Fig. 1.
In vitro activation of recombinantly produced JSiR1 and JSR1. (A and B) Normalized UV-vis spectra of JSiR1 (9-cis retinal, λmax 505 nm, A) and JSR1 (11-cis retinal, λmax 535 nm, B) recorded in solution. Ground states (inactive) are represented by blue (A) and green (B) curves, respectively. Photoproducts (λmax 535 nm) are indicated in black. The Insets of A and B represent normalized UV-vis spectra of JSiR1 (A) and JSR1 (B) recorded in LCP. (C) G protein activation assay, where intrinsic tryptophan fluorescence from the Gα subunit is recorded upon addition of GTPγS (24). Photoactivated recombinant JSiR1 triggers Gi protein dissociation (dark purple curve; K = 5.65 ± 0.24 × 10−3) The dark pink curve indicates inactive JSiR1 whose activity is slightly higher (K = 0.51 ± 0.002 × 10−3) than that of the control (no JSiR1 in light pink; K = 0.32 ± 0.001 × 10−3). The value after the ± sign indicates the SE mean (SEM). All data are measured in triplicates at 20 °C at a pH of 7.3. A concentration of 600 nM of heterotrimeric Gi was used for the assay.