Location of Trp265 in Metarhodopsin II: Implications for the Activation Mechanism of the Visual Receptor Rhodopsin

Crocker, Evan; Eilers, Markus; Ahuja, Shivani; Horn̆ák, Viktor; Hirshfeld, Amiram; Sheves, Mordechai; Smith, Steven O.

doi:10.1016/j.jmb.2005.12.046

Cited by 132 publications

(210 citation statements)

References 59 publications

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“…The breakage of the salt bridge may be facili- In the opsin and opsin* structure, Trp-265 on TM6 is shifted toward the position previously occupied by the retinal β-ionone ring, which in the rhodopsin structure is tethered by a set of hydrophobic residues (Phe-212, Phe-261 and Trp-265) [47,120,134]. As shown in figure 3.11 and 4.6, the highly conserved Trp-265 [148,149] plays an important role in GPCRs as a "toggle switch" to facilitate helix movement and rearrangement of water molecules. The change of the toggle switch, depending on cis/trans isomerisation, may produce a domino effect, i.e.…”

Section: Sequential Activation Process Of Rhodopsinmentioning

confidence: 99%

Crystal structure of the ligand-free G-protein-coupled receptor opsin

Park

Scheerer

Hofmann

et al. 2008

Nature

858

925

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Compared with the known structure of inactive rhodopsin, opsin displays prominent structural changes in the conserved E(D)RY and NPxxY(x) 5,6 F regions and TM5-TM7. At the cytoplasmic side, TM6 is tilted outwards by 6-7 Å, whereas the helix structure of TM5 is more elongated and close to TM6. These structural changes, of which some are attributed to an active GPCR state, reorganize the empty retinal binding pocket to disclose two openings for exit and entry of retinal. The opsin structure thus sheds new light on binding of hydrophobic ligands to GPCRs, GPCR activation and signal transfer to the G protein.

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Section: Sequential Activation Process Of Rhodopsinmentioning

confidence: 99%

Crystal structure of the ligand-free G-protein-coupled receptor opsin

Park

Scheerer

Hofmann

et al. 2008

Nature

858

925

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“…2) suggests that TM3 also does not move. Support for the lack of change at TM2, TM3, and TM4 comes from the comparative sequence analysis (33) and solid-state NMR data (19) of Smith and coworkers, who suggest that these helices are part of a well packed core of rhodopsin that is invariant under photoactivation.…”

Section: Structural Changes At the Cytoplasmic Surface Of Rhodopsin Uponmentioning

confidence: 99%

“…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 support for this mechanism. Recent solid-state NMR studies of Smith and coworkers (19) revealed a separation of opposing residues in TM6 and TM3 at the level of the chromophore upon activation. Taken together, the NMR and SDSL data suggest that a significant length of TM6 of rhodopsin is involved in the conformational change.…”

mentioning

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.

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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“…Two research areas in which high resolution MAS experiments have proved especially successful are studies of amyloid fibrils [37,[51][52][53][54][55][56][57] and membrane proteins [42,[58][59][60][61][62][63][64][65][66][67][68][69][70][71][72][73][74][75]. However, in both of these cases, low sensitivity currently limits the information that can be gleaned from the spectra.…”

Section: Dnp Experiments On the Membrane Protein Bacteriorhodopsinmentioning

confidence: 99%

250GHz CW gyrotron oscillator for dynamic nuclear polarization in biological solid state NMR

Bajaj

Hornstein

Kreischer

et al. 2007

Journal of Magnetic Resonance

162

130

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In this paper, we describe a 250 GHz gyrotron oscillator, a critical component of an integrated system for magic angle spinning (MAS) dynamic nuclear polarization (DNP) experiments at 9T, corresponding to 380 MHz 1 H frequency. The 250 GHz gyrotron is the first gyro-device designed with the goal of seamless integration with an NMR spectrometer for routine DNP-enhanced NMR spectroscopy and has operated under computer control for periods of up to 21 days with a 100% duty cycle. Following a brief historical review of the field, we present studies of the membrane protein bacteriorhodopsin (bR) using DNP-enhanced multidimensional NMR. These results include assignment of active site resonances in [U-13 C, 15 N]-bR and demonstrate the utility of DNP for studies of membrane proteins. Next, we review the theory of gyro-devices from quantum mechanical and classical viewpoints and discuss the unique considerations that apply to gyrotron oscillators designed for DNP experiments. We then characterize the operation of the 250 GHz gyrotron in detail, including its long-term stability and controllability. We have measured the spectral purity of the gyrotron emission using both homodyne and heterodyne techniques. Radiation intensity patterns from the corrugated waveguide that delivers power to the NMR probe were measured using two new techniques to confirm pure mode content: a thermometric approach based on the temperaturedependent color of liquid crystalline media applied to a substrate and imaging with a pyroelectric camera. We next present a detailed study of the mode excitation characteristics of the gyrotron. Exploration of the operating characteristics of several fundamental modes reveals broadband continuous frequency tuning of up to 1.8 GHz as a function of the magnetic field alone, a feature that may be exploited in future tunable gyrotron designs. Oscillation of the 250 GHz gyrotron at the second harmonic of cyclotron resonance begins at extremely low beam currents (as low 12 mA) at frequencies between 320-365 GHz, suggesting an efficient route for the generation of even higher frequency radiation. The low starting currents were attributed to an elevated cavity Q, which is confirmed by cavity thermal load measurements. We conclude with an appendix containing a detailed description of the control system that safely automates all aspects of the gyrotron operation.

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Location of Trp265 in Metarhodopsin II: Implications for the Activation Mechanism of the Visual Receptor Rhodopsin

Cited by 132 publications

References 59 publications

Crystal structure of the ligand-free G-protein-coupled receptor opsin

Crystal structure of the ligand-free G-protein-coupled receptor opsin

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

250GHz CW gyrotron oscillator for dynamic nuclear polarization in biological solid state NMR

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