Dataset Information

Light Dynamics of the Retinal-Disease-Relevant G90D Bovine Rhodopsin Mutant.

ABSTRACT: The RHO gene encodes the G-protein-coupled receptor (GPCR) rhodopsin. Numerous mutations associated with impaired visual cycle have been reported; the G90D mutation leads to a constitutively active mutant form of rhodopsin that causes CSNB disease. We report on the structural investigation of the retinal configuration and conformation in the binding pocket in the dark and light-activated state by solution and MAS-NMR spectroscopy. We found two long-lived dark states for the G90D mutant with the 11-cis retinal bound as Schiff base in both populations. The second minor population in the dark state is attributed to a slight shift in conformation of the covalently bound 11-cis retinal caused by the mutation-induced distortion on the salt bridge formation in the binding pocket. Time-resolved UV/Vis spectroscopy was used to monitor the functional dynamics of the G90D mutant rhodopsin for all relevant time scales of the photocycle. The G90D mutant retains its conformational heterogeneity during the photocycle.

SUBMITTER: Kubatova N

PROVIDER: S-EPMC7496284 | biostudies-literature | 2020 Jun

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Light Dynamics of the Retinal-Disease-Relevant G90D Bovine Rhodopsin Mutant.

Kubatova Nina N Mao Jiafei J Eckert Carl Elias CE Saxena Krishna K Gande Santosh L SL Wachtveitl Josef J Glaubitz Clemens C Schwalbe Harald H

Angewandte Chemie (International ed. in English) 20200813 36

The RHO gene encodes the G-protein-coupled receptor (GPCR) rhodopsin. Numerous mutations associated with impaired visual cycle have been reported; the G90D mutation leads to a constitutively active mutant form of rhodopsin that causes CSNB disease. We report on the structural investigation of the retinal configuration and conformation in the binding pocket in the dark and light-activated state by solution and MAS-NMR spectroscopy. We found two long-lived dark states for the G90D mutant with the ...[more]

PMID: 32602600

Similar Datasets

Project description:Rhodopsin is a canonical member of class A of the G protein-coupled receptors (GPCRs) that are implicated in many of the drug interventions in humans and are of great pharmaceutical interest. The molecular mechanism of rhodopsin activation remains unknown as atomistic structural information for the active metarhodopsin II state is currently lacking. Solid-state (2)H NMR constitutes a powerful approach to study atomic-level dynamics of membrane proteins. In the present application, we describe how information is obtained about interactions of the retinal cofactor with rhodopsin that change with light activation of the photoreceptor. The retinal methyl groups play an important role in rhodopsin function by directing conformational changes upon transition into the active state. Site-specific (2)H labels have been introduced into the methyl groups of retinal and solid-state (2)H NMR methods applied to obtain order parameters and correlation times that quantify the mobility of the cofactor in the inactive dark state, as well as the cryotrapped metarhodopsin I and metarhodopsin II states. Analysis of the angular-dependent (2)H NMR line shapes for selectively deuterated methyl groups of rhodopsin in aligned membranes enables determination of the average ligand conformation within the binding pocket. The relaxation data suggest that the beta-ionone ring is not expelled from its hydrophobic pocket in the transition from the pre-activated metarhodopsin I to the active metarhodopsin II state. Rather, the major structural changes of the retinal cofactor occur already at the metarhodopsin I state in the activation process. The metarhodopsin I to metarhodopsin II transition involves mainly conformational changes of the protein within the membrane lipid bilayer rather than the ligand. The dynamics of the retinylidene methyl groups upon isomerization are explained by an activation mechanism involving cooperative rearrangements of extracellular loop E2 together with transmembrane helices H5 and H6. These activating movements are triggered by steric clashes of the isomerized all-trans retinal with the beta4 strand of the E2 loop and the side chains of Glu(122) and Trp(265) within the binding pocket. The solid-state (2)H NMR data are discussed with regard to the pathway of the energy flow in the receptor activation mechanism.

Project description:The effect of acute exposure to various intensities of white light on visual behavior and retinal structure was evaluated in the T4R RHO dog, a naturally-occurring model of autosomal dominant retinitis pigmentosa due to a mutation in the Rhodopsin gene. A total of 14 dogs (ages: 4-5.5 months) were used in this study: 3 homozygous mutant RHO(T4R/T4R), 8 heterozygous mutant RHO(T4R/+), and 3 normal wild-type (WT) dogs. Following overnight dark adaptation, the left eyes were acutely exposed to bright white light with a monocular Ganzfeld dome, while the contralateral right eye was shielded. Each of the 3 homozygous (RHO(T4R/T4R)) mutant dogs had a single unilateral light exposure (LE) to a different (low, moderate, and high) dose of white light (corneal irradiance/illuminance: 0.1 mW/cm(2), 170 lux; 0.5 mW/cm(2), 820 lux; or 1 mW/cm(2), 1590 lux) for 1 min. All 8 heterozygous (RHO(T4R/+)) mutant dogs were exposed once to the same moderate dose of light. The 3 WT dogs had their left eyes exposed 1, 2, or 3 times to the same highest dose of light. Visual function prior to LE and at 2 weeks and 33 weeks after exposure was objectively assessed in the RHO(T4R/T4R) and WT dogs by using an obstacle-avoidance course. Transit time through the obstacle course was measured under different scotopic to photopic ambient illuminations. Morphological retinal changes were evaluated by non-invasive in vivo cSLO/sdOCT imaging and histology before and at several time-points (2-36 weeks) after light exposure. The analysis of the transit time through the obstacle course showed that no differences were observed in any of mutant or WT dogs at 2 weeks and 33 weeks post LE. The RHO(T4R/T4R) retina exposed to the lowest dose of white light showed no obvious changes in ONL thickness at 2 weeks, but mild decrease was noted 36 weeks after LE. The RHO(T4R/T4R) retina that received a moderate dose (showed an obvious decrease in ONL thickness along the superior and temporal meridians at 2 weeks post LE with more severe damage at 36 weeks post LE in all four meridians. The RHO(T4R/T4R) retina exposed to the high dose showed at 2 weeks after LE extensive ONL damage in all four meridians. This light intensity did not cause any retinal damage in WT dogs even after repeated (up to 3) LE. Analysis of ONL thickness in heterozygous mutant dogs exposed to the moderate dose of light confirmed the increased sensitivity to light damage of the superior/tapetal retina, and the occurrence of an ongoing cell death process several weeks after the acute LE. In conclusion, a short single exposure to a dose of white light that is not retinotoxic in WT dogs causes in the T4R RHO retina an acute loss of ONL in the central to mid peripheral region that keeps progressing over the course of several weeks. However, this severe retinal damage does not affect visual behavior presumably because of islands of surviving photoreceptors found in the area centralis including the newly discovered canine fovea-like area, and the lack of damage to peripheral photoreceptors.

Dataset Information

Light Dynamics of the Retinal-Disease-Relevant G90D Bovine Rhodopsin Mutant.

Publications

Light Dynamics of the Retinal-Disease-Relevant G90D Bovine Rhodopsin Mutant.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets