Phylogenetic relationship between Drosophila melanogaster opsins. The tree was constructed from a ClustalW alignment with bovine rhodopsin as an outgroup. Both neighbor joining and unweighted pair group methods were used. The trees were bootstrapped 1000 replications, and each node was supported 100% by both methods. The class of each pigment is indicated, referred to in Ref. 34. λ_max corresponds to the maximal sensitivity of flies expressing each visual pigment in the R1–6 photoreceptor cells (17, 45, 46). The amino acid (AA) residue present at the site corresponding to bovine rhodopsin Ala-292 is indicated.

Molecular modeling of Drosophila Rh1 wild-type, Rh1A315S, and Rh1A315C mutant pigments. a, model of Drosophila Rh1 wild-type pigment showing all residues within 3.2 Å of the lysine-bound Schiff base chromophore. b–d, close-up views of the Schiff base nitrogen and residue 315 in Rh1 wild type (b), Rh1A315S (c), and Rh1A315C (d). The 3-hydroxy-11-cis-retinal chromophore is shown in red with the Schiff base nitrogen in dark blue and the 3-hydroxyl oxygen in green. The α-carbons are shown in black. Hydrogen atoms are omitted except on water molecules, which are shown in light purple. Wild-type and mutated residues (315) are colored cyan. Sequence numbering is based on Swiss-Prot accession number P06002 (19). e, alignment of the 3-hydroxy-retinal chromophore from Rh1 wild type (purple), Rh1A315S (yellow), and Rh1A315C (blue). The 3-hydroxyl oxygen atoms are shown in green. The arrowheads illustrate the rotation of the chromophore ring in the mutant models Rh1A315S (two o'clock) and Rh1A315C (three o'clock) relative to Rh1 wild type (nine o'clock). f, alignment of the Drosophila Rh1 model (green) and squid rhodopsin crystal structure (PDB entry 2z73; purple). The panel is a ribbon diagram showing the orientation of the helices in the region of the chromophore. g, close-up view of the Schiff base nitrogen and residue Val-301 (corresponding to Rh1 Ala-315) in the region of the chromophore. Sequence numbering is based on Swiss-Prot Accession Number P31356 (47). Higher resolution images of the Drosophila Rh1 model structures and squid rhodopsin crystal structure are shown in supplemental Figs. 1–4.

Rh1 and Rh6 spectral sensitivities and rhodopsin/metarhodopsin difference spectra. a and b, measured spectral sensitivities of flies expressing Rh1, Rh6, or one of the modified rhodopsin pigments in the R1–6 photoreceptor cells (solid lines). Each sensitivity spectrum obtained in this study was also fit to a rhodopsin absorption nomogram (dotted lines). a, mean spectral sensitivities of flies expressing Rh6 S313A (black line) when compared with flies expressing the unmodified Rh6 pigment (gray line). The nomogram curve fit for Rh6 S313A has a λ_max red-shifted from the unmodified pigment 17 nm (dotted lines). For all sensitivity data, the λ_max, correlation coefficient, and number of flies examined are indicated in Table 1. b, mean spectral sensitivities of flies expressing the rhodopsin mutants, Rh1 A315S, and Rh1 A315C (black lines) when compared with the unmodified Rh1 pigment (gray line). The large peak in the UV region occurs because of the action of a sensitizing pigment that absorbs in the UV and activates the Rh1 rhodopsin through energy transfer. The peaks in the visible region are due to direct absorption by the visual pigment itself. The nomogram curve fits for Rh1 A315C and Rh1 A315S λ_max are blue-shifted 4 and 10 nm, respectively (inset). The fine structure noted in the sensitivity spectra in the region of 475 nm is an artifact and results from spectral spikes in xenon lamp output in this region. c–f, difference spectra were measured by in vivo MSP of flies expressing Rh1 or one of the modified rhodopsin pigments. c, mean difference spectra of Rh1 (gray line) when compared with Rh1 A315S and Rh1 A315C (black lines). d–f, calculated R and M absorption spectra based on nomogram curve fitting to the measured difference spectra (DS). In all calculations, the λ_max for R spectra was set to the λ_max measured physiologically. For MSP data, the λ_max, correlation coefficient, and number of flies examined are indicated in Table 1.