Conserved Residues in the Extracellular Loops of Short-Wavelength Cone Visual Pigments

The role of the extracellular loop region of a short-wavelength sensitive pigment, Xenopus violet cone opsin, is investigated via computational modeling, mutagenesis, and spectroscopy. The computational models predict a complex H-bonding network which stabilizes and connects the EC2-EC3 loop and the N-terminus. Mutations which are predicted to disrupt the H-bonding network are shown to produce visual pigments that do not stably bind chromophore and exhibit properties of misfolded protein. The potential role of a disulfide bond between two conserved Cys residues, Cys105 in TM3 and Cys182 in EC2, is necessary for proper folding and trafficking in VCOP. Lastly, certain residues in the EC2 loop are predicted to stabilize the formation of two anti-parallel β strands joined by a hairpin turn, which interact with the chromophore via H-bonding or Van der Waals interactions. Mutations to conserved residues result in a decrease in chromophore binding. These results demonstrate that the extracellular loops are crucial for the formation of this cone visual pigment. Moreover, there are significant differences in structure and function of this region in VCOP compared to rhodopsin.