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Transition metal ion FRET to measure short-range distances at the intracellular surface of the plasma membrane

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) u...

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Detalles Bibliográficos
Autores principales: Gordon, Sharona E., Senning, Eric N., Aman, Teresa K., Zagotta, William N.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: The Rockefeller University Press 2016
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4727948/
https://www.ncbi.nlm.nih.gov/pubmed/26755772
http://dx.doi.org/10.1085/jgp.201511530
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author Gordon, Sharona E.
Senning, Eric N.
Aman, Teresa K.
Zagotta, William N.
author_facet Gordon, Sharona E.
Senning, Eric N.
Aman, Teresa K.
Zagotta, William N.
author_sort Gordon, Sharona E.
collection PubMed
description 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.
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spelling pubmed-47279482016-08-01 Transition metal ion FRET to measure short-range distances at the intracellular surface of the plasma membrane Gordon, Sharona E. Senning, Eric N. Aman, Teresa K. Zagotta, William N. J Gen Physiol Methods and Approaches 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. The Rockefeller University Press 2016-02 /pmc/articles/PMC4727948/ /pubmed/26755772 http://dx.doi.org/10.1085/jgp.201511530 Text en © 2016 Gordon et al. This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/3.0/).
spellingShingle Methods and Approaches
Gordon, Sharona E.
Senning, Eric N.
Aman, Teresa K.
Zagotta, William N.
Transition metal ion FRET to measure short-range distances at the intracellular surface of the plasma membrane
title Transition metal ion FRET to measure short-range distances at the intracellular surface of the plasma membrane
title_full Transition metal ion FRET to measure short-range distances at the intracellular surface of the plasma membrane
title_fullStr Transition metal ion FRET to measure short-range distances at the intracellular surface of the plasma membrane
title_full_unstemmed Transition metal ion FRET to measure short-range distances at the intracellular surface of the plasma membrane
title_short Transition metal ion FRET to measure short-range distances at the intracellular surface of the plasma membrane
title_sort transition metal ion fret to measure short-range distances at the intracellular surface of the plasma membrane
topic Methods and Approaches
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4727948/
https://www.ncbi.nlm.nih.gov/pubmed/26755772
http://dx.doi.org/10.1085/jgp.201511530
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