Subunit counting in membrane-bound proteins

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.

Figure 1

Bleaching steps of single fluorescent protein complexes reveal the number of GFP-labeled subunits. (a) A single image of the acquired sequence shows the selected fluorescent spots in the cell membrane (blue circles). Scale bar, 2 μm. (b) The different labeling schemes include 1, 2 or 4 subunits of a membrane protein complex fused to GFP. (c) Time courses of fluorescence emission for the labeling schemes in b (two examples each from spots bleaching with the expected number of bleaching steps). Green arrows indicate the bleaching steps. The y-axis is scaled in photons per second for comparison purposes (for calibration see Supplementary Methods).

Figure 2

Distribution of bleaching steps for channels with different numbers of labeled subunits. (a) Numbers A × B in the sectors indicate that A spots with B bleaching steps were observed. Green sector, number of bleaching steps expected from biochemical and functional studies. Red sectors, number of bleaching steps different from what is expected. (b) The observed numbers of spots having 1, 2, 3 and 4 bleaching steps in the experiment with the tetrameric CNG channel (red) match closely a calculated binomial distribution (blue), assuming a probability of 77.5% that the GFP is fluorescent.