title: Thermal stability of rhodopsin and progression of retinitis pigmentosa: comparison of S186W and D190N rhodopsin mutants.
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.
authors.affiliation: Department of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, Connecticut 06520, USA.
authors.forename: Monica Yun
authors.initials: MY
authors.lastname: Liu
authors.forename: Jian
authors.initials: J
authors.lastname: Liu
authors.forename: Devi
authors.initials: D
authors.lastname: Mehrotra
authors.forename: Yuting
authors.initials: Y
authors.lastname: Liu
authors.forename: Ying
authors.initials: Y
authors.lastname: Guo
authors.forename: Pedro A
authors.initials: PA
authors.lastname: Baldera-Aguayo
authors.forename: Victoria L
authors.initials: VL
authors.lastname: Mooney
authors.forename: Adel M
authors.initials: AM
authors.lastname: Nour
authors.forename: Elsa C Y
authors.initials: EC
authors.lastname: Yan
mesh_terms: D020134:Catalytic Domain; D002462:Cell Membrane / Q000378:metabolism; D057809:HEK293 Cells; D006207:Half-Life; D006801:Humans; D006860:Hydrogen Bonding; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007536:Isomerism; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D020125:Mutation, Missense; D011489:Protein Denaturation; D055550:Protein Stability; D021381:Protein Transport; D012174:Retinitis Pigmentosa / Q000235:genetics; D012243:Rhodopsin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012545:Schiff Bases / Q000737:chemistry; D013056:Spectrophotometry, Ultraviolet
chemical_list: D012545:Schiff Bases; D012243:Rhodopsin
keywords: 7-Helix Receptor; Dark Noise; G Protein-coupled Receptors; Hydrogen Bond Network; Hydrolysis; Isomerization; Membrane Proteins; Mutant; Rhodopsin; Thermal Decay