PURPOSE: To isolate and characterize the rhodopsin cDNA from the fish, Astyanax fasciatus, and to determine the effect of tyrosine 261 on its spectral tuning. METHODS: The rhodopsin cDNA was cloned using reverse transcription-polymerase chain reaction amplification and then sequenced. A mutant, Y261F, was generated by site-directed mutagenesis. Both wild type and mutant were transiently expressed in COS-1 cells, regenerated with 11-cis retinal, and purified by immunoaffinity chromatography. Ultraviolet-visible spectrophotometry was used to determine wavelength of maximum absorption. RESULTS: A fasciatus rhodopsin cDNA exhibits 80% amino acid identity with bovine rhodopsin. In contrast to all known rhodopsins, this rhodopsin contains a tyrosine instead of a phenylalanine at amino acid position 261. Indeed, this particular amino acid replacement has been implicated in the long wavelength absorption of the red cone pigment. Site-directed mutagenesis was used to change the Astyanax amino acid 261 to phenylalanine (Y261F). Expression of the Y261F mutant in COS-1 cells showed an absorbance maximum of 496 nm, compared to 504 nm for the wild type pigment. CONCLUSIONS: A naturally occurring fish rhodopsin is red shifted about 8 nm due to one critical amino acid substitution.
PURPOSE: To isolate and characterize the rhodopsin cDNA from the fish, Astyanax fasciatus, and to determine the effect of tyrosine 261 on its spectral tuning. METHODS: The rhodopsin cDNA was cloned using reverse transcription-polymerase chain reaction amplification and then sequenced. A mutant, Y261F, was generated by site-directed mutagenesis. Both wild type and mutant were transiently expressed in COS-1 cells, regenerated with 11-cis retinal, and purified by immunoaffinity chromatography. Ultraviolet-visible spectrophotometry was used to determine wavelength of maximum absorption. RESULTS: A fasciatus rhodopsin cDNA exhibits 80% amino acid identity with bovinerhodopsin. In contrast to all known rhodopsins, this rhodopsin contains a tyrosine instead of a phenylalanine at amino acid position 261. Indeed, this particular amino acid replacement has been implicated in the long wavelength absorption of the red cone pigment. Site-directed mutagenesis was used to change the Astyanax amino acid 261 to phenylalanine (Y261F). Expression of the Y261F mutant in COS-1 cells showed an absorbance maximum of 496 nm, compared to 504 nm for the wild type pigment. CONCLUSIONS: A naturally occurring fish rhodopsin is red shifted about 8 nm due to one critical amino acid substitution.
Authors: Emma E Tarttelin; Elena Frigato; James Bellingham; Viviana Di Rosa; Roberto Berti; Nicholas S Foulkes; Robert J Lucas; Cristiano Bertolucci Journal: J Exp Biol Date: 2012-08-15 Impact factor: 3.312