Acquiring snapshots of the orientation of trans-membrane protein domains using a hybrid FRET pair

One challenge in studying the function of membrane-embedded proteins is determining the orientation of key domains in the context of the changing and dynamic membrane environment. We describe a confocal microscopy setup that utilizes external electric field pulses to direct dipicrylamine (DPA) to a membrane leaflet. The detection of FRET between DPA and a fluorescent probe attributes it to the inner or outer leaflet of a membrane. By utilizing short acquisition times and confocal imaging, this attribution could be made even in changing membrane environments. Our setup adds versatility to the study of the biological activity of membrane-embedded proteins. Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies.