Table 1.
Properties of constructs examined in this study determined by resonance energy transfer.
| Actuator | Maximal dequench or quench1 (% of Flimit) | Apparent activation rate2 (µmol m−2) | Relaxation time constant in dark3 (s) | (µmol m−2) | Dequench or quench limit5 (%) | Sensitivity6 (µmol m−2) |
|---|---|---|---|---|---|---|
| A | ||||||
| mTurquoise2 fusions | ||||||
| optoNES | 15.9 ± 0.3 | 115 ± 5 | 6.8 ± 0.6 | 5.78 ± 0.22 | 18.9 ± 0.2 | 162 ± 4 |
| spacer-optoNES | 4.6 ± 0.1 | 131 ± 18 | 7.2 ± 1.1 | 6.41 ± 0.77 | 6.5 ± 0.2 | 192 ± 15 |
| dC7-408LOV2-NES21 | 25.7 ± 0.8 | 121 ± 7 | 11.5 ± 1.5 | 2.24 ± 0.29 | 23.6 ± 0.4 | 124 ± 8 |
| dC8-408LOV2-NES21 | 20.1 ± 0.6 | 134 ± 2 | 12.4 ± 1.6 | 2.29 ± 0.07 | 19.3 ± 0.1 | 137 ± 2 |
| dC9-408LOV2-NES21 | 16.9 ± 0.6 | 148 ± 4 | 13.3 ± 2.1* | 2.20 ± 0.13 | 16.6 ± 0.1 | 146 ± 4 |
| dC10-408LOV2-NES21 | 23.3 ± 0.8 | 148 ± 9 | 17.2 ± 2.3* | 1.77 ± 0.2 | 21.2 ± 0.3 | 146 ± 8 |
| dC11-408LOV2-NES21 | 21.1 ± 0.6 | 122 ± 7 | 11.0 ± 1.3 | 2.57 ± 0.33 | 20.4 ± 0.4 | 130 ± 8 |
| optoJNKi wt | 16.1 ± 0.6 | 163 ± 10.9 | 13.7 ± 1.8 | 3.7 ± 0.5 | 17 ± 0.5 | 206 ± 18 |
| optoJNKiC450A | n/a | n/a | n/a | n/a | n/a | n/a |
| optop38i3 | 16.4 ± 0.6 | 162 ± 5.9 | 13.8 ± 1.8 | 3.7 ± 0.3 | 17.2 ± 0.3 | 204 ± 10 |
| optop38i5 | 16.9 ± 0.6 | 163 ± 5.1 | 14.4 ± 1.8 | 3.5 ± 0.2 | 17.6 ± 0.3 | 203 ± 9 |
| Ypet fusions | ||||||
| optoNES | 54.5 ± 0.8 | 493 ± 9 | 8.0 ± 0.5 | 18.6 ± 1.1 | 53.5 ± 0.7 | 559 ± 12 |
| spacer-optoNES | 29.8 ± 1.1 | 430 ± 65 | 7.6 ± 1.4 | 15.9 ± 7.4 | 33.2 ± 3.7 | 479 ± 83 |
| optoJNKi | 65.6 ± 0.9 | 785 ± 4 | 23 ± 1.1 | 10.1 ± 0.1 | 60.4 ± 0.2 | 843 ± 5 |
| mScarlet fusions | ||||||
| optoNES | 39.4 ± 0.8 | 1140 ± 159 | 7.2 ± 0.7 | 58.3 ± 22.7 | 53.2 ± 2.1 | 1619 ± 348 |
| spacer-optoNES | 51.9 ± 0.8 | 955 ± 5 | 7.3 ± 0.5 | 44.7 ± 0.9 | 64.2 ± 0.2 | 1263 ± 12 |
| optoJNKi | 61.7 ± 1.6 | 1315 ± 33 | 15.5 ± 1.6 | 28.9 ± 1.6 | 68.8 ± 0.5 | 1594 ± 49 |
| Actuator (Ypet fusion with LOV2 mutation) | Max quench at 140 µmol m−2 (% of F0) | Apparent activation rate (µmol m−2) at 140 µmol m−2 | Relaxation time constant in situ (s) | Relaxation time constant reported cell-free (s) | ||
|---|---|---|---|---|---|---|
| B | ||||||
| optoNES wt | 57.4 ± 1.0 | 492 ± 5 | 7.6 ± 0.62 | 80 (Zoltowski et al. [23]) | ||
| optoNES V416I | 54.1 ± 1.6 | 584 ± 7 | 84.1 ± 4.7 | 821 (Zoltowski et al. [23]) | ||
| optoNES I427V | 28.2 ± 0.6 | 272 ± 9 | 0.81 ± 0.14 | 6 (Zayner et al. [3]) | ||
| optoNES F434L | 23.8 ± 1.0 | 237 ± 8 | 0.58 ± 0.25 | 12 (Zayner et al. [3]) | ||
Values shown are estimated best-fit ± standard error from curve fitting as described in the methods.
1. Maximal dequench or quench—this is the equilibrium impact on emission from the fluorescent protein on repeated illumination with 438 nm light (cf Fig. 2e). It is caused by resonance energy transfer to or from the nearby LOV2, but the equilibrium point depends also on continuous relaxation. For this reason the value depends on the rate (~4 Hz) and intensity of 438 nm light flashes, which is indicated. mTurquoise2 fusions were dequenched at 14 µmol m−2 whereas Ypet and mScarlet fusions were less sensitive and were quenched at 140 µmol m−2.
2. Apparent activation rate is the measure of apparent sensitivity of switching, again influenced by concomitant relaxation and dependent on photon flux, which is indicated for each group. mTurquoise2 fusions were activated at 14 µmol m−2 whereas Ypet and mScarlet fusions were less sensitive and were activated at 140 µmol m−2.
3. Relaxation time in darkness is calculated from periods of darkness and is independent of activating photon flux. In Table 1B, the measured values of relaxation mutants in the optoNES context are compared to literature values of recombinant cell-free LOV2 mutant without actuator or fluorescent protein fusion.
4. ED50 is the estimated photon dose at the rate used (~4 Hz) predicted to achieve half-maximal switching at equilibrium, taking concomitant relaxation into account.
5. Dequench or quench limit—this is the estimated relaxation independent of maximal possible quench (for acceptors Ypet and mScarlet) or dequench (for mTurquoise2).
6. Sensitivity is the estimated sensitivity value in the absence of relaxation derived from curve fit formulae that take the concomitant relaxation rate into account.
An asterisk denotes statistically significant difference in relaxation between optoNES and dC9 (P = 0.0364) and dC10 (P = 0.0004), and spacer-optoNES and dC10 (P = 0.001) constructs by one-way ANOVA and post-hoc Tukey test. No other significant difference was found among the mTurquoise-optoNES construct in situ relaxation rates.