Formation of the meta II photointermediate is accompanied by conformational changes in the cytoplasmic surface of rhodopsin

Jf, Resek; Zt, Farahbakhsh; Wl, Hubbell; Hg, Khorana

doi:10.1021/bi00096a012

Cited by 149 publications

(183 citation statements)

References 43 publications

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“…The expression construct, pMT4-Rho, containing the base mutant ⌽ (C140S/C316S) is described in ref. 45. Cysteine double mutations were introduced in the ⌽, and DEAE dextran-mediated transient transfections of the construct in mammalian COS1 cells were used to induce expression of the mutant opsin (46).…”

Section: Methodsmentioning

confidence: 99%

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High-resolution distance mapping in rhodopsin reveals the pattern of helix movement due to activation

Altenbach

Kusnetzow

Ernst

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

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431

454

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Site-directed spin labeling has qualitatively shown that a key event during activation of rhodopsin is a rigid-body movement of transmembrane helix 6 (TM6) at the cytoplasmic surface of the molecule. To place this result on a quantitative footing, and to identify movements of other helices upon photoactivation, double electron-electron resonance (DEER) spectroscopy was used to determine distances and distance changes between pairs of nitroxide side chains introduced in helices at the cytoplasmic surface of rhodopsin. Sixteen pairs were selected from a set of nine individual sites, each located on the solvent exposed surface of the protein where structural perturbation due to the presence of the label is minimized. Importantly, the EPR spectra of the labeled proteins change little or not at all upon photoactivation, suggesting that rigid-body motions of helices rather than rearrangement of the nitroxide side chains are responsible for observed distance changes. For inactive rhodopsin, it was possible to find a globally minimized arrangement of nitroxide locations that simultaneously satisfied the crystal structure of rhodopsin (Protein Data Bank entry 1GZM), the experimentally measured distance data, and the known rotamers of the nitroxide side chain. A similar analysis of the data for activated rhodopsin yielded a new geometry consistent with a 5-Å outward movement of TM6 and smaller movements involving TM1, TM7, and the C-terminal sequence following helix H8. The positions of nitroxides in other helices at the cytoplasmic surface remained largely unchanged.DEER ͉ G protein-coupled receptor ͉ photoreceptor ͉ spin labeling G protein-coupled receptors (GPCRs) constitute a large family of membrane proteins that mediate cellular responses to a variety of both physical and chemical signals.Interaction of a GPCR with a cognate signal produces a conformational change leading to an activated receptor. The activated state subsequently binds to and activates a corresponding heterotrimeric G protein, which in turn triggers events that ultimately modulate an amplified cellular response (for recent reviews, see refs. 1 and 2). The nature of the conformational switch or switches that underlie receptor activation is a subject of much interest (3, 4). Crystal structures of an inactive state of the GPCRs rhodopsin (5-9) and ␤ 2 -adrenergic receptor (10, 11) have been reported, and they provide a crucial foundation for designing and interpreting the results of spectroscopic experiments aimed at revealing conformational changes associated with activation.Site-directed spin-labeling (SDSL) studies of rhodopsin provided the first direct structural evidence for an outward tilt of the cytoplasmic end of transmembrane helix 6 (TM6) as a major event in the activation of rhodopsin, both in solutions of dodecyl maltoside (DM) and in phospholipid bilayers (12-15). Data from site-directed fluorescent labeling and chemical reactivity (16), UV spectroscopy (17), zinc cross-linking of histidines (18), and disulfide cross-linking (13) offered s...

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Section: Methodsmentioning

confidence: 99%

“…Recombinant rhodopsin was regenerated with 11-cisretinal (National Eye Institute), solubilized in DM (Anatrace), and purified and spin-labeled on a 1D4 monoclonal antibody (University of British Columbia) immobilized on Sepharose as described in ref. 45. DEER Spectroscopy.…”

Section: Methodsmentioning

confidence: 99%

High-resolution distance mapping in rhodopsin reveals the pattern of helix movement due to activation

Altenbach

Kusnetzow

Ernst

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

431

454

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show abstract

“…Subsequent to PCR mutagenesis, the PCR fragments were subcloned into the pMT4 vector containing the synthetic gene of rhodopsin using XhoI and PstI restriction sites, and double mutants were constructed using the BsrI restriction site. Cysteine mutants were subcloned into the pMT4 plasmid theta, a synthetic gene of rhodopsin in which the potentially reactive background cysteine residues 140, 316, 322 and 323 were replaced with serines (31,32). All mutations were confirmed by the dideoxynucleotide sequencing method.…”

Section: Construction and Expression Of Rhodopsin Mutantsmentioning

confidence: 99%

Stability of Dark State Rhodopsin Is Mediated by a Conserved Ion Pair in Intradiscal Loop E-2

Janz

Fay

Farrens

2003

Journal of Biological Chemistry

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The rhodopsin crystal structure reveals that intradiscal loop E-2 covers the 11-cis-retinal, creating a "retinal plug." Recently, we noticed the ends of loop E-2 are linked by an ion pair between residues Arg-177 and Asp-190, near the highly conserved disulfide bond. This ion pair appears biologically significant; it is conserved in almost all vertebrate opsins and may occur in other G-protein-coupled receptors. We report here that the Arg-177/Asp-190 ion pair is critical for the folding and stability of dark state rhodopsin. We find ion pair mutants that regenerate with retinal are functionally and spectrally wild-type-like yet thermally unstable in their dark state because of rapid hydrolysis of the retinal Schiff base linkage. Surprisingly, Arrhenius analysis indicates that the activation energies for the hydrolysis process are similar between the ion pair mutants and wild-type rhodopsin. Furthermore, the ion pair mutants do not show increased reactivity toward hydroxylamine, suggesting that their instability is not caused by an increased exposure to bulk solvent. Our results indicate that the loop E-2 ion pair is important for rhodopsin stability and thus suggest that retinitis pigmentosa observed in patients with Asp-190 mutations may in part be the result of thermally unstable rhodopsin proteins.

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“…2). Some conformational change in the cytoplasmic domains accompanied by the helical movement is monitored by the site-directed spin-labeled reagent 73 and fluorescent label 74 but details are still unknown.…”

Section: The Interaction Of Rhodopsin and G Proteinmentioning

confidence: 99%

Diversity of visual pigments from the viewpoint of G protein activation—comparison with other G protein-coupled receptors

Shichida

Yamashita

2003

Photochem Photobiol Sci

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Formation of the meta II photointermediate is accompanied by conformational changes in the cytoplasmic surface of rhodopsin

Cited by 149 publications

References 43 publications

High-resolution distance mapping in rhodopsin reveals the pattern of helix movement due to activation

High-resolution distance mapping in rhodopsin reveals the pattern of helix movement due to activation

Stability of Dark State Rhodopsin Is Mediated by a Conserved Ion Pair in Intradiscal Loop E-2

Diversity of visual pigments from the viewpoint of G protein activation—comparison with other G protein-coupled receptors

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