Figure 3: RP mutants are functional visual pigments.

(a) Ultraviolet-visible spectra of purified WT rhodopsin and RP-linked rhodopsin mutants. The solid traces correspond to spectra measured in the dark, whereas the dashed traces represent spectra obtained after illuminating the samples for 90 s with >515 nm light. (b) G protein activation assay. The traces show the time course of intrinsic fluorescence of the G protein transducin. Fluorescence increases on GDP–GTP exchange in the Gα subunit, corresponding to its activation. The sample containing transducin and a catalytic amount of rhodopsin was illuminated for 60 s, starting at t=0 s. Nucleotide exchange was initiated by addition of GTPγS (arrrow). At t=400 s, an excess amount of light-activated WT rhodopsin was added to test for the completeness of transducin activation. (c) Transducin fluorescence measurement as in b, conducted before and after light activation of rhodopsin (arrow hν). After adding GTPγS at t=60 s, the intrinsic fluorescence of transducin did not change due to lack of active rhodopsin. At t=200 s, the sample was illuminated (arrow) to activate rhodopsin and initiate GDP–GTP exchange. (d) Retinal release. The traces show the intrinsic fluorescence increase on retinal Schiff base hydrolysis and retinal release from light-activated rhodopsin. The reaction was initiated by illumination of rhodopsin at t=90 s. At t=3,100 s, NH₂OH was added to hydrolyse the remaining retinal Schiff base, thereby facilitating the release of any remaining retinal. For b–d, the fluorescence was normalized to the level at the start of the recording and the traces in each panel are vertically displaced for clarity (the displacements are 0.02 units in b, 0.05 units in c and 0.2 units in d).