Acquiring snapshots of the orientation of trans‐membrane protein domains using a hybrid FRET pair. Issue 8 (3rd March 2015)

• Record Type: Journal Article
• Authors: Gahl, Robert F.; Tekle, Ephrem; Zhu, Gefei Alex; Taraska, Justin W.; Tjandra, Nico
• Is Part Of: FEBS letters. Volume 589:Issue 8(2015)
• Page Start: 885
• Page End: 889
• Publication Date: 2015-03-03
• Subjects: Fluorescence -- Förster resonance energy transfer (FRET) -- Confocal microscopy -- Membrane proteins -- Live cells -- Giant unilamellar vesicles (GUV); Biochemistry -- Periodicals; Biophysics -- Periodicals; Molecular biology -- Periodicals; Biochimie -- Périodiques; Biochemistry; Biophysics; Molecular biology; Periodicals; 572.05
• DOI: 10.1016/j.febslet.2015.02.030
• Languages: English
• ISSNs: 0014-5793

Abstract

Abstract : 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. Abstract : Determining the orientation of macromolecules in a membrane is very difficult. Integrating a confocal microscope with a pulsed electric field is a powerful solution. This setup can determine the orientation of molecules rapidly with spatial specificity. The orientation of a transmembrane helix in a pore forming protein was determined. This approach can be applied to a variety of systems including live cells.