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

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.