localization microscopy (BALM) 14 and transient amyloid binding (TAB) imaging 15 utilize amyloidophilic dyes, such as p-TFAA, Thioflavin T (ThT), Thioflavin X 16 , Nile red, SYPRO orange, and LDS772 17 , whose fluorescence quantum yields and/or emission spectra change when the dyes are in proximity of amyloid fibers. These forms of SMLM can image amyloid aggregates for hours to days without immunostaining or covalently modifying the peptides themselves.…”