Figure 8: Improving smFRET–ALEX measurements by using NPA-based fluorophore as FRET acceptor on the protein GlnPQ–SBD2.

(a) Crystal structures of the SBD2 (T369C/S451C) open (left panel, PDB: 4KR5) and closed state (right panel, PDB:4KQP, after binding of the ligand glutamine shown in red) with label positions of donor (D) and acceptor (A). (b) Corresponding one-dimensional histograms of E*-values for increasing amounts of ligand glutamine where an increased E*=0.55±0.1 (dashed line, closed conformation) becomes visible (as opposed to the open conformation, E*=0.36±0.1, dashed line). Excitation intensities of 30 kW cm⁻² at 532 nm and 20 kW cm⁻² at 640 nm; data evaluated with dual colour burst search and displayed with additional per-bin thresholds of F(DD)+F(DA)+F(AA)>75 and minimal number of counts per bin in ALEX histogram of 3. (c,d,e) 2D histograms of joint pair values of S (labelling stoichiometry) and E* (FRET efficiency, that is, interprobe distance) of Cy3B/ATTO647N in the presence (c) and absence (d) of 2 mM TX and Cy3B/NPA–ATTO647N (e) without ligand glutamine. Excitation intensities of 30 kW cm⁻² at 532 nm and 20 kW cm⁻² at 640 nm; data evaluated with all photon burst search and displayed with additional per-bin thresholds of F(DD)+F(DA)+F(AA)>100 and minimal number of counts per bin in ALEX histogram of 3. (f,g,h) Histogram of fluorophore brightness values as determined from photon-counting histograms (PCHs) on single-molecule transits of labelled SBD2 diffusing through the observation volume, comparison of donor brightness F(DD) (f), acceptor brightness when excited via FRET, F(DA) (g) and acceptor brightness via direct red excitation F(AA) (h). Excitation intensities of 30 kW cm⁻² at 532 nm and 20 kW cm⁻² at 640 nm. (i,j,k) Dependence of the mean count rate of F(DD), F(DA) and F(AA) of the different samples for increasing excitation intensity, respectively.