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

Fluorescence readouts for an amyloidfibril sensor critically dependon its molecular interaction and local environment offered by theavailable structural motifs. Here we employ polarized points accumulationfor imaging in nanoscale topography with intramolecular charge transferprobes transiently bound to amyloid fibrils to investigate the organizationof fibril nanostructures and probe binding configurations. Besidesthe in-plane (theta approximate to 90 degrees) mode for binding on the fibrilsurface parallel to the long fibril axis, we also observed a sizablepopulation of over 60% out-of-plane (theta < 60 degrees) dipolesfor rotor probes experiencing a varying degree of orientational mobility.Highly confined dipoles exhibiting an out-of-plane configuration probablyreflect tightly bound dipoles in the inner channel grooves, whilethe weakly bound ones on amyloid enjoy rotational flexibility. Ourobservation of an out-of-plane binding mode emphasizes the pivotalrole played by the electron donor amino group toward fluorescencedetection and hence the emergence of anchored probes alongside conventionalgroove binders.

Automated summary

This article investigates the molecular interaction and local environment of an amyloid fibril sensor by using fluorescence readouts. The study focuses on the organization of fibril nanostructures and the binding configurations of the sensor. The observation of both in-plane and out-of-plane binding modes emphasizes the important role of the electron donor amino group in fluorescence detection.