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K.R. Babu, L. Ramos, H. Zapata, R.R. Birge, B.E. Knox; Extracellular Loop 2 (EC2) Plays a Central Role in Structure-function Relationship of Short Wavelength Visual Pigments . Invest. Ophthalmol. Vis. Sci. 2003;44(13):5116.
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Purpose :The extracellular loop 2 (EC2) connecting transmembrane helices 4 and 5 plays a crucial role in structure-function of visual pigments, forming part of the chromophore-binding pocket. The purpose of this study is to explore the role of EC2 loop in a short wavelength (λmax = 427 nm) cone opsin (VCOP) from Xenopus. Methods: Selected conserved amino acid residues in the EC2 loop was replaced by site-directed mutagenesis. The plasmid with the desired mutation was expressed in COS1 cells by transient transfection. The expressed opsin was reconstituted with 11-cis retinal and purified on immunoaffinity chromatography. The purified pigment was then analyzed by UV-Vis/Cryogenic/ FTIR spectroscopy and biochemical techniques. Results: Single amino acid mutants (D, H, Q, S) at residue E176 (equivalent to position 181 in bovine rhodopsin) did not stably bind 11-cis retinal. Whereas two substitutions (T and C) at S181 (equivalent position 186 in bovine rhodopsin) bound retinal to a limited degree and exhibited a blue shift in the absorption spectrum. Three other mutants (S181A, S181D, S181E) failed to stably bind retinal. Interchanging the positions of the conserved Glu and Ser (E176S/S181E) eliminated 11-cis retinal binding. Coordinated changes in the side chains (E176D/S181T) were not able to rescue the deleterious effect of substitution of Glu with Asp. Movement of E176 closer to S181 (E176G/L178E) also failed to stable form a pigment. A substitution of the conserved cysteine at position 182 to alanine was not tolerated in VCOP where as a similar change at C180 produced a blue shifted pigment. Conclusions: In contrast to bovine rhodopsin, where substitutions in EC2 loop are tolerated, equivalent mutants drastically alter VCOP structure. Thus it is seems likely that in cone pigments there are additional interactions with other amino acid residues. Together, these data indicate a crucial role of EC2 loop in maintaining the structural and functional integrity in the violet cone opsin.
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