Real-time imaging of structure and dynamics of transmembrane biomolecules by FRET-induced single-molecule fluorescence attenuation

Tracking the transmembrane topology and conformational dynamics of membrane proteins is key to understand their functions.It is however challenging to monitor position changes of individual pro-teins in cell membranes with high sensitivity and high resolution.We review on three single-molecule fluorescence imaging methods—SIFA,LipoFRET and QueenFRET—recently developed in our lab for studying the dynamics of membrane proteins.They can be applied,progressively,to investigate mem-brane proteins in solid-supported lipid bilayers,artificial liposome membranes and live-cell plasma membranes.The techniques take advantage of the energy transfer from a fluorophore to a cloud of quenchers and are able to extract in real time positions and position changes of a single fluorophore-labeled protein in the direction normal to the membrane surface.The methods have sub-nanometer precision and have proved powerful to investigate biomolecules interacting with bio-membranes.