Three types of cone cells exist in the human retina， each containing a different pigment responsible for the initial step of phototransduction. These pigments are distinguished by their specific absorbance maxima: 425 nm (blue)， 530 nm (green)， and 560 nm (red). Each pigment contains a common chromophore， 11-cis-retinal covalently bound to an opsin protein via a Schiff base. The 11-cis-retinal protonated Schiff base has an absorbance maxima at 440 nm in methanol. Unfortunately， the chemistry that allows the same chromophore to interact with different opsin proteins to tune the absorbance of the resulting pigments to distinct λ values is poorly understood. Rhodopsin is the only pigment with a native s... More
Three types of cone cells exist in the human retina， each containing a different pigment responsible for the initial step of phototransduction. These pigments are distinguished by their specific absorbance maxima: 425 nm (blue)， 530 nm (green)， and 560 nm (red). Each pigment contains a common chromophore， 11-cis-retinal covalently bound to an opsin protein via a Schiff base. The 11-cis-retinal protonated Schiff base has an absorbance maxima at 440 nm in methanol. Unfortunately， the chemistry that allows the same chromophore to interact with different opsin proteins to tune the absorbance of the resulting pigments to distinct λ values is poorly understood. Rhodopsin is the only pigment with a native structure determined at high resolution. Homology models for cone pigments have been generated， but experimentally determined structures are needed for a precise understanding of spectral tuning. The principal obstacle to solving the structures of cone pigments has been their innate instability in recombinant constructs. By inserting five different thermostabilizing proteins (BRIL， T4L， PGS， RUB， and FLAV) into the recombinant green opsin sequence， constructs were created that were up to 9-fold more stable than WT. Using cellular retinaldehyde-binding protein (CRALBP)， we developed a quick means of assessing the stability of the green pigment. CRALBP testing also confirmed an additional 48-fold increase in pigment stability when varying the detergent used. These results suggest an efficient protocol for routine purification and stabilization of cone pigments that could be used for high-resolution determination of their structures， as well as for other studies.
