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Wavelength [nm]

Figure 7. Spectral absorptions of the rhodopsin and metarhodopsin states of visual pigments expressed in the Drosophila compound eye. Calculated nomograms of visual pigments. Top: rhodopsin (P-) states, maximum absorption (nm) Rhl 493, Rh3 - 341, Rh4 - 358, Rh5 - 492, Rh6 - 513. Bottom: metarhodopsin (M-) states: Rhl - 565, Rh3 - 468, Rh4 - 470, Rh5 - 492, Rh6 - 472.

negatively charged amino acid side chain, which is properly positioned in the 3D structure of the respective opsin to serve as a counter-ion for the posively charged retinylidene Schiff base. In the absence of a distinct charged counter-ion, the required electron density may be provided by several properly positioned amino acid side chains [14]. Spectral tuning of rhodopsins in invertebrates is a complex challenge as one has not only to explain the bathochromic and hypsochromic shifts in the absorption spectra of rhodopsins but also the absorption shifts that occur upon the formation of stable metarhodopsin states. The rhodopsin systems present in the compound eye of Drosophila cover a wavelength range of about 350 nm (Figure 7), while the absorption of the corresponding stable metarhodopsins spreads over about 400 nm from the red to the UV spectral range. Rh2, the rhodopsin expressed in the photoreceptors of ocelli, absorbs light within the same range and is characterized by a maximum absorption of rhodopsin at 418 nm and of metarhodopsin at 506 nm [43,106,107]. So far, there is no evidence that 7TM proteins of the rhodopsin type are responsible for photon absorption outside the wavelength limits given by the rhodopsins/metarhodopsins expressed in Drosophila. Thus, the visual system of Drosophila provides the full set of wavelenth regulations realized in other invertebrate visual systems. To obtain information on the mechanism of spectral tuning of rhodopsins in Drosophila, Britt et al. [108] used germline transformation to generate transgenic flies that express chimeric rhodopsin molecules. By systematically replacing transmembrane domains of Rhl with the corresponding regions of Rh2 and vice versa, they were able to shift the spectral properties between the absorption limits given by Rhl and Rh2, as determined by ERG recordings and microspectrophotometry. Tuning of the native rhodopsins to other wavelengths was only observed with chimeric rhodopsins in which multiple novel transmembrane segments were introduced. The study revealed that the absorption of the rhodopsin and metarhodopsin state is tuned independently, and that spectral tuning of rhodopsin occurs as a coordinated process involving more than one region of opsin.

Octopus rhodopsin is the only invertebrate visual pigment for which highly resolved 3D structures are available, which could help to select amino acids possibly involved in wavelength regulation [74]. However, since Octopus is not accessible to mutagenesis studies, one is restricted to the analysis of primary, secondary and tertiary structure data. In an intraspecific comparison of 2D structures, as shown in Figure 2, one expects to find candidates involved in spectral tuning among the non-conserved amino acids located near the chromophore. A more refined selection may be achieved by comparing the structure of phylogenetically closely related insect rhodopsins with similar absorption characteristics. Gärtner [14] has provided such an analysis in which the distinct variations in the primary/secondary structure revealed by sequence alignments of invertebrate rhodopsins were evaluated together with spectral tuning data obtained for vertebrate rhodopsins. This evaluation included results of computer simulations, of studies using polyene model compounds, as well as biochemical and biophysical studies of recombinantly expressed rhodopsins. Such comparative studies help to define amino acid residues which possibly account for spectral shifts, e.g. those allowing the detection of UV-light [14]. They do not, however, eliminate the need to test the deduced function of particular amino acids in spectral tuning by heterologous expression of correspondingly mutated rhodopsins.

Variations in the chromophore structure of fly rhodopsins, i.e. exchange of 3-hydroxyretinal versus retinal, do not lead to a significant change in the absorption [109], However, 4-hydroxyretinal, the chromophore of rhodopsin from the cephalopod Watsenia scintillans, may well contribute to a blue-shift in the absorption of this rhodopsin [53]. Instead, flies have invented remarkable alternative mechanisms to optimize the wavelength range for light absorption in a photoreceptor cell. The spectral absorption of the rhodopsin expressed in the photoreceptors Rl-6 is enhanced in the UV spectral range by the interaction of rhodopsin with a sensitizing pigment identified as 3-hydroxyretinol [110-112], reviewed in [113]. The interaction is not restricted to Rhl, as other rhodopsins, if ectopically expressed in photoreceptors Rl-6 of Drosophila, also show this interaction [43]. Analysis of the fine structure of the sensitivity peak emerging in the UV leads to the proposal that the rhodopsin undergoes a rigid interaction with the 13-cis isomer of 3-hydroxyretinol [114], the exact binding sites are, however, not yet known. Finally, there remains the possibility that the spectral absorption of photoreceptor cells is broadened by the expression of more than one rhodopsin, as has been reported for the butterfly Papilio xuthus [115]. Whether dual expression of rhodopsins is realized in other invertebrates or occurs as the result of a defect in the control of rhodopsin patterning remains to be investigated.

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