Figure 6
Single-molecule burst analysis of FlAsH-FCMybbR-CoA-AF647. (a) Schematic depiction of single FlAsH-FCMybbR-CoA-AF647 diffusing through the confocal detection volume illuminated with blue and red light. (b) Burst brightness distribution of the donor (FlAsH) and acceptor (CoA-AF647) for dually-labelled peptides upon 480 nm excitation. (c,d) The distribution of MLE donor lifetimes in the absence of the acceptor (τDo), and in the presence of the acceptor (τDA). (e) FRET histogram calculated from intensity bursts of FlAsH-FCMybbR-CoA-Alexa647 using equations (6,7) and fitted to a sum of Gaussian components with major peaks at E = 0% (red) and E = 90% (green) with side peaks at E = 19% (blue) and E = 67% (purple) which most likely describe bridging effects between the two major FRET peaks. (f) 2D plot of donor-acceptor stoichiometry calculated using equation (8) as a function of FRET efficiency. The measurements were performed in the presence of 20 mM cysteamine and 0.01% Tween20 in a PBS buffer (0 M NaCl, pH 7.0).
