It is a big challenge to measure position changes of biomolecules in the direction normal to the plasma membranes of living cells. We developed a one donor-multiple quenchers Fӧrster resonance energy transfer method by using non-fluorescent quenchers in the extracellular environment. It senses subnanometer position changes of a fluorophore-labeled biomolecule in the plasma membrane. The method was validated by monitoring flip-flops of individual lipid molecules incorporated in plasma membranes. We studies membrane perforation by a host defense peptide from the extracellular side and found that the pore-forming peptide is dynamic, switching among different insertion depths. The method is especially useful in studying interactions of membrane proteins with the inner surfaces of plasma membranes. Our method will find wide applications in systematic analysis of fundamental cellular processes at plasma membranes.

Tracking membrane-interacting molecules and visualizing their conformational dynamics are key to understanding their functions.I ti s, however,c hallenging to accurately probe the positions of amolecule relative to amembrane. Herein, as ingle-molecule method, termed LipoFRET,i s reported to assess interplay between molecules and liposomes. It takes advantage of FRET between as ingle fluorophore attached to abiomolecule and many quenchers in aliposome. This method was used to characterizei nteractions between asynuclein (a-syn) and membranes.T hese results revealed that the N-terminus of a-syn inserts into the membrane and spontaneously transitions between different depths.Incontrast, the C-terminal tail of a-syn is regulated by calcium ions and floats in solution in two conformations.LipoFRET is apowerful tool to investigate membrane-interactingbiomolecules with sub-nanometer precision at the single-molecule level.Supportinginformation and the ORCID identification number(s) for the author(s) of this article can be found under: https://doi.