Unusual Thermal and Conformational Properties of the Rhodopsin Congenital Night Blindness Mutant Thr-94 → Ile

Abstract

Naturally occurring point mutations in the opsin gene cause the retinal diseases retinitis pigmentosa and congenital night blindness. Although these diseases involve similar mutations in very close locations in rhodopsin, their progression is very different, with retinitis pigmentosa being severe and causing retinal degeneration. We report on the expression and characterization of the recently found T94I mutation associated with congenital night blindness, in the second transmembrane helix or rhodopsin, and mutations at the same site. T94I mutant rhodopsin folded properly and was able to bind 11-cis-retinal to form chromophore, but it showed a blue-shifted visible band at 478 nm and reduced molar extinction coefficient. Furthermore, T94I showed dramatically reduced thermal stability, extremely long lived metarhodopsin II intermediate, and highly increased reactivity toward hydroxylamine in the dark, when compared with wild type rhodopsin. The results are consistent with the location of Thr-94 in close proximity to Glu-113 counterion in the vicinity of the Schiff base linkage and suggest a role for this residue in maintaining the correct dark inactive conformation of the receptor. The reported results, together with previously published data on the other two known congenital night blindness mutants, suggest that the molecular mechanism underlying this disease may not be structural misfolding, as proposed for retinitis pigmentosa mutants, but abnormal functioning of the receptor by decreased thermal stability and/or constitutive activity.Naturally occurring mutations in rhodopsin (most of them single amino acid replacements) are associated with retinal disease. Most of these are the cause of retinitis pigmentosa (RP), 1 a group of inherited retinal degenerative diseases (1-3) that leads to blindness by causing photoreceptor cell death (4). Over 100 mutations have been found to date in the opsin gene associated with RP, most of them being inherited as an autosomal dominant trait (1). These are located in all the three domains of rhodopsin, namely the intradiscal, the transmembrane, and the cytoplasmic domains of the protein (5). Mutations in the transmembrane and intradiscal domains of rhodopsin that cause RP have been shown to cause misfolding of the mutant proteins (6 -8). Only a very small number of mutations have been associated with the retinal disease characterized by a congenital night blindness (CNB) phenotype. CNB appears to be a stable condition that does not seem to cause photoreceptor degeneration resulting mainly in night vision impairment. Two of these mutations were previously studied, namely G90D (9) and A292E (10) in transmembrane helices II and VII of rhodopsin, respectively. The mechanism of action of the G90D and A292E mutations was proposed to be persistent activation of the phototransduction pathway by constitutive activity of the mutant proteins (9 -11). Another possible explanation for the observed G90D mutant phenotype has been proposed, i.e. enhanced rate of thermal isomerization due to lo...

A Naturally Occurring Mutation of the Opsin Gene (T4R) in Dogs Affects Glycosylation and Stability of the G Protein-coupled Receptor

“…Janz et al (51) In addition, congenital night blindness mutants that are characterized by an open conformation of the chromophorebinding site for nucleophiles have been identified. For example, T94I opsin folds properly and binds 11-cis-retinal to form pigment, but T94I Rho displays reduced thermal stability, has a long lived Meta II photostate, and shows highly increased reactivity toward NH 2 OH in the dark compared with WT Rho (54,55). In addition, it has been shown that T94I opsin is constitutively active (55).…”

Structural and Functional Role of Helices I and II in Rhodopsin

“…The G90D mutant is associated with the retinal disease congenital night blindness, and the aspartate in the mutant would be in the retinal binding pocket next to the Schiff base (55). In the same region, another mutation related to congenital night blindness, T94I, is reported to have very unusual thermal and conformational properties (44). Gly-89 forms a cavity together with glycine 90, and a water molecule is located very close to the carbonyl group of Gly-89 at hydrogen-bonding distance (10).…”

“…The mutant proteins in the dark are less stable than WT, with the stability decreasing with increasing size of the side chain. A number of factors are shown to affect the thermal stability of dark-state rhodopsin, including retinal disease-causing mutations (44) or zinc binding (45). Other factors mediating the stability of dark-state rhodopsin are proposed, like a conserved ion-pair interaction in the intradiscal loop E-2 of rhodopsin (46).…”

Retinal Attachment Instability Is Diversified among Mammalian Melanopsins

“…We used this approach because retinal absorbance decreases after Schiff base hydrolysis (52), and this type of analysis is widely used to assess retinal-protein stability in purified rod and cone visual pigments (30,53,54).…”