title: Transition metal ion FRET to measure short-range distances at the intracellular surface of the plasma membrane.
abstract: Biological membranes are complex assemblies of lipids and proteins that serve as platforms for cell signaling. We have developed a novel method for measuring the structure and dynamics of the membrane based on fluorescence resonance energy transfer (FRET). The method marries four technologies: (1) unroofing cells to isolate and access the cytoplasmic leaflet of the plasma membrane; (2) patch-clamp fluorometry (PCF) to measure currents and fluorescence simultaneously from a membrane patch; (3) a synthetic lipid with a metal-chelating head group to decorate the membrane with metal-binding sites; and (4) transition metal ion FRET (tmFRET) to measure short distances between a fluorescent probe and a transition metal ion on the membrane. We applied this method to measure the density and affinity of native and introduced metal-binding sites in the membrane. These experiments pave the way for measuring structural rearrangements of membrane proteins relative to the membrane.
authors.affiliation: Department of Physiology and Biophysics, University of Washington, Seattle, WA 98195 seg@uw.edu zagotta@uw.edu.
authors.forename: Sharona E
authors.initials: SE
authors.lastname: Gordon
authors.affiliation: Department of Physiology and Biophysics, University of Washington, Seattle, WA 98195.
authors.forename: Eric N
authors.initials: EN
authors.lastname: Senning
authors.affiliation: Department of Physiology and Biophysics, University of Washington, Seattle, WA 98195.
authors.forename: Teresa K
authors.initials: TK
authors.lastname: Aman
authors.affiliation: Department of Physiology and Biophysics, University of Washington, Seattle, WA 98195 seg@uw.edu zagotta@uw.edu.
authors.forename: William N
authors.initials: WN
authors.lastname: Zagotta
mesh_terms: D000818:Animals; D001665:Binding Sites / Q000502:physiology; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods; D005456:Fluorescent Dyes / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D008565:Membrane Proteins / Q000378:metabolism; D008670:Metals / Q000378:metabolism*; D011485:Protein Binding / Q000502:physiology; D051381:Rats
chemical_list: D005456:Fluorescent Dyes; D008565:Membrane Proteins; D008670:Metals