Solution structure of the sixth transmembrane helix of the G-protein-coupled receptor, rhodopsin11This work was supported by National Institutes of Health Grant EY03328 and in part by CA16056.

Chopra, A.; Yèagle, Philip L.; Alderfer, James A.; Albert, Arlene D.

doi:10.1016/s0005-2736(99)00212-6

Cited by 38 publications

(24 citation statements)

References 13 publications

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“…Subsequently, a low resolution view of the helices was obtained from cryo-EM studies on two-dimensional crystals of frog rhodopsin (34). In addition, information was obtained by NMR (35)(36)(37)(38), spin label EPR (39,40), and disulfide formation rates (41,42). Most detailed information we now gain from the crystal structure of rhodopsin, which was solved in 2000 (1), * The costs of publication of this article were defrayed in part by the payment of page charges.…”

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The Three-dimensional Structure of Bovine Rhodopsin Determined by Electron Cryomicroscopy

Krebs

Edwards

Villa

et al. 2003

Journal of Biological Chemistry

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G-protein-coupled receptors are integral membrane proteins that respond to environmental signals and initiate signal transduction pathways, which activate cellular processes. Rhodopsin, a well known member of the G-protein-coupled receptor family, is located in the disk membranes of the rod outer segment, where it is responsible for the visualization of dim light. Rhodopsin is the most extensively studied G-protein-coupled receptor, and knowledge about its structure serves as a template for other related receptors. We have gained detailed structural knowledge from the crystal structure (1), which was solved by x-ray crystallography in 2000 using three-dimensional crystals. Here we report a three-dimensional density map of bovine rhodopsin determined by electron cryomicroscopy of two-dimensional crystals with p22 1 2 1 symmetry. The usage of relatively small and disordered crystals made the process of structure determination challenging. Special attention was paid to the extraction of amplitudes and phases, since usable raw data were limited to a maximum tilt of 45°. In the refinement process, an improved unbending procedure was applied. This led to a final resolution of 5.5 Å in the membrane plane and ϳ13 Å perpendicular to it, making our electron density map the most accurate map of a G-protein-coupled receptor currently available by electron microscopy. Most important is the information we gain about the center of the membrane plane and the orientation of the molecule relative to the bilayer. This information cannot be retrieved from the three-dimensional crystals. In our electron density map, all seven transmembrane helices were identified, and their arrangement is in agreement with the arrangement known from the crystal structure (1). In the retinal binding pocket, a density peak adjacent to helix 3 suggests the position of the ␤-ionine ring of the chromophore, and in its vicinity several of the bigger amino acids can be identified. G-protein-coupled receptors (GPCRs)1 are a large group of integral membrane proteins that provide molecular links between extracellular signals and intracellular processes (2-7). Many neurotransmitters, hormones, and drugs produce their intracellular signaling through the mediation of G-protein-coupled receptors. The binding of a hormone or neurotransmitter causes a change in the structure of the receptor, which then activates a G-protein. Different members of the receptor family respond to different ligands, and the binding site for these ligands is in the membrane-embedded part of the protein (8, 9).One of the most widely studied GPCRs is rhodopsin (1, 10 -18). Rhodopsin, located in the retina disc membrane of the eye, is responsible for the visualization of dim light. It is composed of the protein opsin (ϳ40 kDa) covalently linked to 11-cis-retinal through Lys 296 of helix 7 (19). The chromophore 11-cis-retinal isomerizes upon light activation to its all-trans conformation, which changes the arrangement of the transmembrane helices (20). This triggers the signal transduction c...

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confidence: 99%

The Three-dimensional Structure of Bovine Rhodopsin Determined by Electron Cryomicroscopy

Krebs

Edwards

Villa

et al. 2003

Journal of Biological Chemistry

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“…[11][12][13] In most cases the approach used by these authors was to synthesize peptides containing 15-20 residues representing overlapping portions of the transmembrane domain, and to determine the domain structure by superposing the structures of the shorter fragments. This approach, while successful in some cases, experienced complications due to end effects, which for example, prevented the reconstruction of the entire fifth transmembrane domain of rhodopsin from the two peptide fragments.…”

Section: Discussionmentioning

confidence: 99%

High resolution NMR analysis of the seven transmembrane domains of a heptahelical receptor in organic‐aqueous medium

et al. 2002

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The NMR properties of seven peptides representing the transmembrane domains of the alpha-factor receptor from Saccharomyces cerevisiae were examined in trifluoroethanol/water (4:1) at 10 to 55 degrees C. The parameters extracted indicated all peptides were helical in this membrane mimetic solvent. Using chemical shift indices as the criterion, helicity varied from 64 to 83%. The helical residues in the peptides corresponded to the region predicted to cross the hydrocarbon interior of the bilayer. A study of a truncated 25-residue peptide corresponding to domain 2 gave evidence that the helix extended all the way to the N-terminus of this peptide, indicating that sequence and not chain end effects are very important in helix termination for our model peptides. Both nuclear Overhauser effect spectroscopy (NOESY) connectivities and chemical shift indices revealed significant perturbations around prolyl residues in the helices formed by transmembrane domains 6 and 7. Molecular models of the transmembrane domains indicate that helices for domains 6 and 7 are severely kinked at these prolyl residues. The helix perturbation around proline 258 in transmembrane domain 6 correlates with mutations that cause phenotypic changes in this receptor.

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“…[54][55][56][57][58][59][60][61][62][63] Because membrane proteins exert their functions in an amphiphilic membrane environment, these proteins can exhibit some of their native local character when studied under isotropic lipophilic or amphiphilic conditions. This approach has been used successfully to study single transmembrane helices of bacteriorhodopsin, 50 rhodopsin, 17,18,24,41,49,53 and the subunit C of F1F0 ATP synthase. 64 Nevertheless, the use of DMSO to study membrane proteins remains controversial.…”

Section: Nmr Characterization Of Cb2(27-101)mentioning

confidence: 99%

A transmembrane helix‐bundle from G‐protein coupled receptor CB2: Biosynthesis, purification, and NMR characterization

et al. 2006

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The cannabinoid receptor subtype 2 (CB2) is a member of the G-protein coupled receptor (GPCR) superfamily. As the relationship between structure and function for this receptor remains poorly understood, the present study was undertaken to characterize the structure of a segment including the first and second transmembrane helix (TM1 and TM2) domains of CB2. To accomplish this, a transmembrane double-helix bundle from this region was expressed, purified, and characterized by NMR. Milligrams of this hydrophobic fragment of the receptor were biosynthesized using a fusion protein overexpression strategy and purified by affinity chromatography combined with reverse phase HPLC. Chemical and enzymatic cleavage methods were implemented to remove the fusion tag. The resultant recombinant protein samples were analyzed and confirmed by HPLC, mass spectrometry, and circular dichroism (CD). The CD analyses of HPLC-purified protein in solution and in DPC micelle preparations suggested predominant alpha-helical structures under both conditions. The 13C/15N double-labeled protein CB2(27-101) was further verified and analyzed by NMR spectroscopy. Sequential assignment was accomplished for more than 80% of residues. The 15N HSQC NMR results show a clear chemical shift dispersion of the amide nitrogen-proton correlation indicative of a pure double-labeled polypeptide molecule. The results suggest that this method is capable of generating transmembrane helical bundles from GPCRs in quantity and purity sufficient for NMR and other biophysical studies. Therefore, the biosynthesis of GPCR transmembrane helix bundles represents a satisfactory alternative strategy to obtain and assemble NMR structures from recombinant "building blocks."

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Solution structure of the sixth transmembrane helix of the G-protein-coupled receptor, rhodopsin11This work was supported by National Institutes of Health Grant EY03328 and in part by CA16056.

Cited by 38 publications

References 13 publications

The Three-dimensional Structure of Bovine Rhodopsin Determined by Electron Cryomicroscopy

The Three-dimensional Structure of Bovine Rhodopsin Determined by Electron Cryomicroscopy

High resolution NMR analysis of the seven transmembrane domains of a heptahelical receptor in organic‐aqueous medium

A transmembrane helix‐bundle from G‐protein coupled receptor CB2: Biosynthesis, purification, and NMR characterization

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