Single-molecule orientation-localization microscopy resolves the nanoscale organization of self-assembled peptides.

Abstract

Self-assembling peptides have remarkable applications in biomaterials and therapeutics. Amphipathic peptides with alternating polar and nonpolar residues tend to self-assemble into cross-β-rich fibrils, which are usually signatures of various protein-misfolding pathologies. Key to understanding the mechanisms of self-assembly is the ability to visualize and quantify the complex, heterogeneous organization of these peptides with single-aggregate sensitivity. Here, we utilize single-molecule orientation-localization microscopy (SMOLM), a variant of super-resolution microscopy, to measure amphipathic KFE8 (Ac-FKFEFKFE-NH2) assemblies. Using the transient binding of Nile red (NR) as a blinking mechanism, we find that the orientation distribution of NR on KFE8 is significantly different from that bound to amyloid-beta fibrils, where NR is simply oriented parallel to the coverslip and along the long axis of each fiber. A detailed analysis of NR orientations on KFE8 shows that they bind in a manner consistent with a helical ribbon model of the underlying peptide assembly. Further, the 3D orientations of NR enable us to quantify the pitch-to-diameter ratio of the underlying helix. Finally, we show that SMOLM can resolve a variety of nanoscale structures formed by model self-assembling peptides. Due to limited spatial resolution, standard localization-based super-resolution microscopy cannot resolve these details. These experiments are the first demonstration of utilizing fluorophore orientation “spectra”, i.e., the 3D orientations of dye molecules, to resolve the complex nanoscale organization of self-assembled peptides in solution.