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.
Biochemistry and Biophysics Center, Laboratory of Molecular Biophysics, National Heart, Lung and Blood Institute, National Institutes of Health, 50 South Drive, Bethesda, MD 20892, USA.
Robert F
RF
Gahl
Biochemistry and Biophysics Center, Laboratory of Molecular Biophysics, National Heart, Lung and Blood Institute, National Institutes of Health, 50 South Drive, Bethesda, MD 20892, USA.
Ephrem
E
Tekle
Biochemistry and Biophysics Center, Laboratory of Molecular Biophysics, National Heart, Lung and Blood Institute, National Institutes of Health, 50 South Drive, Bethesda, MD 20892, USA.
Gefei Alex
GA
Zhu
Biochemistry and Biophysics Center, Laboratory of Molecular Biophysics, National Heart, Lung and Blood Institute, National Institutes of Health, 50 South Drive, Bethesda, MD 20892, USA.
Justin W
JW
Taraska
Biochemistry and Biophysics Center, Laboratory of Molecular Biophysics, National Heart, Lung and Blood Institute, National Institutes of Health, 50 South Drive, Bethesda, MD 20892, USA. Electronic address: tjandran@nhlbi.nih.gov.
Nico
N
Tjandra
D000818:Animals; D004247:DNA / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism; D008958:Models, Molecular; D016716:PC12 Cells; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D051381:Rats
D005456:Fluorescent Dyes; D008565:Membrane Proteins; D004247:DNA
Confocal microscopy; Fluorescence; Förster resonance energy transfer (FRET); Giant unilamellar vesicles (GUV); Live cells; Membrane proteins