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SG Britt, E Salcedo; Spectral Tuning of Ultraviolet and Blue Absorbing Visual Pigments in Invertebrates . Invest. Ophthalmol. Vis. Sci. 2002;43(13):4550.
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© ARVO (1962-2015); The Authors (2016-present)
The genes encoding six Drosophila opsins have previously been isolated and characterized (Rh1-6). Each of these pigments is expressed in a distinct class of photoreceptor cells and differs in their absorption properties. Purpose: To identify residues important for regulating the absorption differences between the ultraviolet (UV) and short-wavelength (SW) subfamilies of invertebrate opsins. Methods: We performed a sequence alignment of the known members of these families of invertebrate visual pigments. We identified 27 residues that were conserved within the UV and SW groups, but that differed between them. We further restricted these sites to include only amino acids that differed in charge or polarity. Based on this analysis, we constructed a series of nine mutant genes encoding variants of the Drosophila Rh5 pigment (a SW pigment with λmax = 437 nm) containing single or multiple amino acid changes corresponding to those within the Drosophila Rh3 pigment (a UV pigment with λmax = 345 nm). The modified genes were expressed in the R1-6 photoreceptor cells of y w; ninaE mutant flies. These animals are a suitable host strain because they lack the Rh1 visual pigment normally expressed in these photoreceptor cells. We characterized the spectral sensitivity of the transgenic flies electrophysiologically. Results: We found that of the nine constructs and twelve amino acids that were modified, all but one caused little or no change in the spectral sensitivity profile of the animals expressing them. Interestingly, we found that a single amino acid change, Rh5 N104K, caused a dramatic 52 nm blue shift, in the spectral sensitivity of animals expressing this construct. Conclusion: These findings suggest that the introduction of a lysine residue at this position in the invertebrate UV pigments is responsible for a large part of the spectral difference between the SW and UV subfamilies of invertebrate pigments. This position corresponds to G90 in bovine rhodopsin, a residue in close proximity to the chromophore. Amino acids at this position have been shown to play a role in the tuning of avian UV and violet visual pigments. The introduction of a positively charged lysine at this position would be expected to disrupt the interaction between the neighboring counter-ion and the positively charged Schiff's base, and potentially produce a pigment having a dramatically blue shifted absorption maximum.
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