Fig. 5: smFRET trajectories of immobilized substrates.
From: Direct observation of subunit rotation during DNA strand exchange by serine recombinases
Donor (green) and acceptor (red) intensity traces are shown in the top panels, and FRET traces (black) and fit (red) after Hidden Markov Modelling (HMM) are shown in the bottom panels. A Stitched (after every 25 s, indicated by dividers and red lines) time trajectories of surface-immobilized SUL25 incubated with Sin Q115R at 37 °C. The red overlaid line shows the result from HMM analysis fit to 4 states. The FRET histograms of all filtered traces (on the right) were fit to Gaussian curves giving E values of 0.09, 0.19, 0.47, and 0.77. These values are in good agreement with predicted E values for Sin Q115R ‘inversion’ shown in Fig. 3A. B Analysis as in (A) is shown for UL25 recombination with Tn3 NM. The Gaussian fit gave E values E = 0.05, 0.20, 0.56, 0.81, which also had good correlation with predicted E values shown in Fig. 3B. C–F TIRF time trajectories of surface-immobilized SUL25 in the presence of Sin Q115R at 37 °C and their respective FRET histograms. (C) lnr \(\leftrightarrow\) cnr transitions, (D) cnr \(\leftrightarrow\) cr, (E) cr \(\leftrightarrow\) lr, (F) one molecule showing transitions between all states.
