Detergent-solubilized bovine rhodopsin produces mixed detergent/lipid/protein micelles. The effect of dodecyl maltoside detergent on the thermal stability of dark-state rhodopsin, and upon formation of the different intermediates after rhodopsin photobleaching (metarhodopsin II and metarhodopsin III), and upon transducin activation has been studied. No significant effect is observed for the thermal stability of dark-state rhodopsin in the range of detergent concentrations studied, but a decrease in the stability of metarhodopsin II and an increase in metarhodopsin III formation is observed with decreasing detergent concentrations. The transducin activation process is also affected by the presence of detergent indicating that this process is dependent on the lipid micro-environment and membrane fluidity, and this stresses the importance of the native lipid environment in rhodopsin normal function.
The cytoplasmic sides of transmembrane helices 3 and 6 of G-protein-coupled receptors are connected by a network of ionic interactions that play an important role in maintaining its inactive conformation. To investigate the role of such a network in rhodopsin structure and function, we have constructed single mutants at position 134 in helix 3 and at positions 247 and 251 in helix 6, as well as combinations of these to obtain double mutants involving the two helices. These mutants have been expressed in COS-1 cells, immunopurified using the rho-1D4 antibody, and studied by UV-visible spectrophotometry. Most of the single mutations did not affect chromophore formation, but double mutants, especially those involving the T251K mutant, resulted in low yield of protein and impaired 11-cisretinal binding. Single mutants E134Q, E247Q, and E247A showed the ability to activate transducin in the dark, and E134Q and E247A enhanced activation upon illumination, with regard to wild-type rhodopsin. Mutations E247A and T251A (in E134Q/E247A and E134Q/T251A double mutants) resulted in enhanced activation compared with the single E134Q mutant in the dark. A role for Thr 251 in this network is proposed for the first time in rhodopsin. As a result of these mutations, alterations in the hydrogen bond interactions between the amino acid side chains at the cytoplasmic region of transmembrane helices 3 and 6 have been observed using molecular dynamics simulations. Our combined experimental and modeling results provide new insights into the details of the structural determinants of the conformational change ensuing photoactivation of rhodopsin.
The natural ligand of the retinal photoreceptor rhodopsin, 11-cis-retinal, is isomerized to its all-trans configuration as a consequence of light absorption in the first step of the visual phototransduction process. Here we show, by means of difference spectroscopy and high-performance liquid chromatography analysis, that thermal denaturation of rhodopsin induces the same type of isomerization. This effect is likely due to thermally induced conformational rearrangements of amino acid residues in the retinal-binding pocket--possibly implying helical movements--and highlights the tight coupling between 11-cis-retinal and opsin. This effect could have implications in the instability and functional changes seen for certain mutations in rhodopsin associated with retinal disease, and in the stability of the different conformers induced by mutations in other G protein-coupled receptors.
G-protein-coupled receptors (GPCR) are a major class of membrane proteins belonging to a continuously growing superfamily. These receptors play a critical role in signal transduction, and are among the most important pharmacological drug targets. The first structural model for the GPCR superfamily was the bacterial protein bacteriorhodopsin with its characteristic seven transmembrane (TM) helical architecture. The visual photoreceptor rhodopsin is a better model for GPCR, and the recent elucidation of the crystal structure of bovine rhodopsin has renewed the interest in this receptor as a template for molecular modeling of other GPCR, particularly for the implications in ligand design and drug discovery. In this work different specific structural elements of rhodopsin are reviewed and the role of conserved motifs, like those associated with receptor function, is analyzed. The specific characteristics of the membrane-embedded ligand-binding domain are described. Other aspects, like receptor dimerization or the constitutive activity mechanism, are also outlined. The importance of acquiring knowledge of the active conformation of the receptor by means of both modeling and experimental techniques is also highlighted. In this regard, the model of the activated form of rhodopsin is currently under investigation, and it may provide useful information for pharmaceutical design. Rhodopsin will continue to be a widely used model for GPCR but rhodopsin-based approaches have to be complemented by other theoretical and experimental approaches -while waiting for the crystal structure of other members of the superfamily- if these want to be successfully used for drug discovery.
The naturally occurring mutations G51A and G51V in transmembrane helix I and G89D in the transmembrane helix II of rhodopsin are associated with the retinal degenerative disease autosomal dominant retinitis pigmentosa. To probe the orientation and packing of helices I and II a number of replacements at positions 51 and 89 were prepared by using site-directed mutagenesis, and the corresponding proteins expressed in COS-1 cells were characterized. Mutations at position 51 (G51V and G51L) bound retinal like wild-type rhodopsin but had thermally destabilized structures in the dark, altered photobleaching behavior, destabilized metarhodopsin II active conformations, and were severely defective in signal transduction. The effects observed can be correlated with the size of the mutated side chains that would interfere with specific interhelical interaction with Val-300 in helix VII. Mutations at position 89 had sensitivity to charge, as in G89K and G89D mutants, which showed reduced transducin activation. G89K showed a second absorbing species in the UV region at 350 nm, suggesting a charge effect of the introduced lysine. Increased formation of non-active forms of rhodopsin, like metarhodopsin III, may have some influence in the molecular defect underlying retinitis pigmentosa in the mutants studied. At the structural level, the effect of the mutations analyzed can be rationalized assuming a very specific set of tertiary interactions in the interhelical packing of the transmembrane segments of rhodopsin.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.