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. 2007 May 18;93(6):2178–2187. doi: 10.1529/biophysj.106.101220

FIGURE 2.

FIGURE 2

smFRET assay for characterization of interactions between complexin and the SNARE complex. (a) Transient binding of complexin to the SNARE complex yields smFRET events characterized by increased fluorescence emission from the FRET acceptor on the SNARE complex during excitation of the FRET donor on complexin by green laser illumination. Measurements were made with complexin-39 (labeled with Alexa 555)/synaptobrevin-41 (labeled with Alexa 647). (b) Simultaneous measurements of fluorescence emission of both FRET acceptor and donor during smFRET. The FRET pair of Fig. 2 a was used. The sudden transitions upon binding and unbinding, as well as the overall intensity levels, are indicative of single-molecule level detection. (c) Simultaneous measurements of fluorescence emission of both FRET acceptor and donor during smFRET. Rare events are specially selected to illustrate the recovery of the donor emission after acceptor photobleaching or blinking. (d) Distribution of FRET efficiency for individual binding events between complexin and the SNARE complex. Labeled pair combinations are the same as in Fig. 2 a. The efficiency histogram was fit to a Gaussian distribution with a main peak of 0.65.