Figure 3. Photoswitching of LOV2Jα in OptoJNKi measured by spectrophotometry.

(a) Absorption spectra of recombinant OptoJNKi (AsLOV2Jα-JIP11, 1 mg ml⁻¹), shown at 30 s intervals at 20 °C after bleaching (using 470 nm light, 6.4 mW cm⁻² for 1 min), show a recovery consistent with FMN adduct decay with a t_½ in the order of 60 s. Representative spectra are shown (n=3). (b) A corresponding absorbance spectral time series was acquired for recombinant AsLOV2Jα (1 mg ml⁻¹), showing high similarity with optoJNKi, suggesting minimal disturbance of the photocycle by fusion of the JIP11 peptide. Representative spectra are shown (n=3). (c) Absorbance recovery kinetics at 455 nm is shown for recombinant OptoJNKi and AsLOV2Jα at both 20 and 37 °C. Traces are means of three replicates for each condition. Each individual replicate was fitted to exponentials and time constants, shown as mean±s.e.m. (n=3), indicate OptoJNKi has recovery kinetics marginally faster than AsLOV2Jα. Strikingly however, adduct decay is >three times faster at 37 °C than 20 °C.