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. 2020 Oct 9;11:5107. doi: 10.1038/s41467-020-18816-8

Fig. 8. RET allows mutational relaxation time optimisation to be monitored in situ.

Fig. 8

a Raw (vector-corrected) fluorescence quench-recovery cycles captured from intact living HEK293 cells using the protocol used in Figs. 2–6 for quantitation of LOV2 switching and relaxation of optoNES constructs with wild-type LOV2 or photocycle mutants V416I, I427V and F434L. These are tagged with Ypet because the LOV2 FRET responses are larger and clearer. The dotted line shows the changes in the Ypet vector that does not contain LOV2, which is used to correct all traces. Data are means from n = 3 wells. b Curve-fits of vector-normalised quench-recovery data from a are shown. Curve fitting is performed as described in the methods and provides activation time constants and dark dequench values plotted in c. c Relaxation time constants are estimated by exponential fits of the % of dequench values from b, and best-fit values (±S.E., n = 3 wells) plotted against dark time preceding the dequench for each construct. The red crosses represent the initial dequench value of the first measurement (taken as representing the dark state because the cells had not previously been exposed to light). Averaged parameters based on the experiments in parts a–c are listed in Table 1B. Relaxation time constants for the fast photocycle mutants I427V and F424L are 10–20 times shorter than the wild-type and V416I is about 10× longer. Relaxation is a consistent 10–15-fold faster than has been reported under cell-free conditions3,23. The extents of activation of the fast relaxing mutants are low, because of the considerable concurrent relaxation taking place during the activation conditions used. For the same reason apparent activation sensitivity and maximum dequench change inversely with relaxation rate. This is expected because a slower relaxation allows a more complete activation during the illumination phases while faster relaxation counteracts apparent activation and reduces the possible level of dequench at limiting photon flux. Source data are provided as a Source Data file.