Detecting Single-Molecule Dynamics on Lipid Membranes with Quenchers-in-a-Liposome FRET

Tracking membrane-interacting molecules and visualizing their conformational dynamics are key to understanding their functions. It is, however, challenging to accurately probe the positions of a molecule relative to a membrane. Herein, a single-molecule method, termed LipoFRET, is reported to assess interplay between molecules and liposomes. It takes advantage of FRET between a single fluorophore attached to a biomolecule and many quenchers in a liposome. This method was used to characterize interactions between alpha-synuclein (alpha-syn) and membranes. These results revealed that the N-terminus of alpha-syn inserts into the membrane and spontaneously transitions between different depths. In contrast, the C-terminal tail of alpha-syn is regulated by calcium ions and floats in solution in two conformations. LipoFRET is a powerful tool to investigate membrane-interacting biomolecules with sub-nanometer precision at the single-molecule level.