Fig. 6. Activation rates and dark state relaxation rates of LOV2-based actuators fused to Ypet.

a Fluorescence emission of Ypet-optoNES, detected through a 542/26 nm filter, on excitation pulses with 14–140 µmol m⁻² of 438 nm excitation light (as Fig. 3) shows reducing intensity or quenching at a rate depending on the photon dose. This is consistent with a FRET from LOV2* to Ypet that is diminished as LOV2-390 is formed. Note 5x more light is used here than for mTq2 fusions. Similar results are obtained with b Ypet-spacer-optoNES and c Ypet-optoJNKi. Means (n = 3) are shown. d shows the best-fit maximal quench increases (±S.E., n = 3 wells) with illumination intensity increases, allowing a quench limit to be estimated by fitting to a single site model (dotted line). Quench limits, activation rate constants and relaxation time constants during illumination for the Ypet constructs, and corresponding data from mScarlet constructs, are shown in Supplementary Fig. 13, indicating FRET from LOV2 to the fluorescent tags in all cases and to a reduced extent in the spacer construct (the Ypet-spacer construct with limited energy transfer was too dim for parameter estimation). e The constructs (means of n = 3 wells shown) were subjected to cycles of activation and darkness as in Fig. 4 and fluorescence (438 nm excitation, 500 nm emission, corrected to control Ypet-C1 signal shown as dotted line) measured and f fitted to repeated exponential decays with a common time constants for each construct in each experiment; g The best-fit dequench ± S.E. from f is plotted against the preceding dark time and fitted to an exponential to derive a relaxation time in darkness. The best-fit parameters—max. quench at 140 µmol m⁻² photons, apparent activation during concurrent relaxation and relaxation time during darkness—estimated from activation-relaxation cycles are listed for both Ypet and mScarlet fusions, together with derived parameters, in Table 1A. The quench limits and sensitivities are in general consistent with d and dark relaxation times are slightly slower than lit relaxation times, suggesting a contribution of photochemical back reaction in d. Source data are provided as a Source Data file.