Abstract
The subunit number and stoichiometry of membrane-bound proteins are difficult to determine without disrupting their membrane environment. Here we describe a single-molecule technique for counting subunits of proteins in live cell membranes by observing bleaching steps of GFP fused to a protein of interest. After testing the method with proteins of known stoichiometry expressed in Xenopus laevis oocytes, we resolved the composition of NMDA receptors composed of NR1 and NR3 subunits.
Publication types
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Research Support, N.I.H., Extramural
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Research Support, Non-U.S. Gov't
MeSH terms
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Animals
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Calcium Channels / chemistry
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Cells, Cultured
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Cloning, Molecular
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Cyclic Nucleotide-Gated Cation Channels
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Green Fluorescent Proteins / chemistry
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Ion Channels / chemistry
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Membrane Proteins / biosynthesis
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Membrane Proteins / chemistry*
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Oocytes / metabolism
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Protein Subunits / biosynthesis
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Protein Subunits / chemistry*
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Receptors, N-Methyl-D-Aspartate / biosynthesis
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Receptors, N-Methyl-D-Aspartate / chemistry
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Recombinant Fusion Proteins / biosynthesis
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Recombinant Fusion Proteins / chemistry*
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Xenopus laevis
Substances
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Calcium Channels
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Cyclic Nucleotide-Gated Cation Channels
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Ion Channels
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Membrane Proteins
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Protein Subunits
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Receptors, N-Methyl-D-Aspartate
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Recombinant Fusion Proteins
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Green Fluorescent Proteins