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Acquiring snapshots of the orientation of trans‐membrane protein domains using a hybrid FRET pair
Author(s) -
Gahl Robert F.,
Tekle Ephrem,
Zhu Gefei Alex,
Taraska Justin W.,
Tjandra Nico
Publication year - 2015
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/j.febslet.2015.02.030
Subject(s) - membrane , confocal , förster resonance energy transfer , context (archaeology) , biophysics , membrane protein , confocal microscopy , chemistry , nanotechnology , materials science , fluorescence , biology , microbiology and biotechnology , physics , biochemistry , optics , paleontology
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.