Journal
JOURNAL OF GENERAL PHYSIOLOGY
Volume 147, Issue 2, Pages 189-200Publisher
ROCKEFELLER UNIV PRESS
DOI: 10.1085/jgp.201511530
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Funding
- National Eye Institute of the National Institutes of Health (NIH) [R01EY017564, R01EY010329]
- National Institute of Mental Health [R01MH102378]
- National Institute of General Medical Sciences of the NIH [R01GM100718]
- National Institute of Neurological Disease and Stroke [F32NS077622]
- National Heart Lung Blood Institute of the NIH [T32HL007312]
- American Heart Association [14IRG18770000]
- NIH [S10RR025429, P30DK017047, P30EY001730]
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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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