Abstract
Over 100 point mutations in the rhodopsin gene have been associated with retinitis pigmentosa (RP), a family of inherited visual disorders. Among these, we focused on characterizing the S186W mutation. We compared the thermal properties of the S186W mutant with another RP-causing mutant, D190N, and with WT rhodopsin. To assess thermal stability, we measured the rate of two thermal reactions contributing to the thermal decay of rhodopsin as follows: thermal isomerization of 11-cis-retinal and hydrolysis of the protonated Schiff base linkage between the 11-cis-retinal chromophore and opsin protein. We used UV-visible spectroscopy and HPLC to examine the kinetics of these reactions at 37 and 55 °C for WT and mutant rhodopsin purified from HEK293 cells. Compared with WT rhodopsin and the D190N mutant, the S186W mutation dramatically increases the rates of both thermal isomerization and dark state hydrolysis of the Schiff base by 1-2 orders of magnitude. The results suggest that the S186W mutant thermally destabilizes rhodopsin by disrupting a hydrogen bond network at the receptor's active site. The decrease in the thermal stability of dark state rhodopsin is likely to be associated with higher levels of dark noise that undermine the sensitivity of rhodopsin, potentially accounting for night blindness in the early stages of RP. Further studies of the thermal stability of additional pathogenic rhodopsin mutations in conjunction with clinical studies are expected to provide insight into the molecular mechanism of RP and test the correlation between rhodopsin's thermal stability and RP progression in patients.
Highlights
The S186W mutation in rhodopsin causes retinitis pigmentosa (RP)
Immunocytochemistry—Before investigating the thermal processes of the S186W mutant and comparing them with those of wild type (WT) rhodopsin and the D190N mutant, we examined the subcellular localization of the proteins to exclude misfolding and mistrafficking as the mechanism of RP for these mutations
The S186W mutation is associated with RP, it has not been previously characterized at the molecular level
Summary
Results: The mutation expedites thermal isomerization and hydrolysis of the Schiff base by orders of magnitude. We measured the rate of two thermal reactions contributing to the thermal decay of rhodopsin as follows: thermal isomerization of 11-cis-retinal and hydrolysis of the protonated Schiff base linkage between the 11-cisretinal chromophore and opsin protein. Compared with WT rhodopsin and the D190N mutant, the S186W mutation dramatically increases the rates of both thermal isomerization and dark state hydrolysis of the Schiff base by 1–2 orders of magnitude. The decrease in the thermal stability of dark state rhodopsin is likely to be associated with higher levels of dark noise that undermine the sensitivity of rhodopsin, potentially accounting for night blindness in the early stages of RP. Further studies of the thermal stability of additional pathogenic rhodopsin mutations in conjunction with clinical studies are expected to provide insight into the molecular mechanism of RP and test the correlation between rhodopsin’s thermal stability and RP progression in patients
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