Structural and functional alterations associated with deutan N94K and R330Q mutations of green cone opsin

Abstract

Deuteranopia is an X-linked congenital dichromatic condition in which single point mutations in green cone opsin lead to defective non-functional cone photoreceptor cells. Green cone opsin belongs to the G protein-coupled receptor superfamily and consists of a seven transmembrane helical apoprotein covalently bound to 11-cis-retinal, by means of a protonated Schiff base linkage, in its inactive dark state. Several point mutations in green cone opsin have been reported to cause deuteranopia, but the structural details underlying the molecular mechanisms behind the malfunction of mutated opsins have not been clearly established. Here, deutan N94K and R330Q mutants were studied by introducing these substitutions into the native green cone opsin gene by site-directed mutagenesis. The mutant proteins were purified and analyzed using UV-vis spectroscopy and transducin activation assay. We find that the N94K mutant binds the retinal chromophore by means of an unprotonated Schiff base linkage in contrast to previous studies that reported no chromophore regeneration. The other mutant studied, R330Q, showed impaired functionality as measured by its reduced transducin activation ability when compared to wild-type green cone opsin. A double Cys mutant that could form a stabilizing disulfide bond was used in an attempt to address the instability of the green opsin mutants. Our results suggest the presence of key intramolecular networks which may be disrupted in deuteranopia, and these findings could help in finding therapeutic solutions for treating color blindness. Furthermore, our results can also have implications for the study of other visual pigments and other rhodopsin-like G protein-coupled receptors.

Human Blue Cone Opsin Regeneration Involves Secondary Retinal Binding with Analog Specificity

“…The presence of retinylidene SB in visual pigments can be measured by acidifying the sample using 2 N H 2 SO 4 to pH 1.9 (21). In the dark state, acidification disrupts the spectral tuning arising from retinal and opsin interactions, but not the protonated SB that shifts the absorbance maximum to 440 nm (21,22). Here, we used acidification to measure the regeneration of photoactivated blue cone opsin.…”

Whale shark rhodopsin adapted to its vertically wide-ranging lifestyle

“…The reduced thermal stability of the whale shark RHO is reminiscent of cone opsins that have lower thermal stability than RHOs to enable daylight vision. In fact, compared to the human RHO (Janz et al, 2003), the whale shark RHO has lower thermal stability, which is rather closer to that of mammalian LWS/MWS (Alexander et al, 2017; Srinivasan et al, 2017) and chicken RH2 (Sato et al, 2018). Near the sea surface, the cone opsin-like low thermal stability of whale shark RHO in high-temperature water (Figure 2C) can facilitate the photoreceptor light adaptation suitable for the bright light condition.…”

Disease mechanisms of X‐linked cone dystrophy caused by missense mutations in the red and green cone opsins

“…In rhodopsin, conformational changes in Helix 8 are intimately involved in the binding process of visual arrestin, and chemical modifications of H8 inhibit arrestin binding 50 . The R330Q mutant expressed in COS‐1 cells showed decreased phototransduction activation but maintained similar thermal and chemical stabilities as normal green cone opsin 38 . The G338 residue is well conserved in vertebrate opsins.…”

“…50 The R330Q mutant expressed in COS-1 cells showed decreased phototransduction activation but maintained similar thermal and chemical stabilities as normal green cone opsin. 38 The G338 residue is well conserved in vertebrate opsins. In vitro study showed that the corresponding rhodopsin mutant G324E gave an absorbance spectrum similar to normal rhodopsin, however, G338E mutant green opsin gave no absorbance.…”